Molecular Phylogenetics and Evolution 40 (2006) 856–865www.elsevier.com/locate/ympev

Across Siberia and over Europe: Phylogenetic relationships of the freshwater Wsh genus Rhodeus in Europe and the phylogenetic position

of R. sericeus from the River Amur

Jörg Bohlen a,¤, Vendula Klechtová a,b, Nina Bogutskaya c, Jörg Freyhof d

a Institute of Animal Physiology and Genetics of the Academy of Sciences of the Czech Republic, Rumburská 89, 27 721 Lib5chov, Czech Republicb Department of Zoology, Faculty of Biological Sciences, University of South Bohemia, Braninovská 31, 37005 Beské Bud5jovice, Czech Republic

c Zoological Institute, Russian Academy of Sciences, Universitetskaya nab, 1, St. Petersburg, 199034, Russiad Leibniz-Institute of Freshwater Ecology and Inlands Fisheries, Müggelseedamm 310, 12586 Berlin, Germany

Received 9 February 2006; revised 21 April 2006; accepted 25 April 2006Available online 5 May 2006

Abstract

We present the phylogenetic relationships of the freshwater Wsh genus Rhodeus from 45 localities in Europe and three localities in theAmur basin using the mitochondrial cytochrome b sequence. The study aims to (1) test the colonisation history between Europe and EastAsia and (2) to reconstruct the evolutionary history of Rhodeus in Europe. Our analyses revealed Wve monophyletic clades which corre-spond to four species: R. sericeus in the Amur basin, R. meridionalis in the River Vardar, R. colchicus in western Caucasus and two sisterlineages in most of Europe which together represent R. amarus. The reconstructed colonisation scenario assumes a continuous distribu-tion of Rhodeus from Europe through Siberia until East Asia during Pliocene, depletion of the Siberian population in late Pliocene, isola-tion of the population in River Vardar during early Pleistocene, refuges during late Pleistocene glaciations in the Danubian basin, in thelowlands of western Black Sea and in western Caucasus Mountains and a post-glacial expansion from the Danubian refuge towards west-ern Europe and from the Black Sea refuge across Eastern Europe.© 2006 Elsevier Inc. All rights reserved.

Keywords: Acheilognatinae; Biogeography; Freshwater Wshes; Eurasia; Cytochrome b; Pleistocene glaciations; Bitterling

1. Introduction

The genus Rhodeus is a member of the subfamily Achei-lognatinae (bitterlings) within the family Cyprinidae, whichoccurs with three genera and about 40 species in East andSoutheast Asia and with one genus and two species in theEuro-Mediterranean zoogeographic subregion (EMZS, fordeWnition see Baÿnaÿrescu, 1992) (Arai and Akai, 1988;Bogutskaya and Komlev, 2001). For a long time, bitterlingshave attracted scientiWc interest, mainly due to their uniquereproductive strategy that includes spawning into the man-tle cavity of living clams (Bivalvia) (Smith et al., 2004).

* Corresponding author. Fax.: +420 315 639510.E-mail address: bohlen@iapg.cas.cz (J. Bohlen).

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.04.020

Bitterlings inhabit a wide variety of lowland habitats likelakes, lowland rivers and slow-Xowing streams (Lelek, 1987).

Within the EMZS, bitterlings are found in the basins ofNorth, southern Baltic, Black, western and southern Caspianand Aegean Seas (south to Pinios drainage and western Ana-tolia). In the Mediterranean basin bitterlings are only foundin northern Rhône (France) and Drin drainages (Albania,Montenegro, Macedonia) and in the rivers of northernAegean drainage. Bitterlings are highly invasive in France(southern Rhône and west of Seine) and in southern Russia(Don and Kuban drainages, maybe Moscow). They are intro-duced to Crimea peninsula, Great Britain, northern Italy andelsewhere (Maitland and Campbell, 1992; Holbik, 1999).

Rhodeus sericeus was described from the Amur basinand is also known from Sakhalin Island in Northeast Asia

J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865 857

(Bogutskaya and Komlev, 2001; Holbik, 1999). Tradition-ally, bitterlings from the EMZS are also identiWed asR. sericeus (Holbik and Jedlibka, 1994; Holbik, 1999) or aredistinguished as an own subspecies R. s. amarus (Berg,1949; Bogutskaya and Komlev, 2001; Lelek, 1987), which isunder the phylogenetic species concept a distinct species(Kottelat, 1997). Although the disjunct distribution area ofthis species reveals a gap of 4000 km across Siberia, all com-parative studies have shown that East Asian and Europeanpopulations are morphologically very similar (Bogutskayaand Komlev, 2001; Holbik and Jedlibka, 1994) suggesting avery close relationship between both taxa.

Very recently, a second species of bitterling was describedin the EMZS, R. colchicus, which is morphologically distinctfrom the European R. amarus as well as from the East AsianR. sericeus and restricted to western Caukasus (Bogutskayaand Komlev, 2001). The absence of East Asian populationsof R. colchicus evokes questions regarding its origin and thezoogeographic history of the European and East Asian bit-terlings. Since morphologic characters suggest the Europeanand East Asian populations to be more closely related toeach other than either of them to R. colchicus or any Asianspecies, only two colonisation scenarios (Fig. 1) are possible:

Fig. 1. Two alternative colonisation scenarios for Eurasian bitterlings. (A)Speciation of R. colchicus (col) and R. sericeus (ser) in East Asia and asubsequent parallel colonisation of Europe by both lineages with subse-quent extinction of the ancestral lineage of R. colchicus; (B) Colonisationof Europe by a common ancestor of R. colchicus and R. sericeus (anc)with subsequent split into R. colchicus and R. sericeus and a subsequentre-colonisation of Eastern Asia by R. sericeus. Aterisks indicate speciationevents. The present day distribution range is indicated by grey Welds.

(1) two diVerent bitterlings (ancestors of R. colchicus andR. amarus/sericeus) colonised Europe in parallel from EastAsia with subsequent extinction of the ancestral lineage ofR. colchicus, or (2) a colonisation of Europe by a commonancestor of R. colchicus, R. amarus and R. sericeus with sub-sequent split into R. colchicus and R. amarus/sericeus and asubsequent re-colonisation of Eastern Asia by R. sericeus.The second scenario would suggest for the Wrst time a recip-rocal faunal exchange between Europe and East Asia, ahypothesis that would provide new insights into interconti-nental zoogeography.

Its occurrence in Aegean and Adriatic river systems isworth mentioning since the eVective barriers provided bythe Alpidic mountain range (Alps, Dinarics, Balkan) iso-lated most Mediterranean rivers since Miocene; thereforemany freshwater Wshes are represented by endemic species.It has been shown recently that some cold-adapted rheo-philic Wsh species were able to cross the dividing mountainranges via river capture (Durand et al., 1999; Klechtováet al., 2004), but this mechanism is not available for lowlandspecies like bitterlings. However, the degree of endemismamong freshwater Wshes in the Mediterranean drainagearea is not completely understood and new endemic taxaare regularly found or re-established (Kottelat, 1997). Inthe case of bitterlings, Karaman (1924) had described thebitterling from River Vardar as R. amarus var. meridionalis,but later authors did not consider it as valid. This situationleaves us with two alternative hypotheses: (1) that bitter-lings in the Mediterranean drainage represent, like mostother freshwater Wsh species in these areas, endemic speciesdistinct from the species in Central and Eastern Europe,but just were not identiWed as such; (2) that bitterlings inthe Mediterranean drainage are conspeciWc with the speciesin Central and Eastern Europe, but this would need to Wndan explanation on which route they managed to colonisethese generally well isolated areas.

In the present study, we analyse the phylogenetic rela-tionships of bitterlings from the EMZS and from the Amurbasin in East Asia using the sequences of mitochondrialcytochrome b gene. The analyses were designed to test onthe one hand the relationships between the East Asian andthe European populations of R. sericeus and R. colchicus todiVerentiate between the competing colonisation hypothe-ses. On the other hand, we focussed on the phylogeneticrelationships of bitterlings within the EMZS to identifygenetic lineages, to test if the populations in the Mediterra-nean drainage represent distinct lineages and to reconstructthe evolutionary history of bitterlings on the basis of thepresent distribution area of genetic lineages and the appli-cation of a molecular clock.

2. Materials and methods

2.1. Sampling strategy

Altogether, we analysed 114 specimens of bitterlingsfrom 48 localities, covering most of the distribution area

858 J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865

within the EMZS as well as from three localities in theAmur basin (Table 1). As outgroup we used R. amurensisand Acheilognathus chankaensis from the Amur basin aswell as of R. sinensis, a species that was formerly suggestedto be closely related to R. sericeus (Svetovidov andEremeyev, 1935).

2.2. Laboratory procedures

DNA was extracted from Wn or muscle tissue by DneasyTissue Kit (Qiagen) or phenol–chloroform method (Sam-brook et al., 1989). The cytochrome b gene was ampliWedusing the primers Glu L. Ca14337–14359: GAA GAA CCACCG TTG TTA TTC AA and Thr H. Ca15568–15548:ACC TCC RAT CTY CGG ATT ACA (Klechtová, 2006).PCRs were performed in 50�l volume containing 10 mMTris–HCl, 50 mM (HN4)2SO4, 0.1% Triton X-100, 1.2–1.8 mM MgCl2, 2 mM TMA oxalate (PCR enhancer),10 nmol of each nucleotide, 2.5 U of Taq polymerase (allchemicals by Top–Bio) and 25 pmol of each primer. ThePCR proWle on a MJ Research PTC-200 thermocyclerstarted with a 2 min period of initial denaturation at 95 °C,followed by 35 cycles each consisting of a denaturation stepat 94 °C (30 s), a primer annealing step at 54 °C (30 s) and anelongation step at 72 °C (45 s). The PCR was completed bya Wnal elongation period of 10 min at 72 °C. PCR productswere puriWed by ethanol precipitation or with QIAquickPCR PuriWcation Kit (Qiagen). Direct sequencing of puri-Wed PCR products was performed with BigDye™ Termina-tor Cycle Sequencing Ready Reaction Kit 3.1 (AppliedBiosystems) according to manufacturer’s instructions andpuriWed with DyeEx Spin Kit (Qiagene). The sequencingwas performed on ABI Prism 310 and ABI Prism 3130 GA.

2.3. Phylogenetic analyses

Chromatograms were assembled and edited manuallyusing the SeqMan II module of the DNAStar softwarepackage and aligned with Clustal X (Thompson et al.,1997). The sequences of all haplotypes were deposited inGenBank under the accession numbers (sequences are sub-mitted to Genbank, accession numbers will be Wlled hereafter arrival).

Statistical information on the dataset and estimates ofhaplotype (h) and nucleotide (�) diversities (Nei, 1987)across the dataset as well as within the identiWed lineageswere obtained with DNsp 4.10.3 (Rozas et al., 2005) and themean pairwise nucleotide diVerences between and withinthe indicated mitochondrial lineages were obtained inMEGA 2.1 (Kumar et al., 2001). Using the computer soft-ware Modeltest 3.06 (Posada and Crandall, 1998), theGTR+�+I model of nucleotide substitution has been deter-mined under Akaike information criterion to Wt best thegiven dataset. An estimation of the phylogenetic relation-ships of the haplotypes was performed using neighbourjoining (NJ) algorithm and Bayesian inference (BI). NJ treewas constructed with the best-Wt model in PAUP 4.0b10

(SwoVord, 2002) and the statistical support of the internalbranches was assessed by nonparametric bootstrappingwith 1000 replicates. Bayesian analyses were performed inMrBayes 3.01 (Huelsenbeck and Ronquist, 2001). Startingfrom a random tree, six Markov chains were run for1,000,000 generations with sampling frequency of 100. TheGTR+�+I model was speciWed. The number of trees to dis-card was estimated from log-likelihood plots. From theresulting trees, 500 were discarded as “burnin” and theremaining trees were used to build a 50% majority rule con-sensus tree.

A haplotype network was constructed to estimate the“Wne scaled” intraspeciWc relationships employing the sta-tistical parsimony (Templeton et al., 1992) implemented inthe TCS 1.21 program (Clement et al., 2000). Haplotypeswere disconnected when more than ten mutational stepsdivided them.

To examine population range expansions within themtDNA lineages we conducted Tajima’s D (Tajima,1989a) test of neutrality in Arlequin 2.000 (Schneideret al., 2000). Although this test is originally designed toexamine for departure of selective neutrality, some eVectscan be also due to population substructure or changes inpopulation size, e.g. signiWcantly positive value can resultfrom population decline and conversely, in expandingpopulation there would be a tendency of Tajima’s D valueto be negative (Tajima, 1989b). We take into account thepossibility of obtaining biased results due to the insuY-

cient numbers of representative sequences available forthe lineages I, II and VI, and focus in detail only on thelineages III and IV. Number of representative sequencesrestricted this analysis to the lineages III and IV. Addi-tionally, we examined the demographic history of thesetwo clades using mismatch distribution of pairwise geneticdiVerences (Slatkin and Hudson, 1991; Rogers and Har-pending, 1992). The observed distribution was tested forgoodness-of-Wt to a model of sudden population expan-sion using parametric bootstrapping with 1000 replicatesin Arlequin.

To estimate the times of divergence between the identi-Wed lineages from the molecular data, we conducted likeli-hood ratio test by comparing the two likelihood scores ofthe maximum likelihood (ML) trees calculated with molec-ular clock enforced and with molecular clock not enforcedin PAUP* under the best-Wt model.

3. Results

Our phylogenetic analyses showed that East AsianR. sericeus and all bitterlings from the EMZS form a mono-phyletic clade (ingroup clade) (Fig. 2), which is sister cladeto the remaining bitterlings from Asia (not shown in Fig. 2).Five distinct monophyletic lineages can be found within theingroup clade (labelled I–V in Fig. 2). The most prominentdivision within the ingroup clade is between the bitterlingsfrom the EMZS and R. sericeus from the Amur basin (line-age I). Among the bitterlings from the EMZS those from

J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865 859

(continued on next page)

Table 1Origin of samples of Rhodeus sericeus from East Asia and Rhodeus spp. from the Euro-Mediterranean zoogeographic subunit analised in the present study

River, drainage, country Locality No. No. indiv. Haplo-type Accession No.

Rhodeus amarusR. Moskwa at Moskow, Volga, RUSa 1 2 25 DQ396649R. Don at Bolshoy Goluboy, Don, RUSa 2 1 16 DQ396640R. Kuban at Korzhevsky, Kuban, RUSa 3 1 12 DQ396638

2 5 DQ396632R. Sluch at Chizhovka, Dnjepr, UA 5 1 4 DQ396631R. Guiva at Oziornoe, Dnjepr, UA 6 1 23 DQ396647

1 4 DQ3966311 24 DQ396648

Dniprovskij liman at Stara Zburijevka, Dnjepr, UA 7 1 46 DQ3966701 47 DQ3966711 5 DQ396632

R. Dnjestr at Yampol, Dnjestr, UA 8 1 5 DQ396632R. Chornaya at Khmetnitskoje, Chornaya, UAa 9 1 5 DQ396632

1 17 DQ396641R. Alma at Dorozhnoje, Alma, UAa 10 3 8 DQ396634R. Western Bug at Busk, Vistula, UA 11 1 9 DQ396635

1 11 DQ396637R. Southern Bug at Peski, Southern Bug, UA 12 1 7 DQ396633

1 13 DQ396639R. Kyovka at Týnec u Lanqhota, Danube, CZ 13 1 2 DQ396630

1 33 DQ3966571 34 DQ3966582 35 DQ396659

R. Ubljanka at Ubla, Danube,SK 14 1 2 DQ3966301 36 DQ396660

R. Danube at Banske Rameno, Danube, SK 15 1 22 DQ3966461 2 DQ3966301 18 DQ3966421 19 DQ396643

R. Jantra at Bjala, Danube, BG16 1 29 DQ396653

1 30 DQ3966542 2 DQ396630

R. Danube at Archar, Danube, BG 17 1 53 DQ3966731 54 DQ3966741 2 DQ396630

R. Lom at Lom, Danube, BG 18 1 44 DQ396668R. Zlatica at Gagavica, Danube, BG 19 1 2 DQ396630R. Suganica at Krapbene, Danube, BG 20 5 2 DQ396630R. Kazabevskij at Kazabevo, Danube, BG 21 1 45 DQ396669R. Katuneska at Berhanovo, Danube, BG 22 2 2 DQ396630R. Vidima at Berievo, Danube, BG 23 1 37 DQ396661R. Birlat at Vaslui, Danube, ROM 24 1 24 DQ396648R. Prut at Murgeni, Danube, ROM 25 1 24 DQ396648R. Danube at Oltenita, Danube, ROM 26 1 31 DQ396655R. Mures at Sarmas, Danube. ROM 27 1 2 DQ396630Glogovnica channel at Mostari, Danube, HR 28 1 39 DQ396663

1 2 DQ3966301 40 DQ396664

R. Dravinja at Ptuj, Danube, HR 29 2 32 DQ396656R. Elbe at Melnik, Elbe, CZ 30 5 2 DQ396630

1 37 DQ396661R. Sázava at Na Mariánce, Elbe, CZ 31 4 2 DQ396630R. Barycz, Odra, PL 32 1 43 DQ396667Lake Vierer at Plön, Schwentine, Da 33 1 20 DQ396644

1 21 DQ396645Ditch, Alte Wettern‘ at Oldenburg, Weser, Da 34 2 2 DQ396630Ditch, WeidenXeet‘ at Bremen, Weser, D 35 1 38 DQ396662Creek, Mark‘ at Zevenbergen, Rhine, NL 36 3 41 DQ396665Grietherorther Altrhein at Rees, Rhine, D 37 1 2 DQ396630

1 41 DQ396665Bienener Altrhein at Rees, Rhine, D 38 2 42 DQ396666

860 J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865

the River Vardar in Greece form the sister lineage (lineageII) to a group that form a trichotomy: lineage III collectsbitterlings from River Vistula, the Carpathian Mountainsand eastwards areas, lineage IV is represented only by theanalysed specimens of R. colchicus from central Caucasus,while lineage V is composed of most bitterlings from theDanube basin and from the western parts of the Europeandistribution area (Fig. 3).

Our haplotype network (Fig. 4) reveals four discon-nected groups of haplotypes similar to the lineages identi-Wed in the phylogenetic tree: One group contains thehaplotypes 1, 3, 6 and 14 from the River Amur (lineage I),another group haplotypes 48, 49, 50 and 51 from the RiverVardar (lineage II), one group is represented solely by hap-lotype 15 found in the specimens of R. colchicus (lineageIV) and the fourth group collects the remaining haplotypesfrom the lineages III and V. Within the last group, the hap-lotypes of lineage III and V formed two distinct starburststhat were separated by six mutational steps. The haplotypegroup corresponding to the lineage III shows a “starburst”pattern with the most common haplotype at its core, whichis assigned to populations or lineages that have experiencedrather recent expansion in size from smaller number offounders (Avise, 2000).

The result of Tajima’s test of neutrality showed a statis-tically signiWcant departure from neutrality for the lineagesIII and V with strongly negative values (Table 2), whichindicates a demographic expansion of these two lineages.These results were in agreement with mismatch analyses,which showed unimodal distributions for both clades(Fig. 5). The trends of the observed distributions were closeto the expectations under a model of sudden populationexpansion, especially for the lineage III (pD 0.95 and 0.56for lineage III and V, respectively).

Genetic divergence among all bitterling samples was0.92%; pairwise distances are given in Table 3.

The likelihood ratio test did not detect signiWcant diVer-ences between the trees estimated with and without enforc-ing molecular clock (�2D 42.4698, dfD58, pD 0.94), hencesuggesting that within the studied group sequences evolvedin a clock-like manner. Rates of diversiWcation have beencalculated several times for cyprinid Wshes and criticallydiscussed by Doadrio and Carmona (2004). These authorssuggested a mutation rate of 1.05% per million years as bestWtting for cyprinid Wshes. Applying this mutation rate, theseparation between lineage I and the rest of the ingroupclade took place about 2.4–2.9 MYA, between lineage IIand the lineages III, IV and V about 1.4–1.8 MYA and

Table 1 (continued)

Haplotypes numbers found in each locality correspond to numbers in Fig. 2; locality numbers correspond to numbers in Fig. 3. AL, Albania; BG,Bulgaria; CHN, China; CZ, Czech Republic; D, Germany; F, France; GEO, Georgia; GR, Greece; HR, Croatia; NL, Netherlands; PL, Poland; ROM,Romania; RUS, Russia; SK, Slovakia; TR, Turkey; and UA, Ukraine.

a Introduced population.

River, drainage, country Locality No. No. indiv. Haplo-type Accession No.

R. Saone, Rhone, Fa 39 4 32 DQ396656R. Veleka at road 9, Veleka, BG 40 1 26 DQ396650

1 27 DQ3966511 28 DQ396652

Omerli Reservoir at Istanbul, Rive, TR 41 1 52 DQ3966722 28 DQ396652

R. Struma at Blageovrad, Struma, BG 43 2 55 DQ3966751 2 DQ396630

Lake Skadar, Drin, AL 44 1 56 DQ3966761 57 DQ396677

Lake Ohrid, Drin, AL 45 1 2 DQ396630

Rhodeus meridionalisR. Vardar at Axioupolis, Vardar, GR 42 2 48 DQ396679

1 49 DQ3966801 50 DQ3966812 51 DQ396682

Rhodeus colchicusR. Notanebi, Notabeni, GEO 4 2 15 DQ396678

Rhodeus sericeusLake Khanka, Amur, RUS 46 1 1 DQ396683R. Slistaja, Amur, RUS 47 1 6 DQ396685

1 3 DQ396684R. Ussuri, Amur, RUS 48 1 14 DQ396686

Outgroup samplesSpecies River, drainage Country No. indiv. Accession No.

Rhodeus amurensis Lane Khanka, Amur RUS 1 DQ396627Rhodeus sinensis Pond in Shanghai, Yangtse CHN 1 DQ396629Acheilognathus khankaensis Lane Khanka, Amur RUS 1 DQ396628

J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865 861

between the lineages III, IV and V about 1.3–1.4 MYA(Table 3).

4. Discussion

4.1. Major lineages

The monophyly of the ingroup clade supports theassumption from morphologic studies that R. sericeus fromthe Amur is closely related to the bitterlings in the EMZS.Consequently, the disjunct distribution area of the ingroupclade is in need of explanation and information about thiscan be revealed from the phylogenetic relationships ofgenetic lineages inside the ingroup clade. Five distinct mono-phyletic lineages can be found within the ingroup clade onthe basis of molecular genetic data, which is in sharp contrastto former studies on morphologic characters, since thesestudies concluded that bitterlings of our ingroup clade do notdiVer from each other with the exception of R. colchicus.

4.2. Reconstructed biogeography of bitterlings

The sister relationship of R. sericeus from the Amurregion and all Rhodeus from the EMZS suggests thatboth are descendants of the same ancestor, but their com-parably large genetic distance does not support the possi-bility of a recent colonisation of East Asia from Europe,as the morphologic similarity of the bitterlings may sug-gest. In opposite, their common ancestor inhabited bothEast Asia and Europe before the diversiWcation ofbitterlings in the EMZS began. The calculated time ofdivergence suggests a separation of the East Asian fromthe EMZS bitterlings for at least 2.36 MYA, this meanssince the end of Pliocene, a time when the world’s temper-ature was declining rapidly towards the approaching gla-cial cycles (Baÿnaÿrescu, 1992; Bradley, 1999). Largecontinental areas like Siberia have been more severelyaVected by the decreasing temperatures and becameunsuited for many freshwater Wshes (Baÿnaÿrescu, 1992),

Fig. 2. Phylogenetic relationships of identiWed haplotypes of the cyt b gene among Rhodeus sericeus from East Asia and Rhodeus spp. from the Euro-Med-iterranean zoogeographic subunit. The Asian species of Rhodeus sinensis, R. amurensis and Acheilognathus chankaensis were used as outgroup. Upper val-ues on the branches represent NJ bootstrap values; lower values are Byesian posterior probabilities. Lineages are indicated as ‘clade I’ to ‘clade V’.

862 J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865

leading to the present vicariance in the distribution of bit-terlings.

The diVerentiation of the bitterlings within the MEZSinto the four lineages II–V occurred much later than theseparation from East Asia: the separation of the bitterlingsin the Aegean drainage from the non-Mediterranean bitter-lings can be dated back to about 1.8 MYA, this means earlyPleistocene, while the age of the trichotomy of the lineagesA III to A V was calculated back to 1.1–1.5 MYA intomiddle Pleistocene.

The glacial maxima made vast areas of Europe unsuitedfor freshwater animals and drove them into refuge areas

(Baÿnaÿrescu, 1992; Kotlík and Berrebi, 2001; Perdiceset al., 2003). From the present distribution of the geneticlineages it is possible to infer the position of these refugesand the post-glacial spreading of the lineages: populationsin the Vardar River and in western Caucasus have mostlikely endured glacial maxima in the areas they inhabit nowand had few possibilities to disperse afterwards. On thecontrary, the wide distribution of lineages III and V indi-cates a post-glacial dispersal. We suggest that the ‘eastern’lineage III had its glacial refuge in the western BlackSea drainage, while the ‘western’ lineage V found refugein the Danube drainage. From here lineage V expanded

Fig. 3. Geographic origin of samples and distribution of the four identiWed ingroup lineages in the Euro-Mediterranean Zoogeographic Subunit. Localitynumbers correspond to locality numbers in Table 1. The distribution area of the genus Rhodeus in the Euro-Mediterranean zoogeographic subunit is indi-cated in grey.

Fig. 4. Unrooted haplotype network based on the cyt b gene of the Rhodeus spp. from the Euro-Mediterranean zoogeographic subunit. The haplotypenumbers refer to numbers in Table 1. The circle area is proportional to the haplotype frequencies.

J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865 863

post-glacially to the north and westwards until River Vis-tula, while lineage III expanded through the whole basin ofRiver Danube and westwards to the coast of the North Sea.A Pleistocene refuge in the Danubian basin and post-gla-cial expansion over western Europe was postulated for Bar-bus barbus (Kotlík and Berrebi, 2001), while Cobitis taeniacolonised Central and Eastern Europe from refugia in theBlack Sea basin (Culling et al., 2006).

Fig. 5. Mismatch-distribution of cytochrome b haplotypes in the western(A) and eastern (B) lineage of R. amarus.

The bitterlings in the Mediterranean drainage are ofdiVerent origin: the population in the Aegean River Vardaris a long-term isolated independent lineage, while the sam-ples from the Adriatic basin belonged to the western lineageIII, indicating a post-glacial expansion of the western line-age III across the Dinaric Mountains. The population inthe River Struma is of mixed origin: on the one hand, itshaplotype 55 was quite distant among the haplotypes of theeastern lineage V indicating a local isolation since last inter-glacial, on the other hand its second haplotype 2 is the mostcommon haplotype in the western lineage III and must bethe result of a post-glacial expansion from the Danubianbasin. However, our data proof the population in the RiverVardar to represent a distinct isolated lineage, which mayrepresent a separate taxononomic unit diVerent from theone in non-Mediterranean Europe.

4.3. Taxonomic implications

Four diVerent names are available for our Wve distinctlineages of bitterlings: the oldest available name isR. sericeus, described from the Onon River in the upperAmur basin. It applies to the East Asian Rhodeus of thelineage I. Karaman (1924) described R. meridionalis fromthe River Vardar, and we apply this name to our mono-phyletic and distinct lineage II. The species status ofR. colchicus for the south Caucasian bitterling is ahypothesis by Bogutskaya and Komlev (2001) basing onmorphologic data and supported by our haplotype net-work and the calculation of the genetic divergence. Thelineages III and V represent the species R. amarus. How-ever, we suggest a revision of the morphologic investiga-tion of European bitterlings with the background of thepresent phylogeny.

4.4. Reconstructed colonisation scenario (Fig. 6)

Our data refuse the hypothesis of a reciprocal faunalexchange between Europe and East Asia, because the popu-lation from East Asia is not a descendant of the populationsfrom the EMZS but their sister lineage, while the morpho-logically derived R. colchicus is nested in the lineage of

Table 2Molecular diversity in the main mtDNA lineages of Rhodeus identiWed in the present study as indicated by nucleotide diversity (�), haplotype diversity(Hd) and Tajima’s D value

N gives the number of specimens.Statistical signiWcance of Tajima’s D test (1989): NS, departure from neutrality non-signiWcant (p > 0.1).

a Departure from neutrality signiWcant at p D 0.05.b Departure from neutrality signiWcant at p D 0.01.

mtDNA lineage N No. of haplotypes �§ SD Hd § SD Tajima’s D

Lineage V R. amarus ‘eastern’ 59 22 0.001§ <0.001 0.705§ 0.067 ¡2.5 a

Lineage III R amarus ‘western’ 42 26 0.003§ 0.008 0.967§ 0.013 ¡2.01b

Lineage II R. meridionalis 6 4 0.001§ 0.002 0.867§ 0.129 ¡0.68 NSLineage I R. sericeus 5 4 0.005§ 0.005 0.900§ 0.161 ¡0.45 NSLineage IV R. colchicus 2 1 — — —

Overall 114 57 0.008§ <0.001 0.917§ 0.022

864 J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865

bitterlings from the EMZS. In the case of Rhodeus, the larg-est morphologic diVerence does not occur together with thelargest genetic diversity. Alternatively, our results suggest:

(1) A continuous distribution of a common ancestor ofthe recent Rhodeus from Europe through Siberia untilEast Asia during Pliocene.

(2) Extirpation of bitterlings from Siberia in latePliocene or early Pleistocene and consequent separa-tion of the East Asian from the Europeanpopulation.

(3) Isolation of the population in the River Vardar by aglacial maximum during early Pleistocene.

(4) Survival of non-Mediterranean bitterlings duringlate Pleistocene glaciations in three refuges: one inthe Danubian basin, one in the lowlands of westernBlack Sea and one in the western Caucasus Moun-tains.

(5) Post-glacial expansions from these refuges: from theDanubian refuge through all Danubian basin, toElbe, Odra, western Europe and southwards into theDrim and Struma basins; and from the northern partsof the Black Sea refuge across Eastern Europe andwestwards until River Vistula.

(6) Development of distinct morphologic characters inthe population in central Caucasus (R. colchicus).

5. Conclusions

The reconstruction of the phylogenetic relationships ofbitterlings from the EMZS and the Amur basin revealedthat the bitterlings from the Amur form the sister-clade toall bitterlings from the EMZS. This Wnding contradicts theformer hypothesis that based on morphological investiga-tions that the populations in East Asia derived from Euro-pean populations. Our genetic data show four majorlineages within the EMZS, indicating the existence of ahigher diversity than previously known. We suggest to usedfor the East Asian populations the scientiWc name Rhodeussericeus, for the population in the River Vardar in GreeceR. meridionalis, for the population from River Notabeni inGeorgia R. colchicus and for the populations from Centraland Eastern Europe R. amarus.

Acknowledgments

For help with obtaining samples we are thankful toG. Augustin, H. Brunken, L. Choleva, P. Economidis, M.Hein, K. Janko, L. Kalous, J. Kotusz, Z. Lajbner, A. Nas-eka, H. Persat, M. Reichard, R. Kanda, C. Schütz, M. Soes,U. Weibel. The study was supported by the Grants IAA600450508 of the GAAS, LC06073 of the Ministry of Edu-cation CZ, VaV-SM/6/3/05 of the Ministry of Environment

Table 3Genetic distances between and within the identiWed mtDNA lineages of Rhodeus from East Asia and from the Euro-Mediterranean zoogeographicsubunit

The mean pairwise diVerences in percents of nucleotide divergence § SD are given below diagonal, values on diagonal (underlined with grey) indicatewithin-group divergences and the estimated times of divergence in millions years are given above the diagonal.

Lineage I R. sericeus

Lineage II R. meridionalis

Lineage III R. amarus ‘western’

Lineage IV R. colchicus

Lineage V R. amarus ‘eastern’

Lineage I R. sericeus 0.49 § 0.13 2.68 2.42 2.85 2.36Lineage II R. meridionalis 2.81 § 0.43 0.14 § 0.06 1.37 1.80 1.36Lineage III R. amarus ‘western’ 2.54 § 0.41 1.44 § 0.34 0.13 § 0.02 1.30 1.36Lineage IV R. colchicus 2.99 § 0.46 1.89 § 0.4 1.36 § 0.33 — 1.26Lineage V R. amarus ‘eastern’ 2.48 § 0.39 1.43 § 0.4 1.43 § 0.4 1.32 § 0.31 0.35 § 0.06

Fig. 6. Reconstructed Xow of genetic information of bitterlings through Eurasia. Aterisks indicate speciation events. ser, sericeus; anc, ancestral form; col,colchicus; mer, meridionalis; and ama, amarus.

J. Bohlen et al. / Molecular Phylogenetics and Evolution 40 (2006) 856–865 865

CZ and by IRP IAPG AVOZ50450515. BNG was sup-ported by a grant from the Presidium of RAS Programme‘Dinamics of Gene Pools.’

References

Arai, R., Akai, Y., 1988. Acheilognathus melanogaster, a senior synonym ofA. moriokae, with a revision of the genera of the subfamily Acheilogna-thinae (Cypriniformes, Cyprinidae). Bull. Natl. Sci. Mus. Ser. A (Zool.)14, 199–213.

Avise, J.C., 2000. Phylogeography. The history and formation of species.Harward University Press, Cambridge and London.

Baÿnaÿrescu, P.M., 1992. Zoogeography of Fresh Waters, vol. 2. Distribu-tion and Dispersal of Freshwater Animals in North America and Eur-asia. Aula, Wiesbaden.

Berg, L.S., 1949. Fishes of the USSR and Adjacent Countries. Nauka,Moscow and Leningrad.

Bogutskaya, N.G., Komlev, A.M., 2001. Some new data to morphology ofRhodeus sericeus (Cyprinidae: Acheilognathinae) and a description of anew species, Rhodeus colchicus, from west Transcaucasia. Proc. Zool.Inst. 287, 81–97.

Bradley, R.S., 1999. Paleoclimatology: Reconstructing Climates of theQuaternary, 2nd ed. Academic Press, San Diego.

Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer programto estimate gene genealogies. Mol. Ecol. 9, 1657–1659.

Culling, M.A., Janko, K., Boroj, A., Vasil’ev, V., Cøté, I.M., Hewitt, G.M.,2006. European colonization by the spined loach (Cobitis taenia) fromPonto-Caspian refugia based on mitochondrial DNA variation. Mol.Ecol. 15, 173–190.

Doadrio, I., Carmona, J.A., 2004. Phylogenetic relationships and biogeog-raphy of the genus Chondrostoma inferred from mitochondrial DNAsequences. Mol. Phylogenet. Evol. 33, 802–815.

Durand, J.D., Persat, H., Bouvet, Y., 1999. Phylogeography and postglacialdispersion of the chub (Leuciscus cephalus) in Europe. Mol. Ecol. 8,989–997.

Holbik, J., 1999. Rhodeus sericeus. In: Baÿnaÿrescu, P.M. (Ed.), The fresh-water Wshes of Europe 5 I. Cyprinidae. Aula, Wiesbaden, pp. 1–32.

Holbik, J., Jedlibka, L., 1994. Geographic variation of some taxonomicallyimportant characters in Wshes: the case of the bitterling Rhodeus seric-eus. Environm. Biol. Fish. 41, 147–170.

Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference ofphylogenetic trees. Bioinformatics 17, 754–755.

Karaman, S., 1924. Pisces Macedoniae. Hrvatska Stampaija, Split. 1–90.Kotlík, P., Berrebi, P., 2001. Phylogeography of the barbel (Barbus barbus)

assessed by mitochondrial DNA variation. Mol. Ecol. 10, 2177–2185.Kottelat, M., 1997. European freshwater Wshes. An heuristic checklist of

the freshwater Wshes of Europe (exclusive of former USSR), with anintroduction for non-systematists and comments on nomenclature andconservation. Biologia (Bratislava) 52 (Supplement 5), 1–271.

Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: MolecularEvolutionary Genetics Analysis software. Arizona State University,Tempe.

Lelek, A., 1987. Freshwater Wshes of Europe, vol. 9. Threatened Wshes ofEurope. Aula Verlag, Wiesbaden.

Maitland, P.S., Campbell, R.N., 1992. Freshwater Fishes of the BritishIsles. HarperCollins Publishers, London.

Nei, M., 1987. Molecular Evolutionary Genetics. Columbia UniversityPress, New York.

Perdices, A., Doadrio, I., Economidis, P.S., Bohlen, J., Banarescu, P., 2003.Pleistocene eVects on the European freshwater Wsh fauna: double ori-gin of the cobitid genus Sabanejewia in the Danube basin (Osteichth-yes: Cobitidae). Mol. Phylogenet. Evol. 26, 289–299.

Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNAsubstitution. Bioinformatics 14, 817–818.

Rogers, A.R., Harpending, A., 1992. Population growth makes waves indistribution of pairwise genetic diVerences. Mol. Biol. Evol. 9, 552–569.

Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X., Rozas, R., 2005. DNASequence Polymorphism version 4.10.2. Departament de Genetica,Universitat de Barcelona, Barcelona.

Sambrook, J., Fritsch, E.F., Manitais, T., 1989. Molecular Cloning. A Lab-oratory Manual, second ed. Cold Spring Harbor Laboratory, ColdSpring Harbor.

Schneider, S., Roessli, D., ExcoYer, L., 2000. Arlequin ver. 2.000: A Soft-ware for Population Genetics Data Analysis. Genetics and BiometryLaboratory, University of Geneva, Switzerland.

Slatkin, M., Hudson, R.R., 1991. Pairwise comparison of mitochondrialDNA sequences in stable and exponentially growing populations.Genetics 129, 555–562.

Klechtová, V., Bohlen, J., Freyhof, J., Persat, H., Delmastro, G.B., 2004.The Alps as barrier to dispersal in cold-adapted freshwaterWshes? Phylogeographic history and taxonomic status of the bull-head in the Adriatic freshwater drainage. Mol. Phylogenet. Evol. 33,225–239.

Klechtová, V., Bohlen, J., Freyhof, J., Ráb, P., 2006. Molecular phylogenyof the Southeast Asian freshwater Wsh family Botiidae (Teleostei: Cobi-toidea) and the origin of polyploidy in their evolution. Mol. Phyloge-net. Evol. 39, 529–541.

Smith, C., Reichard, M., Jurajda, P., Przybylski, M., 2004. The reproduc-tive ecology of the European bitterling (Rhodeus sericeus). J. Zool.Lond. 262, 107–124.

Svetovidov, A.N., Eremeyev, G.K., 1935. On the European and Amur bit-terlings (Rhodeus sericeus). Doklady Akadem. Nauk SSSR 1, 582–587.in Russian.

SwoVord, D.L., 2002. PAUP*: Phylogenetic Analysis Using Parsimony(and other Methods) Version 4. Sinauer Associates, Sunderland, Mas-sachusetts.

Tajima, F., 1989a. Statistical method for testing the neutral mutationhypothesis by DNA polymorphism. Genetics 123, 585–595.

Tajima, F., 1989b. DNA polymorphism in a subdivided population: theexpected number of segregating sites in the two-subpopulation model.Genetics 123, 229–240.

Templeton, A.R., Crandall, K.A., Sing, C.F., 1992. A cladistic analysis ofphenotypic associations with haplotypes inferred from restrictionendonuclease mapping and DNA sequence data. III. Cladogram esti-mation. Genetics 132, 619–633.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,1997. The ClustalX windows interface: Xexible strategies for multiplesequence alignment aided by uality analysis tools. Nucleic Acids Res.24, 4876–4882.