Molecular Phylogeny of North Mediterranean Freshwater Barbs (Genus Barbus: Cyprinidae) Inferred from...

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Molecular Phylogenetics and EvolutionVol. 14, No. 2, February, pp. 165–179, 2000doi:10.1006/mpev.1999.0702, available online at http://www.idealibrary.com on

Molecular Phylogeny of North Mediterranean Freshwater Barbs(Genus Barbus: Cyprinidae) Inferred from Cytochrome b Sequences:

Biogeographic and Systematic ImplicationsCostas S. Tsigenopoulos and Patrick Berrebi

Laboratoire Genome et Populations, C.N.R.S.—UPR 9060, c. c. 063, Universite Montpellier II, Place E. Bataillon,34 095 Montpellier Cedex 5, France

Received December 16, 1998; revised April 28, 1999

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We investigated phylogenetic relationships amongorth Mediterranean species of the genus Barbus us-

ng sequences of the cytochrome b gene. Our resultsndicate that the species belong to two major cladeshat are consistent with those previously defined fromorphological features. The first clade includes spe-

ies ranging from France to the Black Sea. In thislade, there is a well-supported monophyletic group ofarge-sized fluvio-lacustrine barbs; however, the mono-hyly of the small-sized rheophilic species is not clear.he second clade comprises species found in Spain,reece, and Asia Minor and probably represents theldest group present in the north Mediterranean riv-rs. In general, there is good concordance betweeneography and phylogenetic relationships. These re-ults are compared to those from previous morphologi-al- and allozyme-based studies and demonstrate wide-pread discordance and polyphyly in the traditionalaxonomy of the genus Barbus. This study is one of therst reporting the phylogenetic and biogeographicelationships of a genus that is widely distributed inuropean rivers and contains species that are a majoromponent of the European ichthyofauna. r 2000 Academic

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Key Words: Barbus; Cyprinidae; cytochrome b; bioge-graphy.

INTRODUCTION

The genus Barbus constitutes the dominant compo-ent of Old World cyprinids, with more than 800pecies spread over Europe, Africa, and Asia. In Eu-ope, it is found from Spain to the Black Sea and fromhe Mediterranean Sea to the Dniepr basin in theorth. Howes (1987) assigned all European taxa alongith some species from North Africa and the Middleast to the genus Barbus sensu stricto. Furthermore,pecies that belong to Barbus sensu stricto seem to have

common tetraploid origin, similar karyotypes, and
arasitic fauna (Berrebi, 1995, and references therein). I
165

The European region contains numerous endemicsh species and subspecies, most inhabiting southerneninsulas, where mountain and sea barriers, chang-ng river courses, and postglacial dispersal have playedn important role in their distribution and speciationrocesses. The genus Barbus is, therefore, not unusualn having many species living in the Mediterraneanivers, whereas central and northern Europe are domi-ated by the Danubian Barbus barbus (Banarescu,989). Only the Dalmatian coast does not have anypecies of Barbus. In this karst region, Aulopyge hy-gelii is present. This monotypic genus exhibits manyorphological characters in common with Barbus

Howes, 1987) and has the same number of chromo-omes (reviewed by Collares-Pereira, 1994).On the basis of morphological characters, some of the

pecies appear to be very similar to one another, a facthat persuaded taxonomists to organize them intopecies groups (Banarescu, 1964; Almaca, 1984). Sev-ral authors have also suggested that ecological traitsan be of great importance in defining groups in theenus (Almaca, 1981; Economidis, 1989). In particular,hey noticed that, in general, large-bodied species,hich have a strong and serrated last dorsal ray,ccupy the wide rivers in the plains, whereas smallerpecies, which have a weak (cyclolepis type) or veryeak (meridionalis type) last dorsal ray, are character-

stic of small mountainous streams. This group ofheophilic or strictly riverine small-sized barbs in-ludes many species and the monophyly of this groupas been the subject of controversy (Tsigenopoulos etl., 1999).European cyprinids have recently become the focus

or molecular studies investigating either major diver-ences within the family (Briolay et al., 1998; Gilles etl., 1998; Zardoya and Doadrio, 1998) or, more often,elationships between species and populations in spe-ific European regions (Karakousis et al., 1995; Tsi-enopoulos and Karakousis, 1996; Hanfling and Brandl,998; Brito et al., 1997; Doadrio and Carmona, 1998;
msiridou et al., 1998; Durand et al., 1999). Neverthe-
1055-7903/00 $35.00Copyright r 2000 by Academic PressAll rights of reproduction in any form reserved.

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166 TSIGENOPOULOS AND BERREBI

ess, few studies have investigated the molecular differ-ntiation among widespread freshwater species or popu-ations of the European continent.

Previous molecular studies based on allozyme dataKarakousis et al., 1995; Machordom et al., 1995;sigenopoulos et al., 1999) resolved some taxonomicroblems within the group Barbus sensu stricto, butittle is known about phylogenetic relationships amonguropean species. Furthermore, it is unclear whether

he groups defined from morphological and ecologicaleatures are well supported by molecular data.

In this study we use mitochondrial DNA sequences tonfer phylogenetic relationships among geographicallyistinct populations and species of the widespreadenus Barbus in Europe. We first investigate whetherpecies having the same ecophenotype form monophy-etic groups. A previous allozyme study (Tsigenopoulost al., 1999) did not find clear grouping of either largeuvio-lacustrine or small riverine species. Next, we

nvestigate the evolutionary history of this significantomponent of the European ichthyofauna and its rel-vance to biogeography and climatic history. Using aolecular clock for mtDNA evolution, we estimate

emporal boundaries for major divergence events withinhe lineage. Finally, we test the hypothesis that A.yegelii represents a derived form of the Europeanetraploid barbs.

MATERIALS AND METHODS

ample Collection

Barbus species were collected during fishing expedi-ions between 1993 and 1997. When possible, at least 3ndividuals per taxon were analyzed to assess variabil-ty within species and/or populations and to control forolymerase chain reaction errors. The sample includedtotal of 124 individuals from 39 populations, represent-

ng 26 species and subspecies throughout the northernediterranean basin. Table 1 lists the taxa analyzed,

he number of individuals per taxon, and the collectionites. Figure 1 shows the sample localities of eachpecies. To test whether A. hyegelii is a member of theuropean Barbus sensu stricto (tetraploid barbs,n 5 100), we also collected and sequenced 2 otherpecies of the genus that exhibit different ploidy levels:arbus intermedius (2n 5 150; Golubtsov and Krysa-ov, 1993) and B. anoplus (2n 5 50; Naran, 1997).

NA Extraction, PCR Amplification, and Sequencing

The samples used in this study were either frozenat 280°C) protein extracts used in a previous allozymetudy (Tsigenopoulos et al., 1999) or white musclereserved in 70% ethanol.From ethanol-preserved muscle tissues, total ge-

omic DNA was obtained by digesting 1 µg of tissueith 12 µl of proteinase K (20 µg/µl) in 400 µl of 5%
helex 100 resin (Bio-Rad, Hercules, CA), following the s
anufacturer’s instructions; 10 µl of supernatant weresed per 50-µl PCR. For protein extracts, we used 500l of the extract (muscle or liver) in 500 µl extractionuffer (0.05 M Tris–HCl, pH 8.0, 0.1 M EDTA, and 1%DS). Digestion was carried out overnight with theame quantity of proteinase K as above, and the DNAas purified using the standard phenol–chloroformrotocol (Kocher et al., 1989) and resuspended in 100nd 200 µl of sterile distilled water for muscle and liverrotein extracts, respectively; 5 µl were used for a 50-µlCR.For PCR amplifications we used primers L15267

58-AAT GAC TTG AAG AAC CAC CGT-38), L1580358-TGG GGC GGT TTC TCA GTA G-38), H1589158-GTT TGA TCC CGT TTC GTG TA-38), and H1646158-CTT CGG ATT ACA AGA CC-38) (Briolay et al.,998). Numbers refer to the position of the 38 end of therimers in the complete mitochondrial DNA sequencef the carp Cyprinus carpio (Chang et al., 1994). Theolume of each PCR was 50 µl and consisted of 1.8 mMgCl2, 1 µM each primer, 0.18 mM each dNTP, 1 unit

f Taq polymerase (Promega), and 13 of amplificationuffer (Promega). The combination of L15267 and15891 in the PCRs resulted in the amplification of 660p, which comprised a small part of the Glutamic acidransfer RNA (tRNA-Glu) and about half of the cyto-hrome b gene. Primers L15803 and H16461 were usedor the amplification and sequencing of the remainderf the gene for some species in the sample.Both strands were sequenced using the two initial

CR primers to control sequence accuracy and toesolve any ambiguous bases. Sequencing was per-ormed on a Pharmacia automated sequencer accordingo the manufacturer’s instructions.

Cytochrome b sequences determined in this studyere deposited in the GenBank/EMBL data base under

heAccession Nos.AF112122–AF112134 and AF112405–F112438. Sequences from Romanian populations

AF112431) were kindly provided by P. Kotlik.

ata Analyses and Phylogenetic Reconstruction

Sequences of both strands were compared to eachther and aligned to the sequence of the carp (C. carpio;hang et al., 1994) using the sequence editor ESEE

version 3.1s; Cabot, 1997). Since nuclear copies ofitochondrial genes (pseudogenes) can seriously mis-

ead phylogenetic inference (reviewed by Zhang andewitt, 1996), the mitochondrial origin of the cyto-

hrome b sequences was carefully checked using thebove computer program, which searched for nucleo-ide deletions and/or insertions and stop codons. Theet of unique mtDNA haplotypes was analyzed with theEGA program (version 1.01; Kumar et al., 1993),hich performed pairwise sequence comparisons andetermined the distribution and the amount of varia-ion (number of variable and parsimony-informative
ites) and the levels of saturation by codon position.

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167BIOGEOGRAPHY OF EUROPEAN Barbus SPECIES

he absolute numbers of transitions were plottedgainst the absolute number of transversions. In addi-ion, the transition:transversion ratio for each pair ofequences was calculated and plotted versus correctedKimura two-parameter; Kimura, 1980) genetic dis-ances.

Using the PHYLIP package (Felsenstein, 1993), dis-ance trees were generated with the neighbor-joininglgorithm (Saitou and Nei, 1987) based on Kimura’swo-parameter distances. A maximum-likelihood analy-is (Felsenstein, 1981) was also conducted with theame package, with 10 random additions of taxa andlobal rearrangements. Maximum-parsimony analysesere performed with the PAUP 3.1.1. package (Swof-

ord, 1993), using the heuristic search option andandom addition of taxa, with sites treated as unor-

TAB

List of Samples Included in the Present Analysis, w

Species Number of individuals

arbus bocagei 4. graellsii 1. meridionalis 4. caninus 4. caninus 3. petenyi 3. petenyi 1. petenyi 1. peloponnesius peloponnesius 4. peloponnesius petenyi 3. peloponnesius petenyi 4. peloponnesius rebeli 4. peloponnesius rebeli 3. cyclolepis cyclolepis 4. cyclolepis cyclolepis 4. cyclolepis strumicae 5. cyclolepis strumicae 3. cyclolepis strumicae 3. cyclolepis cholorematicus 4. cyclolepis sperchiensis 3. euboicus 3. prespensis 4. barbus 4. barbus 4. plebejus 4. plebejus 1. tyberinus 4. macedonicus 4. barbus thessalus 4. graecus 4. albanicus 4. plebejus escherichi 3. plebejus escherichi 1. cyclolepis pergamonensis 4. cyclolepis pergamonensis 2. capito pectoralis 2ulopyge hyegelii 2. intermedius 3. anoplus 2

Note. The last column corresponds to the sampling sites mapped in

ered characters. n

Alternative phylogenetic topologies were comparedsing Kishino and Hasegawa’s (1989) test implemented

n the PHYLIP package. This test compares the meannd variance of log-likelihood differences between trees,aken across sites. The relative-rate test of the RRTreerogram (Robinson et al., 1998) was also used toompare substitution rates of sequences grouped inhylogenetically defined lineages. Support values fornternal nodes of the trees were estimated using theootstrap resampling procedure (Felsenstein, 1985)ith 1000 and 100 replicates, for neighbor-joining andarsimony, respectively. Phylogenetic trees were rootedsing cytochrome b sequences of three Old Worldyprinids: C. carpio (X61010), Leuciscus leuciscusY10452), and Gobio gobio (Y10449). C. carpio belongso the same subfamily as the genus Barbus (Cyprini-

1

h Number of Individuals and Sampling Locations

Localities Map

Jerte R., Spain 1Matarrana R., Spain 2Aubaygue R., southern France 3Astico R., Vicenza, northern Italy 4Isonzo R., northeastern Italy 5Toryza R. (middle Danube basin), Slovakia 6Arges R. (lower Danube basin), Romania 7Dimbovita R. (lower Danube basin), Romania 8Alfios R., Peloponnese, Greece 9Axios (Vardar) R., western Macedonia, Greece 10Aliakmon R., western Macedonia, Greece 11Acheron R., western Greece 12Erzen R., central Albania 13Evros (Maritza) R., northeastern Greece 14Nestos (Mesta) R., northeastern Greece 15Strymon (Strouma) R., northern Greece 16Axios (Vardar) R., central Macedonia, Greece 10Pinios R., Thessaly, central Greece 17Cholorema R., Thessaly, central Greece 18Sperchios R., central Greece 19Manikiotiko Stream, Euboea Island, Greece 20Prespa Lake, northwestern Greece 21Drome R., southern France 22Oslava R. (middle Danube basin), Czech Republic 23Astico R., northern Italy 4Isonzo R., northeastern Italy 5Topino R. (Tiber basin), central Italy 24Aliakmon R., central Macedonia, Greece 11Pinios R., Thessaly, central Greece 17Sperchios R., central Greece 19Pinios R., Peloponnese, Greece 25Kirmir Stream (Sakarya basin), northern Turkey 26Karasu Stream (Sakarya basin), Inegol, Turkey 27Gediz R., Usak, Turkey 28Gumuldur Stream (Menderez basin), Izmir, Turkey 29Gediz R., Usak, Turkey 28Krka R., Croatia 30Tana Lake, EthiopiaBuffalo R., East Cape, South Africa

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istant cyprinid groups (Leuciscinae and Gobioninae,espectively) (Briolay et al., 1998; Gilles et al., 1998).

RESULTS

Cytochrome b sequences of 594 nucleotides in lengthcoding for 198 amino acids) were obtained for each ofhe 124 individuals in Table 1. We identified 47 uniqueaplotypes, in which no insertions or deletions (indels)ere detected.

ompositional Bias and Saturation

Mean base composition in cytochrome b sequencesas similar to those previously reported for cyprinids

Briolay et al., 1998; Gilles et al., 1998) and for Actinop-erygian fish in general (Cantatore et al., 1994; Lydeardnd Roe, 1997), with low G content (16.4%) and almostqual A, T, and C content (26.9, 28.3, and 28.4%,espectively). Strong bias in base composition is aeature typical of the cytochrome b gene and of other

itochondrial protein-coding genes (Brown, 1985; Ir-in et al., 1991). Significant compositional biases existt the second and especially the third codon position,here there is a marked underrepresentation of gua-ine (15.7 and 7.0%, respectively).Among 594 nucleotide sites, 245 were variable and

92 were informative for parsimony, and 76 and 82% ofhese substitutions, respectively, occurred in the thirdodon position. Transitional differences were nearlyour times more numerous than transversions. In Fig.A, we plot the absolute number of transitions versusransversions estimated from substitutions at all codonositions. In Fig. 2B, we plot the absolute number ofransitions versus transversions estimated from substi-utions at only the third codon position. The results ofhe analysis show that transitions, mainly at the thirdodon position, are saturated only when Europeanarbus species are compared to the African barbs and

he outgroups. The examination of transition:transver-ion ratio in the data indicated a decline in this ratioith increasing genetic divergence (Fig. 3). This ratiopproached the value of approximately 2.5 for compari-ons among the most divergent taxa, i.e., when thehree outgroups and the two African Barbus species areompared to the European Barbus taxa. To investigateelationships within European Barbus species, noeighting of transversions with respect to transitionsas performed, and thus all substitutions were in-

luded in the phylogenetic analyses.

enetic Divergence

Populations that belong to B. peloponnesius, B. cani-us, B. petenyi, and B. cyclolepis strumicae show veryigh levels of intraspecific genetic variation (correctedenetic distances of 9.5, 9.3, 7.0, and 6.1% between
opulations, respectively). In the case of B. caninus, the b
opulations from the Astico and Isonzo rivers showery divergent haplotypes, whereas B. plebejus popula-ions from these rivers carry the same haplotype.urprisingly, from the opposite sides of the Aegean Sea,he haplotypes of B. graecus (Greece) and B. capitoectoralis (Turkey) differ by only two nonsynonymousubstitutions.The most polymorphic populations, both with three

aplotypes present, were found to be those from thetrymon and Pinios rivers in Greece. The dominantaplotype of the Strymon river (B. cyclolepis strumi-ae-1) was the one found in individuals from the Axiosiver. In the Pinios river, we also detected a mixture ofaplotypes, including haplotypes found in theholorema (B. cyclolepis cholorematicus) and Sperchios

B. cyclolepis sperchiensis) rivers.

hylogenetic Relationships

All analyses recovered slightly different tree topolo-ies, which differ only at weakly supported internalodes. Therefore, we present only the tree from theeighbor-joining analysis (Fig. 4) and discuss differ-nces with other topologies where relevant. All treeopologies supported the monophyly of the Europeanarbus species and a close relationship of A. hyegeliiith these species. Within the European species twoell-supported clades were present. The first clade

ontains species from the western (B. bocagei and B.raellsii) and eastern (B. graecus, B. albanicus, and B.apito pectoralis) sides of the Mediterranean Sea. Theecond clade includes species ranging from France tohe Black Sea (Greece and Turkey included). In thisatter association, we identified one strongly supported

onophyletic group of large-sized fluvio-lacustrine spe-ies, whereas the small riverine species form twoeakly supported associations. Relationships amongost of the subgroups of riverine species are unre-

olved, and this presumably reflects an abundance ofhort internodes.The most heterogeneous group seems to be that of the

iverine species; likewise, it comprises the largestumber of haplotypes. We consider nodes supported byore than 70% of bootstrap replicates to be robust

Zharkikh and Li, 1992; Hillis and Bull, 1993; Lecointret al., 1994). However, we also discuss groups that areresent in all of our phylogenetic reconstructions andhat are supported by more than 50% of bootstrapeplicates. In the riverine species, only three groups areell supported (Fig. 4): the first group contains popula-

ions of B. cyclolepis from central Greece (B. c. strumi-ae, B. c. cholorematicus, and B. c. sperchiensis); theecond consists of taxa from southern and westernreece (B. euboicus, B. p. peloponnesius, and B. pelopon-esius rebeli), and the third comprises taxa from north-rn Greece, northeastern Italy and the central Danube
asin (B. peloponnesius petenyi, B. caninus, and B.

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etenyi, respectively). The groups with poor bootstrapupport comprise the three subspecies of B. cyclolepisrom northern Greece and Asia Minor (B. c. cyclolepis,. c. strumicae, and B. c. pergamonensis) and the

FIG. 2. Absolute number of transitions versus transversions estimosition. Filled circles correspond to comparisons between the Europarpio, B. anoplus, and B. intermedius).

opulations from northwestern Greece, Albania, and s

omania (B. prespensis, B. peloponnesius rebeli, and B.etenyi, respectively). The exact branching positions of. meridionalis from France and B. caninus fromentral Italy were problematic and constitute the main

d from substitutions at (A) all codon positions and (B) the third codonBarbus species and the most distant taxa (L. leuciscus, G. gobio, C.

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ig. 4 and the trees obtained from the MP and MLnalyses.The monophyly of species that belong to the Cyprini-

ae subfamily (C. carpio, A. hyegelii, and Barbus

FIG.2—

pecies) is well supported in all analyses. Representa- T

ives of diploid and hexaploid barbs (African species)eem to form a group distinct from that formed by theuropean species. Surprisingly, the South African dip-

oid B. anoplus displays the longest branch in the tree.

tinued
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he cytochrome b gene of this species seems to have an

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volutionary rate approximately twice as high as thatf the other Barbus species. Moreover, this specieshows a strong bias in base composition, with a markednderrepresentation of G1C (37.7%).Using Kishino and Hasegawa’s (1989) test, we inves-

igated the polyphyly found in certain species, such as. peloponnesius and B. petenyi (Fig. 4). The monophy-

etic clustering of these taxa always leads to signifi-antly worse topologies. Finally, results of the relative-ate test (Robinson et al., 1998) indicate that sequencesn the genus Barbus s. str. evolve at approximately theame rate. Therefore, we can use the rate of cytochromeevolution to date the maximum time of divergence

etween Barbus groups.

hylogenetic Analyses Inferred from CompleteCytochrome b Sequences

The complete sequence of the cytochrome b gene1140 nucleotides) was determined for 13 haplotypesnvestigated in the previous analysis. We chose repre-entatives from each group of European barbs to assesshe effect on resulting topologies of using more data.hese haplotypes consist of A. hyegelii, B. albanicus,

FIG. 3. Transition/transversion ratio versus corrected (Kimura t

nd B. bocagei; B. barbus and B. macedonicus from the e

roup of fluvio-lacustrine barbs; and B. meridionalis, B.aninus (Astico River), B. petenyi (Toryza River), B.eloponnesius petenyi (Axios River), B. peloponnesiusebeli (Erzen River), B. p. peloponnesius (Alfios River),nd B. cyclolepis strumicae (Pinios River), from theroup of riverine species. Phylogenetic trees were rootedsing C. carpio and G. gobio as outgroups.Base composition in complete cytochrome b se-

uences was similar to those calculated for the 594-bpragments, and of the 398 variable sites, 260 werenformative for parsimony. All analyses yielded similaropologies, with European barbs divided into two majorlades, as in Fig. 4, and B. meridionalis occupying a basalosition to the first clade of Barbus species (from France tohe Black Sea). The NJ topology supported a weak mono-hyly of the riverine species, whereas the ML tree indi-ated that the large fluvio-lacustrine species are theister group to the riverine species from southeasternurope (trees not shown). Maximum parsimony yieldednly one most-parsimonious tree with 890 stepsCI 5 0.566), and the majority-rule consensus tree isresented in Fig. 5. This topology may also be consid-

-parameter) genetic distances. Filled circles are as those in Fig. 2.
wo
red as the consensus of the phylogenetic trees ob-

nlB e-sized fluvio-lacustrine species are in boldface.


FIG. 4. Phylogenetic tree of the 45 haplotypes of Barbus specieeighbor-joining method using Kimura’s (1980) two-parameter dist

engths are proportional to the estimated mean number of substitutionarbus s. str. are represented by boxes delimited by dotted lines. Larg

s recovered from cytochrome b sequences (594 bp), estimated by theances. Only bootstrap values higher than 50% are displayed. Branchs per site (see scale bar at bottom). The two principal clades in the genus

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ained from the NJ and ML methods, with a polytomyor the group of fluvio-lacustrine and the two groups ofiverine species. Furthermore, phylogenetic relation-hips among species of the first clade of European barbsfrom France to the Black Sea) are quite similar tohose found with the shorter sequences (Fig. 4). Threeroups were present: a well-supported group of large-ized species and two groups of riverine species. Boot-trap support for riverine species associations wasncreased, especially for the group of riverine speciesrom the Italian peninsula, middle Danube, and north-rn Greece (94 and 96% of bootstraps for MP and NJ,espectively). For the group of riverine species from thealkans, bootstrap values were always relatively low

67 and 55% of bootstraps for MP and NJ, respectively).The branching patterns inferred from the analyses of

omplete cytochrome b sequences (Fig. 5) were notxactly in concordance with those reported for the94-bp region (Fig. 4) and gave better resolution foreeper nodes in the first clade of European species.oreover, the enlargement of the sequenced region

mproved, sometimes considerably, the bootstrap sup-ort of some groupings but gave little insight into the

FIG. 5. Maximum-parsimony tree of the 13 Barbus s. str. speciesarpio and G. gobio as outgroups. Nodes supported by less than 50% ondicate bootstrap values using the entire cytochrome b data, and nusing the initial 594 bp. Dashes (-) show nodes that do not exist in the

ranching patterns among the two groups of small f

iverine barbs and the group of large-sized fluvio-acustrine species.

DISCUSSION

evel of Resolution with Cytochrome b Sequences

Given the results of our phylogenetic analyses, weecognize two well-supported clades among northernediterranean Barbus species. The first clade includes

he two species from Spain (B. bocagei and B. graellsii)nd several species from southeastern Europe (B. al-anicus, B. graecus, and B. capito pectoralis). Theecond clade includes all the other European speciesrom France to Turkey. In this latter clade, only large-ized fluvio-lacustrine barbs form a well-supportedonophyletic group. Indeed, although riverine species

eem to be well differentiated, phylogenetic relation-hips within species are not well resolved with our datasee Fig. 4). When more variable sites were added withequences of the complete cytochrome b gene, there wasetter resolution for riverine species from Italy, theiddle Danube, and northern Greece, which then

uenced for the complete cytochrome b gene region (1140 bp), using C.otstrap values were forced to collapse. Boldface numbers above nodesrs in italics below nodes indicate bootstrap values for the same taxa

4-bp data. Boldface species are as in Fig. 4.

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evertheless, riverine species from the Balkans andastern Europe consistently produced a weakly sup-orted association (Fig. 5).The lack of resolution for the deepest nodes may be

ue to a rapid radiation of lineages in a relatively shorteriod of time, which did not allow the accumulation ofynapomorphies. Alternatively, lack of resolution coulde the result of multiple hits (homoplasy), suggestinghat the cytochrome b may not be a perfect marker fornferring the evolutionary history of European barbs.owever, among European species, there is no evidence

f saturation, even in the third codon position (see Fig.), suggesting that species radiated rapidly in a shorteriod of time.

evels of Congruence with Previous Studies;Do Species Assemblages That Cluster Taxaof a Given Ecophenotype Really Exist?

Phylogenetic analyses using cytochrome b sequencesre in agreement with previous morphology-based stud-es, which suggested that European Barbus species

ay belong to two major lineages. Almaca (1984, 1988)as the first to propose a grouping of some easternediterranean species (B. albanicus, B. graecus, and. capito pectoralis) with species from Iberia, Northfrica, and the Middle East. Moreover, he placed all thether European species into a distinct group. Doadrio1990) also proposed that species of Barbus sensutricto should be attributed to two subgenera: theubgenus Luciobarbus Heckel, 1843, with species fromberia, North Africa, Greece, and the Middle East; andhe subgenus Barbus, which comprises all the otheruropean species. Here, we also find that two Iberianpecies (B. bocagei and B. graellsii) form a well-upported monophyletic group with these eastern Medi-erranean species, while all the other European Barbuspecies included in our study form a well-supportedister group (see Figs. 4 and 5). However, lack of keypecies (from North Africa and the Middle East) in ourample precluded investigation into whether this divi-ion into two subgenera really exists.In the first clade of European barbs (subgenus Bar-

us, sensu Doadrio, 1990), only the large fluvio-acustrine barbs (B. macedonicus, B. barbus, B. plebe-us, B. tyberinus, and B. plebejus escherichii) form aell-supported monophyletic group. In previous allo-

yme studies, in which some of these species werencluded, this grouping was always ambiguous (Machor-om et al., 1995; Tsigenopoulos et al., 1999).Our results indicate that the phylogenetic relation-

hips among riverine species of the genus Barbus sensutricto appear to be more puzzling than previouslyhought. Moreover, phylogenetic relationships amongiverine barbs inferred from mtDNA sequences are inonflict with those inferred in previous studies usingorphological characters and allozyme data. Karaman

1971) proposed that the small riverine taxa can be f

ivided into two groups based on the presence ofenticulations on the fourth dorsal ray and the numberf scales on the lateral line. The first group compriseshe meridionalis-like taxa (no denticulations, fewercales) and the second group comprises the cyclolepis-ike taxa (few denticulations, more scales). B. pelopon-esius and B. petenyi were considered to be part of theeridionalis-like taxa (Tsigenopoulos et al., 1999, and

eferences therein). However, the results of the presenttudy suggest that the phylogenetic relations amonghe small-sized riverine species appear to be moretrongly correlated with their geographic distributionhan with their morphological similarity (Figs. 4 and). A first group, which was better resolved withomplete cytochrome b sequences, consists of taxa fromegions thought to have participated in exchanges withhe Danubian basin (Italian peninsula, central Europe,nd northern Greece); this group was also supported byllozyme studies (Tsigenopoulos et al., 1999). The sec-nd group is weakly resolved and comprises all thether haplotypes found in the Balkans, even thoseresenting a meridionalis-like morphology (B. pelopon-esius from southern and western Greece and B. pete-yi from the lower Danube). Alternative topologiesonstructed to test the monophyly of the above speciesesulted consistently in worse likelihood values thanhe one obtained from the data with Kishino andasegawa’s (1989) test.In general, phylogenetic incongruencies can reflect

rrors in the current taxonomy, random sorting ofncestral polymorphisms among closely related speciesNeigel and Avise, 1986), introgression (Avise, 1994;uvernell and Aspinwall, 1995), or a combination of

hese. Lack of congruence between allozyme data andata generated from mtDNA sequences is a frequenthenomenon in the study of natural populations (Avise,994). That may be due to the different inheritanceodes of the markers, with the latter being more

nfluenced by stochastic events and selection (Harrison,989). In our study, we favor explanations evokingainly the random lineage sorting from a polymorphic

iverine ancestral population that led to taxa with aeridionalis-like or a cyclolepis-like morphology. Phylo-

enetic relationships among riverine taxa seem to beorrelated with their geographic distribution and therere no monophyletic groups clustering meridionalis-ike or cyclolepis-like taxa. Karakousis et al. (1995)ave also suggested the possibility of convergence to

ustify the close morphological resemblance betweenopulations of B. peloponnesius from southern andestern Greece and those of meridionalis-like taxa

rom western and central Europe.

he Role of Southern European Peninsulas as Refuges:Pattern and Timing of Diversification

Primary (or true) freshwater fish are of great value
or zoogeographic studies because, being unable to

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ross saline waters (30–36 ppm), they depend on wateroutes for movement and dispersal and their evolutions therefore strictly correlated with the history ofydrographic networks (Bianco, 1989; Strange andurr, 1997).There are no molecular studies reporting phylogeo-

raphic relationships among widespread Europeanreshwater fish species. On a smaller scale, there aretudies available on the brown trout (Bernatchez et al.,992; Apostolidis et al., 1997) and recently on the chubLeuciscus cephalus) in Greece (Imsiridou et al., 1998;oadrio and Carmona, 1998; Durand et al., 1999).tudies on the chub are limited to the Balkan penin-ula, but we refer to these results because this regionlso harbors a great diversity of Barbus taxa. However,omparison with the trout is complex, since this speciesay use migration routes very different from those of

trictly freshwater fish.Banarescu (1973) proposed that western Palearcticarbus (or Barbus sensu stricto) species originated

rom an east Asian ancestor and reached the Euro-editerranean region by a Siberian route. This migra-

ion is believed to have taken place during the Loweriocene–Upper Oligocene, when there was probably aide geographic and faunistic connection between Eu-

ope and Asia (Steininger and Rogl, 1984).If we use the rate of cytochrome b evolution esti-ated for marine fishes (1–1.2% sequence divergence

er million years; Bermingham et al., 1997), the time ofivergence between the two major clades of the genusarbus (or the two subgenera Barbus and Luciobarbus)

s estimated to be 10.6 to 12.8 million years ago. Thiseriod coincides with the middle Miocene, when Parate-hys, a huge, nearly brackish, interior sea, coveredoutheastern Europe and western Asia, extending asar east as the present Caspian and Aral Seas. Duringhis time migration of Barbus species may have beenossible toward southern peninsulas and western Eu-ope.The Danube basin, which has the richest aquatic

auna on the continent, is quite young in its present-ay shape and size (Banarescu, 1990). The biggest partf this basin was covered by the Paratethys, and whenhe retreat of the sea began in the west and progressedradually eastward (late Miocene), two large freshwa-er lakes were formed: the Panonian Lake, in theresent middle Danube basin, and the Dacian Lake, inhe lower Danube basin.

B. meridionalis is known from the fossil record sincehe Miocene in southern France (Persat and Berrebi,990). It may have been among the first Barbus specieso arrive on the European continent. Using the molecu-ar clock hypothesis, we estimate that these migrationsccurred in the late Miocene. Then, riverine speciesound in tributaries of the middle Danubian basin mayave reached northeastern Italy and northern Greece
y river connections in the Pliocene. Banarescu (1990) c
roposed that this dispersal was more recent (in theuaternary) and was due to river captures of theorava river (Danube basin) and the Axios river.

ndeed, our results support this hypothesis of a recentispersal from the Danube basin to the south, givenhat populations from northern Greece and northeast-rn Italy are genetically very similar to and seem toriginate from a middle Danubian (Panonian) stockFigs. 4 and 5).

Within the Balkan and eastern Europe riverine taxa,hylogenetic relationships seem to be correlated withpecies’ geographic distribution (Fig. 4). Some associa-ions between closely related sequences were found, buthylogenetic resolution in general was weak. Moreover,hen the sequenced region was nearly doubled, repre-

entatives of these associations formed a group, whichas still poorly supported (Fig. 5). The fish fauna oforthern and northeastern Greece consists mainly ofpecies taxonomically close to those of central Europend the Danube (Economidis and Banarescu, 1991).ccording to our data, the star-like structure in theiverine association of Barbus species from the Balkansnd the lower Danube (see Figs. 4 and 5) may indicate aapid radiation of populations in these regions, originat-ng probably from the eastern remnant of the Parate-hys (Dacian lake). Subsequently, isolation of theseaxa by the rise of mountain chains and the separationf landmasses led to the present distribution of haplo-ypes by in situ diversification of mtDNA lineages.

For the large fluvio-lacustrine barbs, genetic differen-iation among species indicates that population diver-ence occurred mainly in the Pleistocene but also in theliocene. In this group, B. plebejus escherichii, a subspe-ies from northern Turkey, is very close to B. barbusFig. 5), which used the Danubian tributaries to colo-ize central and northern Europe. The present situa-ion of the Black Sea is rather exceptional compared tots past, as it was for most of this time a freshwater lakeHsu, 1978; Ryan et al., 1997). Therefore, given theuvio-lacustrine ecotype of these species, populations

rom northern Turkey could probably easily reach theanube delta and migrate toward central Europe.Comparing dispersal in the large fluvio-lacustrine

nd the small rheophilic species, Bianco (personalommunication) suggested that the former may moreffectively disperse via river confluence in the lowland,hile the later may more effectively use changing river

ourses in the mountains. For example, the two B.lebejus populations from northern Italy carry theame haplotype, suggesting that this fluvio-lacustrinepecies may have rapidly used river confluences thatccurred in this region (probably during the lowering ofhe sea level in the last Wurmian glaciation). In con-rast, B. caninus populations from rivers of the sameegion display very distinct haplotypes, suggesting thathis riverine species, confined in high mountain river
ourses, may not have managed to disperse. Likewise,

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n central and northern Greece (from the Pinios to thexios rivers), the fluvio-lacustrine taxa (B. macedoni-us and B. barbus thessalus) have very similar haplo-ypes, whereas the riverine taxa from the same regionre divided into at least three groups (Fig. 4).Previous analyses of chub species from Greece also

ndicated a west-to-east separation of populations, withopulations from northern and central Greek riversarrying Danubian haplotypes (Durand et al., 1999).oreover, in our study there is a Danubian influence on

he northern Greek populations of B. peloponnesiusetenyi, but, in contrast, central Greece (from theinios to the Sperchios rivers) is populated by B.yclolepis taxa.

enetic Difference between European andNon-European Barbus Species withDifferent Ploidy Levels

Our results agree with the previous phylogeneticonclusions of Howes (1987) and Berrebi et al. (1996) tohe extent that Barbus species may form distinctineages based on their geographical distribution andheir ploidy level. Myers (1960) characterized the genuss a ‘‘monstrous aggregation’’ that required a thoroughaxonomic revision. Genetic distances between theuropean and the African barbs can reach valuesreater than those reported between different genera inhe family (Briolay, 1998; Gilles, 1998). This is alsovident in the phylogenetic trees, as the African speciesone hexaploid and one diploid) appeared very diver-ent compared to the European species (Fig. 4). More-ver, a species that does not belong to the genus BarbusA. hyegelii) is grouped with the European tetraploidarbus species. Therefore, we confirm earlier morpho-

ogical studies (Howes, 1987; Skelton, 1988), whichuggested that the so-called genus Barbus is a polyphy-etic assemblage.

axonomic Position of Aulopyge hyegelii

Aulopyge hyegelii Heckel, 1841 is an endangeredpecies, endemic to rivers and lakes of the Dalmatianarst regions, where no Barbus species exist. Thealmatian region contains a very high concentration ofndemic cyprinids and, according to Bianco (1986),epresents a separate zoogeographical region of highnterest. This monotypic genus was initially viewed byaraman (1971) as a relic of an earlier Eurasian barbinssemblage (cyprinids bearing barbs). Later, Howes1987, 1991) proposed that this monotypic genus maye a member either of the European Barbus sensutricto lineage or of the ‘‘stem-group’’ of Eurasian plusfrican barbins. According to our results, A. hyegeliielongs to the Barbus sensu stricto lineage (Fig. 4) andas high divergence compared to the African species.Consequently, we may consider A. hyegelii to be a

art of the first migration wave that reached the
editerranean region and found a refuge in Dalmatia.
ikewise, based on the molecular clock hypothesis forarbus sensu stricto species, we estimate a divergence

ime of 10–12 MYA (middle Miocene) between A. hy-gelii and the European barbs.Overall, we observed important genetic divergence

mong species and subspecies of the genus Barbus andhe present data confirm the necessity for a thoroughaxonomic revision. Phylogenetic relationships amonguropean taxa show concordance with their geographicistribution and cytochrome b sequences seem to pro-ide a powerful source of information that can yield anmproved taxonomic classification. Moreover, a fullevision of the taxonomy and systematics of the polyphy-etic genus Barbus will have to wait until detailed

orphological, karyological, and molecular studies cane achieved for a larger range of taxa than wereampled in this study.

ACKNOWLEDGMENTS

The authors thank the following for their help in sample collection:. S. Economidis, A. P. Apostolidis, and C. Triantaphyllidis fromhessaloniki, Greece; P. G. Bianco from Naples, Italy; E. Unlu fromiyarbakir, Turkey; A. Crivelli from Arles, France; M. Mracovcic fromagreb, Croatia; and P. Kotlik from Prague, Czech Republic forroviding sequences of the Romanian B. petenyi samples. We are alsorateful to F. Bonhomme, N. Galtier, A. Chenuil, E. Douzery, K.awson, J. C. Garza, and one anonymous referee for helpful sugges-

ions on the manuscript, and to E. Desmarais and L. Pouyaud forechnical assistance in the laboratory. C. S. T. was supported by ah.D. grant from the Greek State Scholarships Foundation (IKY).

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