Molecular Phylogenetics and Evolution 38 (2006) 416–425www.elsevier.com/locate/ympev

Phylogenetic position of the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminichthys

(Teleostei: Cyprinidae)

Jörg Freyhof a, Dietmar Lieckfeldt b, Nina G. Bogutskaya c, Christian Pitra b, Arne Ludwig b,¤

a Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, 12587 Berlin, Germanyb Leibniz-Institute for Zoo and Wildlife Research, Department of Evolutionary Genetics, Alfred-Kowalke-Strasse 17, 10315 Berlin, Germany

c Zoological Institute, Russian Academy of Sciences, Universitetskaya emb. 1, St. Petersburg 199034, Russia

Received 21 April 2005; revised 8 June 2005; accepted 1 July 2005Available online 6 October 2005

Abstract

The Dalmatian cyprinid genus Phoxinellus is characterized by reductive characters most likely associated with the environmental con-ditions of small karstic streams, where all species of this genus occur. Based on 33 morphological traits, nuclear and mtDNA sequencesPhoxinellus was found to be paraphyletic and included three not closely related monophyletic units. The scientiWc name Phoxinellusshould therefore be restricted to species having plain coloration, small or absent postcleithrum, no genital papilla and an almost entirelynaked body such as P. alepidotus, P. dalmaticus, and P. pseudalepidotus. Species that also have a small or absent postcleithrum and nogenital papilla but display a dark stripe from the head to the caudal peduncle, and are entirely covered by distinct, not overlapping scalesshould be positioned closely to Telestes. Thus, we suggest inclusion of Phoxinellus croaticus, P. fontinalis and Paraphoxinus metohiensis inthe genus Telestes. The Phoxinellus species that have a irregularly spotted color pattern, a large postcleithrum, an increased number ofprecaudal anal-Wn pterygiophores, and a large genital papilla in females represent its own evolutionary line closely related to the Balkanspecies of Pseudophoxinus. For this monophyletic group, we propose to introduce a new genus: Delminichthys. This genus includes thespecies D. adspersus, D. ghetaldii, D. krbavensis and D. jadovensis. 2005 Elsevier Inc. All rights reserved.

Keywords: Phylogeny; Biogeography; Adriatic freshwater drainage; Freshwater diversity; Phoxinellus; Telestes

1. Introduction

Containing more than 2000 described species, the Tele-ost family Cyprinidae is one of the most species rich Wshfamilies (Nelson, 1994). The phylogenetic structure of thisfamily is still under debate, but several subfamilies arewidely accepted and seem to form strongly supportedmonophyletic clades (Chen et al., 1984; Howes, 1991; Liuet al., 2002).

One of these is the subfamily Leuciscinae, which iswidely distributed throughout northern Eurasia, NorthAmerica and North Africa. In the Palearctic, at least 35

* Corresponding author.E-mail address: Ludwig@izw-berlin.de (A. Ludwig).

1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2005.07.024

genera with more than 180 species are known and in NorthAmerica about 45–50 native genera including more than250 species are recognized (Mayden, 1991).

The subfamily Leuciscinae was subdivided by Boguts-kaya (1988a,b, 1990a,b) into the tribes Abramidini, Albur-nini, Aspinini, Leuciscini, Pseudaspinini, Elopichthyini,Pelecini, and Hypophthalmichthyini. The latter three arevery specialized, monotypic or almost monotypic (Yue,2000). The inclusion of Elopichthyini and Hypophthalm-ichthyini within Leuciscinae is still under debate (Liuet al., 2002). If Elopichthys and Hypophthalmichthys areexcluded, Leuciscinae is clearly divided into two majorgroups, Leuciscini s.l. (including Aspinini + Alburnini +Abramidini + Pelecini) on one hand and Pseudaspinini onthe other. These two groups coincide with the informal

J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 417

groups “leuciscins” ( D Leuciscini) and “phoxinins”( D Pseudaspini) proposed by Cavender and Coburn(1992). This principal dichotomy is also supported bymolecular data (Pyron, 1996; Briolay et al., 1998; Gilleset al., 2001; HänXing and Brandl, 2000; Cunha et al.,2002; Liu et al., 2002) and this is the reason, why Phoxi-nus phoxinus was used as outgroup in this study. Pseu-daspinini have a holarctic distribution, but have theirhighest diversity in the Nearctic. Leuciscini are mostlyrestricted to the palearctic with most species occurringin Europe and Western Asia. Notemigonus chrysoleucasis the only representative of Leuciscini in North Amer-ica, what was already proposed by Berg (1949) and Illick(1956) based on morphological data and is well sup-ported by molecular studies (Pyron, 1996; Schmidt et al.,1998).

In Europe, most native cyprinid genera (24 out of 34)belong to the Leuciscini. According to fossil records, theycolonized Europe from East or Central Asia during theOligocene reaching the Iberian Peninsular by the late Oli-gocene-early Miocene (Sanjur et al., 2003; see also Cav-ender, 1991 for review). The relationships amongpalearctic leuciscins is still far from being settled andmany species were re-grouped during the last decades (seeKottelat, 1997 for overview). This is especially true forthe genus Phoxinellus Heckel (1843) (type species: Phox-inellus alepidotus Heckel, 1843). Various small Mediterra-nean leuciscins were included in this genus and laterre-grouped into other genera (Trewavas, 1971; Karaman,1972; Almaca, 1977; Banarescu, 1977; Banister, 1980;Economidis and Banarescu, 1991; Bogutskaya, 1992;Kottelat, 1997). Four new species of Phoxinellus wererecently discovered (Zupanbib and Bogutskaya, 2000;Zupanbib and Bogutskaya, 2002; Bogutskaya andZupanbib, 2003). In their most recent review, Bogutskayaand Zupanbib (2003) restrict Phoxinellus to 10 species allendemic to Dalmatia, sharing the following set of charac-ters: pharyngeal teeth 5–5 or 5–4, 6 1/2 to 8 1/2 brancheddorsal- and anal-Wn rays, absence of a communicationbetween the preoperculo-mandibular and infraorbitalsensory canals and small size (standard length usuallysmaller than 150 mm). These diagnostic characters can beinterpreted as reductive, most likely associated with theenvironmental conditions of small karstic streams, whereall 10 species occur (Zupanbib and Bogutskaya, 2002). In2003, a Weld expedition to Bosnia-Herzegovina andCroatia gave JF and NB the unique opportunity to studyall species of Phoxinellus in their natural habitat. Itbecame obvious during this Weld work that some Phoxin-ellus were morphologically very similar to Telestes whileothers seemed to be unique. It was therefore the aim ofthis study, to analyze the phylogenetic structure of Phox-inellus within the Palearctic Leuciscinae and to test ifPhoxinellus is monophyletic or includes several phyloge-netic lineages. Similarities within Phoxinellus could bedue to parallel adaptation to similar habitats (karststreams).

2. Materials and methods

2.1. Sample origin and morphological analysis

The location of voucher specimens and the origin of sam-ples are listed in Appendix A. Methods of morphologicalanalysis followed Hubbs and Lagler (1958). All materials andmorphological characters examined and morphologicalresults are presented by Bogutskaya and Zupanbib (2003)and not presented again in the present study. Fish werecaught by handnet or portable electroshockers and preservedin 10% formalin. One Wn of each specimen was preserved in98% ethanol for molecular analysis.

2.2. Sequence analyses

Total genomic DNA from ethanol preserved specimenswas extracted using standard procedures (QIAamp DNABlood and Tissue Kit, Qiagen). The entire mitochondrialcytochrome b gene was ampliWed in two overlapping frag-ments with primers GluDgL; Cb3H (Palumbi et al., 1991) andCytb-F; Thr-R (Zardoya and Doadrio, 1998). PCR reactionmixtures contained 0.8 U AmpliTaq DNA Polymerase (Per-kin–Elmer), 10mM Tris–HCl (pH 8.3), 50mM KCl, 1.5mMMgCl2, 200�M dNTPs, 2�M of each primer and approxi-mately 100 ng of DNA in a Wnal volume of 25�L. Reactionmixtures were subjected to the following PCR cycling proto-col on a GeneAmp PCR System 2400 (Perkin Elmer): initialdenaturation (94°C: 3min), 35 cycles (94°C: 15s; 50°C: 20s;72°C: 1min) and Wnal extension (72°C: 7min). All PCRproducts were puriWed (QIAquick PCR PuriWcation Kit,Qiagen), and directly sequenced with either one of the primersdescribed above, using the Xuorescent Prism BigDye Termi-nator Cycle Sequencing kit (ABI) according to the manufac-turers instructions followed by product separation on anautomated 3100 Genetic Analyzer (ABI).

To evaluate the molecular data from the mitochondrialcytochrome b gene we investigated the phylogenetic informa-tion content of a recently described highly variable noncodingnuclear region according to Lieckfeldt et al., 2006). GenomicDNA was extracted from tissue samples using the DNeasyTissue Kit (Qiagen). Primers for the intron of the RAG1 gene(Venkatesh et al., 1999) were used for several species. Aftercloning and sequencing, speciWc primers (Cyp_unFLP1F 5�-AAGTGGTGCATCGTGTTGTG-3�; Cyp_unFLP1R 5�-CAGCCTGAACAATCAAAACAG-3�) were designed for aconvenient PCR product covering a great portion of the vari-able region. AmpliWcation was carried out in 25�l reaction vol-umes containing 50–100ng of DNA, 1.5mM MgCl2, 10mMTris–HCl (pH 8.3), 50mM KCl, 200�M of each dNTP,10pmol of each primer and 0.5 units of AmpliTaq DNA poly-merase (Perkin–Elmer). Reaction mixtures were subjected tothe following cycling protocol: initial denaturation (94°C:3min), 35 cycles (94°C: 15s; 55°C: 20s; 72°C: 45s) and Wnalextension (72°C: 7min). PCR products were puriWed by treat-ment with ExoSAP-ITTM (USB), and directly sequenced. Thenuclear fragment was sequenced in 26 species (Table 1).

418 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425

2.3. Phylogenetic analyses

We carried out the following types of phylogeneticanalyses to investigate evolutionary relationships: (i)neighbour-joining (NJ) as implemented in MEGA 2.1(Kumar et al., 1993), (ii) maximum parsimony (MP) and

(iii) maximum-likelihood (ML) using PAUP¤ 4.0b10(SwoVord, 2002). For the NJ analyses, distances were cal-culated applying Tamura and Nei’s (1993) method using a� shape parameter of gamma distribution calculated bymaximum likelihood. Non-parametric bootstrap analyses(Felsenstein, 1985) with 1000 pseudo-replicates were

Table 1

a all nuclear sequences resulted from this study or Freyhof et al. (2005).

ScientiWc name in source Accession Nr. cyt b / nuca Reference New name

Phoxinus phoxinus Y10448 Briolay et al., 1998Chondrostoma vardarensis AF090749 Zardoya and Doadrio (1999)Chondrostoma prespensis AF090747 Zardoya and Doadrio (1999)Chondrostoma nasus Z75109 Briolay et al., 1998Chondrostoma oxyrhynchum AF095606 Zardoya and Doadrio (1999)Chondrostoma soetta AF533767 Durand et al., 2003Chondrostoma polylepis AF045982 Zardoya and Doadrio (1998)Chondrostoma duriensis AF045983 Zardoya and Doadrio (1998)Chondrostoma willkommii AF045984 Zardoya and Doadrio (1998)Telestes pleurobipunctatus AF090764 Zardoya and Doadrio (1999)Telestes p. alWensis AF090765 Zardoya and Doadrio (1999) Telestes alWensisTelestes beoticus AF090770 Zardoya and Doadrio (1999)Leuciscus souYa Y10439 Briolay et al., 1998 Telestes souYaRutilus rutilus Y10440 Briolay et al., 1998Leuciscus borysthenicus AF090759 Zardoya and Doadrio (1999) Petroleuciscus borysthenicusLeuciscus keadicus AF090760 Zardoya and Doadrio (1999) Squalius keadicusLeuciscus pyrenaicus AF045991 Zardoya and Doadrio (1998) Squalius pyrenaicusLeuciscus aradensis AF421825 Sanjur et al., 2003 Squalius aradensisLeuciscus cephalus vardarensis AF090754 Zardoya and Doadrio (1999) Squalius vardarensisAlburnoides bipunctatus Y10445 Briolay et al., 1998Tropidophoxinellus hellenicus AF090776 Zardoya and Doadrio (1999)Scardinius erythrophthalmus Y10444/AY831422 Briolay et al., 1998Leucaspius delineatus Y10447 Briolay et al., 1998Alburnus alburnus Y10443 Briolay et al., 1998Anaecypris hispanica AF045978 Zardoya and Doadrio (1998)Notemigonus crysoleucas U01318 Schmidt et al., 1998Blicca bjoerkna Y10442 Briolay et al., 1998Leuciscus leuciscus Y10449 Briolay et al., 1998Pachychilon pictus AF090762 Zardoya and Doadrio (1999) Pachychilon pictumPhoxinellus prespensis AF090763 Zardoya and Doadrio (1999) Pseudophoxinus prespensisPseudophoxinus stymphalicus AF090767 Zardoya and Doadrio (1999)Pseudophoxinus s. marathonicus AF090768 Zardoya and Doadrio (1999) Pseudophoxinus marathonicusPseudophoxinus s. thesproticus AF090769 Zardoya and Doadrio (1999) Pseudophoxinus thesproticusPhoxinellus alepidotus AY838925/AY831414 this studyPhoxinellus pseudalepidotus AY838926/AY831415 this studyPhoxinellus dalmaticus AY838927 this studyPhoxinellus ghetaldii AY838929/DQ077154 this study Delminichthys ghetaldiiPhoxinellus jadovensis AY838924/DQ077155 this study Delminichthys jadovensisPhoxinellus krbavensis AY838930/DQ077156 this study Delminichthys krbavensisPhoxinellus adspersus AY838923/DQ077153 this study Delminichthys adspersusPhoxinellus croaticus AY838932/DQ077158 this study Telestes croaticusPhoxinellus fontinalis AY838928/DQ077157 this study Telestes fontinalisPhoxinellus metohiensis AY838931/DQ077159 this study Telestes metohiensisSqualius cephalus AY549461/AY831424 Freyhof et al., 2005Squalius microlepis AY549462/AY831425 Freyhof et al., 2005Squalius albus AY549460/AY831427 Freyhof et al., 2005Telestes muticellus AY838934/AY831416 this studyTelestes turskyi AY549463/AY831417 Freyhof et al., 2005Telestes polylepis AY838933/DQ077160 this studyAlburnus mento AY838935 this studyAlburnus baliki AY838936 this studyLadigesocypris ghigii AY838937 this studyPelecus cutratus AY838938 this studyPseudophoxinus minutus AY838939/AY831421 this study

J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 419

performed to obtain estimates of support for each node ofthe NJ trees. For MP analyses, we excluded constant anduninformative sites, weighted all characters and charactertransformations equally, and used the TBR branch swap-ping option. Because exhaustive and branch and boundanalyses result in prohibitively long computation times,we used the heuristic search option with 1000 replicates ofrandom sequence addition. Statistical support for recov-ered nodes was assessed using non-parametric bootstrapanalysis with 1000 pseudo-replicates. For the ML analy-ses, we used likelihood ratio tests and the computer appli-cation MODELTEST 3.06 (Posada and Crandall, 1998)to determine the best-suited model of sequence evolution.The accompanying parameter values for these data (basefrequency, instantaneous rates for each substitution type,shape of the distribution used to accommodate theamong-site rate variation, proportion of invariant sites)were then applied to reconstruct phylogenetic trees. Thebest-Wt model selected by MODELTEST for the data setwas the general time-reversible model (Rodrigez et al.,1990) with an allowance for invariant sites and a gammashape for among-site rate variation under the hierarchicallikelihood ratio test method. Heuristic ML searches wereperformed with 10 replicates of random sequence addi-tion and TBR branch swapping. ML bootstraps employed1000 iterations.

A likelihood ratio test for rate constancy (Felsenstein,1988) was performed using PUZZLE 4.02 (Strimmer andvon Haeseler, 1996), where the likelihood of the ML treewas compared with the likelihood of the same treewith the constraint of a strict molecular clock. Becauserate heterogeneity among lineages was highly signiWcant,we dated the nodes by using the nonparametric ratesmoothing (NPRS) method of Sanderson (1997). Thismethod estimates rates and divergence times by using acriterion that maximizes the autocorrelation of rateswithin clades. The ML tree with optimized branch lengthsobtained using PAUP¤ 4.0 was transformed into anultrametric tree by using the NPRS algorithmimplemented in the software TreeEdit (version 1.0 alpha4–61, August 2000, written by Andrew Rambaut andMike Charleston and available at http://evolve.zoo.ox.ac.uk/software/TreeEdit/main.html). This approach doesnot assume a molecular clock, but assumes that ratesof change tend to be similar between adjacent brancheson the tree. It produces an ultrametric tree by minimizingthe sum of squared changes in rate between ancestorand descendant branches across the tree. To transformrelative time to absolute ages we calibrated thetree with a well-dated geological event from the latePliocene: the opening of the strait of Korinthos, whichseparates the Peloponnesus from the mainland (Der-mitzakis, 1990). To compute error estimates for the agesinferred from the cyt b gene, we reapplied the NPRS pro-cedure to 100 bootstrapped matrices obtained byresampling the data using PHYLIP 3.573c (Felsenstein,1993).

3. Results

3.1. Phylogenetic analysis of cytochrome b sequences

The entire cytochrome b (1141 bp) was used for phyloge-netic reconstructions. Topologies were nearly identicalunder MP (Fig. 1), ML and NJ. The parsimonious tree was3551 steps long (CI D 0.221; RI D 0.452).

All three tree constructing methods (MP, ML, NJ) gen-erated six major lineages (clades A–F, Fig. 2) and, eightmonotypic lineages: Notemigonus, Pelecus, Pachychilon,Leuciscus, Blicca, Rutilus, Alburnoides, and Tropidophoxin-ellus. Clade A, supported by high bootstrap values, con-tained species of the genera Chondrostoma and Phoxinellus.Clade B combined several members of the genus Telestes:T. alWensis, T. beoticus, T. metohiensis, T. muticellus, T.pleurobipunctatus, T. polylepis, T. souYa, and T. turskyi.Interestingly, Telestes croaticus and T. fontinalis formed aseparate clade C. Clade D is composed of two lineages:Pseudophoxinus and Delminichthys. This split is supportedby high bootstrap values in the MP and NJ analyses butnot in the ML approach. Furthermore, the newly proposedgenus Delminichthys also consists of at least two groups (D.adspersus/D. ghetaldii and D. jadovensis/D. krbavensis).Clade E is composed of three genera, Squalius, Ladigesocy-pris, and Petroleuciscus, the latter being in basal position.Although with only weak bootstrap support (51% MP/67%NJ/44% ML), a potential additional clade (F) is formed bythe following genera: Scardinius, Leucaspius, Anaecypris,and three species of the genus Alburnus.

3.2. Phylogenetic analysis of nuclear sequences

Taken together sequences of 26 species were analyzed.Seventeen species are listed in Table 1. Additionally, the fol-lowing species were also used for the characterisation of anintergeneric hypervariable region: Alburnoides maculatus(AY831413); Alburnus albidus (AY831418); Leuciscus dani-lewski (AY831419); Rutilus rubilio (AY831420); Rutilus aula(DQ088993); Squalius tenellus (AY831426); Squalius aphipsi(DQ088994); Scardinius graecus (AY831423) and Scardiniusdergle (AY831428). We observed a great length variabilityof the PCR fragment ranging from 120 bp in Pseudophoxi-nus minutus up to 643 bp in Leuciscus danilewski. Intraspe-ciWc variability was not detected as also described previously(Lieckfeldt et al., 2006). Alignments are available uponrequest from the authors. While the number of species ismore limited, the topologies of the diVerent former speciesof Phoxinellus obtained by the nuclear marker are similar tothe mtDNA trees (Fig. 3). In agreement with the mtDNAtrees we observed the following clades: one clade containingthe species of the genera Telestes and Phoxinellus, a secondclade contain the species of the genus Squalius, a third cladeuniWes the Scardinius-species. Most important, in all nucleartrees we found a high bootstrap support for the phyloge-netic aYnity of the new genus Delminichthys as also foundin phylogenetic reconstructions based on mtDNA. In

420 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425

general, the topology of the nuclear trees is concordant withthe results of the mitochondrial data set and of the morpho-logical characters.

3.3. Unequal rates of change and dating divergences

With its interspersed long and short branches our phylo-gram (Fig. 2) clearly violates a molecular clock. A generalclock-like behavior was also rejected because the constrainedand unconstrained analyses were signiWcantly diVerent in a

likelihood ratio test (without clock, ¡lnD18466.95; withclock, ¡lnD19122.76; P<0.005; �D1311.61). Because thetests of rate heterogeneity among lineages were signiWcant, wedated the nodes by using a tree-based methodology (Sander-son, 1997) that relies on geological calibration (Dermitzakis,1990) of nucleotide substitution rates. The molecular clockwas calibrated using a well-dated geological event from thelate Pliocene: the opening of the strait of Korinthos(2.5MYA), which separates the Peloponnesus from the main-land (Dermitzakis, 1990). Telestes pleuropibunctatus and

Fig. 1. Most parsimonious tree calculated in PAUP¤ 4.0b10 (SwoVord, 2002) based on entire cytochrome b sequences. The MP-, ML-, and NJ-topologieswere very similar. Details of calculations are discussed in the text. Bootstrap values (MP/NJ/ML) are shown on branches.

Chondrostoma polylepisChondrostoma duriensisChondrostoma willkommii

Chondrostoma soettaChondrostoma oxyrhynchumChondrostoma nasusChondrostoma prespensisChondrostoma vardarensis

Phoxinellus dalmaticusPhoxinellus alepidotusPhoxinellus pseudalepidotus

Telestes beoticus

Telestes pleurobipunctatusTelestes alfiens

Telestes metohiensis

Telestes souffia

Telestes muticellus

Telestes turskyiTelestes polylepis

Telestes fontinalisTelestes kroaticusPseudophoxinus prespensis

Pseudophoxinus minutusPseudophoxinus thesproticus

Pseudophoxinus marathonicusPseudophoxinus stymphalicus

Dalmatichthys ghetaldiiDalmatichthys jadovensisDalmatichthys krbavensis

Dalmatichthys adspersus

Petroleuciscus borystenicusSqualius keadicusSqualius vardarensisSqualius cephalusSqualius albusSqualius pyrenaicusSqualius microlepisSqualius ardensisLadigesocypris ghigiiTropidophoxinellus hellenicus

Scardinius erythrophthalmusAlburnoides bipunctatus

Alburnus balikiAnaecypris hispanica

Leucaspius delineatusAlburnus alburnusAlburnus mento

Rutilus rutilusBlicca bjoerkna

Pachychilon pictumPelecus cultratus

Leuciscus leuciscus

Notemigonus crysoleucasPhoxinus phoxinus

86/95/9580/87/91

100/100/6452/64/52

100/100/8657/72/92

89/99/86

100/100/100

87/98/78

99/98/99

100/100/99

100/100/9495/98/84

64/59/74

99/100/89

92/98/65

100/100/56

100/100/97

71/63/6765/75/90100

10094

76/98/91

100/99/99

80/86/89

73/64/46

806846

50/56/42

51/67/44

94/99/82979967

557541

A

B

C

D

E

F

DelminichthysDelminichthysDelminichthysDelminichthys

croaticus

alfiensis

J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 421

T. alWensis were separated by the formation of the Strait ofKorinthos (Zardoya and Doadrio, 1999). In the mitochon-drial cytochrome b gene, genetic distance and substitutionplots indicated some degree of saturation. The data set con-tains both deep splitting events (Telestes croaticus/T. fontinalisvs. all other Telestes species) as well as very recent separationevents (genera Telestes and Squalius) (Figs. 1 and 2).

Based on morphological characters, Bogutskaya andZupanbib (2003) suspected, that Phoxinellus might be dividedinto tree groups. Molecular data support theses groupswithin Phoxinellus. Phoxinellus should therefore be restrictedto species with plain coloration, small or absent postclei-thrum, no genital papilla and an almost naked body such asP. alepidotus, P. dalmaticus, and P. pseudalepidotus.

Fig. 2. Clock-constrained ML tree (chronogram) showing the cladogenesis of cyprinid taxa examined. The tree was constructed under the GTR+G+Imodel (I D 0.844, proportion of invariable sites D 0.512). The node ages were estimated according to Sanderson (1997) nonparametric rate smoothing(NPRS) method using TreeEdit and PAUP¤. The scale bar below the tree shows the time scale in millions of years resulting from a calibration of themolecular clock based on the opening of the Strait of Korinthos (see text). The corresponding split between Telestes pleurobipunctatus from the ArachthosRiver and T. alWensis from the Alphios River is indicated (arrow).

Million Years

Chondrostoma polylepisChondrostoma duriensisChondrostoma willkommii

Chondrostoma soettaChondrostoma oxyrhynchumChondrostoma nasusChondrostoma prespensisChondrostoma vardarensis

Phoxinellus dalmaticusPhoxinellus alepidotusPhoxinellus pseudalepidotus

Telestes beoticus

Telestes pleurobipunctatusTelestes alfiens

Telestes metohiensis

Telestes souffia

Telestes muticellus

Telestes turskyiTelestes polylepis

Telestes fontinalisTelestes kroaticus

Pseudophoxinus prespensisPseudophoxinus minutus

Pseudophoxinus thesproticus

Pseudophoxinus marathonicusPseudophoxinus stymphalicus

Dalmatichthys ghetaldii

Dalmatichthys jadovensisDalmatichthys krbavensis

Dalmatichthys adspersus

Petroleuciscus borystenicusSqualius keadicus

Squalius vardarensis

Squalius cephalusSqualius albus

Squalius pyrenaicus

Squalius microlepisSqualius ardensis

Ladigesocypris ghigii

Tropidophoxinellus hellenicusScardinius erythrophthalmus

Alburnoides bipunctatus

Alburnus balikiAnaecypris hispanica

Leucaspius delineatusAlburnus alburnusAlburnus mento

Rutilus rutilus

Blicca bjoerkna

Pachychilon pictumPelecus cultratusLeuciscus leuciscus

Notemigonus crysoleucas

25 20 15 10 5 0

croaticus

Delminichthys adspersusDelminichthys jadovensisDelminichthys krbavensisDelminichthys ghetaldii

alfiensis

422 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425

The monophyly of this group is supported by high bootstrapvalues. Similar bootstrap support is given for Phoxinellus assister taxon of Chondrostoma, and for both as sister taxa ofTelestes forming a species-rich monophyletic group withinLeuciscini. Morphologically, Phoxinellus is distinguishedfrom other genera by a set of leuciscine plesiomorphic char-acter states in combination with autapomorphic reductiveelements (reduction of scales and postcleithrum). Specieswith a dark stripe from the head to the caudal peduncle,small or absent postcleithrum, no genital papilla and with abody covered by distinct, not overlapping scales should bepositioned into the genus Telestes. Such species are T. croati-cus, T. metohiensis, and T. fontinalis. Species such as Phoxin-ellus adspersus, P. ghetaldii, P. jadovensis and P. krbavensisare only distantly related to Phoxinellus. These species allhave an irregularly spotted color pattern, a large postclei-thrum, an increased number of precaudal anal-Wn pterygio-phores, and a large genital papilla in females. We propose tojoin these species in a new genus, named Delminichthys. Sup-ported by high bootstrap values, Delminichthys was posi-tioned as sister genus of Pseudophoxinus. Interestingly, thesetwo groups may form a sister group to all other Leuciscini.

3.4. Description of the new genus

3.4.1. Delminichthys, new genus3.4.1.1. Type species. Leucos adspersus Heckel (1843)

3.4.2. DiagnosisLower jaw never with a trenchant horny sheath; mouth

terminal or inferior; no midventral keel in front of anus;lower lip without median lobe; scales very thin, not over-lapping; 3 (rarely 4) anal-Wn pterygiophores in front of Wrstcaudal haemal spine; postcleithrum very strong, thickenedand long, its lower end reaches much below the pectoral-Wnbase; entire dorsal surface and Xanks covered by numerousdark spots of irregular shape and size; spots dense on backand may be fused together forming larger irregularlyshaped blotches; large genital papilla formed like a thick-ened triangular fold with anus at base on its ventral surfacein females; in adult females (larger than 53 mm SL) genitalpapilla base wide, extending over lowermost part of the Wrstanal-Wn rays; pectoral Wn almost reaches or reaches the pel-vic-Wn base in males; and the pelvic Wn reaches way beyondthe anal-Wn origin in males.

3.4.3. EtymologyDerived from Delminium, the capital of the pre-Roman

Dalmatia and greek ichthys (Wsh). Gender masculine.

3.4.4. RemarksDelminichthys includes the following named species: D.

adspersus (Heckel, 1843), D. ghetaldii (Steindachner, 1882),D. jadovensis (Zupanbib and Bogutskaya, 2002), D. krbav-ensis (Zupanbib and Bogutskaya, 2002). For description of

Fig. 3. Neighbour-joining tree of the nuclear intergeneric region based on Kimura-2 Parameter distance values calculated in MEGA 2.1 based on thenuclear sequences. Bootstrap percentages >50% (Neighbour-Joining/Minimum Evolution/Maximum Parsimony) are shown on branches.

0.01

Squalius tenellusSqualius microlepisSqualius cephalus

Squalius albusSqualius aphipsii

Scardinius graecus

Scardinius erythrophthalmusScardinius dergle

Rutilus aulaRutilus rubilio

Alburnus maculatusAlburnus albidus

Pseudophoxinus minutusDalmatichthys krbavensis

Dalmatichthys jadovensisDelminichthys adspersus

Delminichthys ghetaldii

Leuciscus danilewskiPhoxinellus pseudalepidotusPhoxinellus alepidotus

Telestes fontinalisTelestes croaticus

Telestes metohiensisTelestes turskyi

Telestes muticellusTelestes polylepis

95/97/9170/67/69

91/92/87

79/76/95

89/91/9568/69/--

99/99/99

96/96/98

68/72/69

62/51/69

70/63/69

75/60/64

89/93/8993/93/89

70/76/55

627181

Delminichthys krbavensisDelminichthys jadovensis

Alburnoides maculatus

J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 423

species see Zupanbib and Bogutskaya, 2002 and Boguts-kaya and Zupanbib (2003). Delminichthys is distinguishedfrom other European leuciscine cyprinids by the followingcombination of characters: scales very thin, not overlap-ping (vs. scales well ossiWed, overlapping in all genera, butin Phoxinellus and Telestes in part); lower jaw never with ahorny sheath (vs. present in Chondrostoma); 8–12 gill rakers(vs. 84–130 in Anaecypris); no midventral keel in front ofanus (vs. presence in Abramis, Alburnus [including Chalcal-burnus], Alburnoides, Ballerus, Blicca, Leucaspius, Pelecus,Tropidophoxinellus; Vimba); lower lip without median lobe(vs. present in Pachychilon); 3 (rarely 4) anal-Wn pterygio-phores in front of Wrst caudal haemal spine (vs. 1–2 in Leu-ciscus [including Aspius], Squalius, Petroleuciscus,Phoxinellus, Telestes); postcleithrum very strong, thickenedand long, its lower end reaches much below the pectoral-Wnbase (very thin or absent in Phoxinellus, Telestes croaticus,T. metohiensis, T. fontinalis); entire dorsal surface andXanks covered by numerous dark spots of irregular shapeand size (vs. body coloration plain in Phoxinellus, with awide lateral stripe from the eye to the caudal Wn in Teles-tes); large genital papilla (vs. no genital papilla in Phoxinel-lus, Telestes); scales embedded in skin (vs. not embedded inPseudophoxinus).

4. Discussion

Both molecular data sets and morphological characterswell agree in the grouping of species of Phoxinellus asshown by Bogutskaya and Zupanbib (2003). Ketmaier et al.(2004) already postulated a close relationship of Phoxinel-lus croaticus and P. metohiensis to species of the genusTelestes by mtDNA data. The position of other Phoxinellusspecies within Leuciscini was never truly investigated due toa mosaic of plesiomorphic leuciscine traits and apomor-phic, but reductive characters. Some character states such

as increased number of deeply embedded scales, character-istic for Delminichthys and Dalmatian Telestes croaticus, T.fontinalis, T. polylepis, and T. turskyi are also found inendemic Dalmatian leuciscine cyprinids such as Chondros-toma phoxinus, Squalius microlepis, and S. tenellus whichare not closely related to any above mentioned species. Thereduction of scales as in Phoxinellus is also found in theendemic Dalmatian barbine cyprinid Aulopyge huegelii andin the barbine cyprinid genus Sinocyclocheilus that inhabitskarstic waters in China (Yue, 2000). These characters mayreXect an adaptation on their speciWc environmental condi-tions in the karstic waters of Dalmatia.

The limited resolving power of the molecular data couldbe caused by diVerent rates of substitutions among taxa.The detected lower rate of evolution in Delminichthys com-pared to other leuciscine cyprinids (Fig. 4) might be relatedto a very long generation time, all species of this genusinhabit cold, karstic springs and migrate to subterraneanwaters during winter and summer droughts (Curcic, 1913;Trgovcevic, 1905; Vukovic and Ivanovic, 1971; Vukovic,1977). Because detailed ecological data from species of thegenus Delminichthys are absent, it must remain speculativehow physiological parameters (for example a lower meta-bolic rate) might inXuence the evolutionary rate. The lim-ited resolving power of the molecular data could also be aneVect of long branch attraction between taxa with a longhistory of isolated evolution, such as Telestes croaticus, andT. fontinalis. A further cause could also be a fast radiationrelative to the substitution rate of sequences analyzed. Itcan not be excluded that cyprinids radiated within a rela-tively short time frame. This rapid radiation following along time of stasis may have resulted in the partly low reso-lution of the data set (Fig. 1).

Banarescu and Herzig-Straschil (1998) reviewed Teles-tes (as a subgenus of Leuciscus) based on morphologicaldata. Telestes as a valid genus was re-established by

Fig. 4. Plot of DNA sequence divergence based on uncorrected p-distance values calculated in MEGA 2.0 (Kumar et al., 1993) vs. time (MYA). The Wguredemonstrates the lower evolutionary rate of the species from the genus Delminichthys (lower line) in relation to the other Leuciscinae-species included inthis study. Time values were taken from Fig. 2.

y = 0.0085x

R2= 0.7391

y = 0.0052x

R2 = 0.9385

0

0.05

0.10

0.15

0.20

0.25

0 5 10 15 20 25

time in million years

DN

A s

equ

ence

div

erg

ence

424 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425

Ketmaier et al. (1998) based on enzyme electrophoreticdata. The distinctness of Telestes from Leuciscus (includ-ing Aspius) and Squalius is also strongly supported bymtDNA data (Gilles et al., 2001; Zardoya and Doadrio,1999). Squalius and the recently described genus Petroleu-ciscus can be clearly diagnosed by apomorphic morpho-logical characters (Bogutskaya, 1996, 2002), whereasTelestes and Leuciscus are diYcult to distinguish morpho-logically. Both show a set of plesiomorphic leuciscinecharacters and almost no autapomorphies.

Our data suggest the ancestor of the widespread generaChondrostoma (see Durand et al., 2003 for overview) andTelestes occurred within Dalmatian water bodies. Followingthe molecular data, Telestes croaticus and T. fontinalis maybe interpreted as the most basic Telestes, and Phoxinellusmost likely forms the sistergroup of Chondrostoma. BasicTelestes and all Phoxinellus are strictly endemic to Dalmatia.Some descendants from this group may have remained inDalmatia and have formed geographically very restrictedspecies (T. metohiensis, T. polylepis, T. turskyi, and unstudiedT. ukliva). Following the molecular clock attempt by Zard-oya and Doadrio (1999), Delminichthys forms its own line-age, separated from European Pseudophoxinus since middleMiocene (13MY). Delminichthys is a remnant of the Wrstmajor radiation within leuciscines during mid-Miocene(Zardoya and Doadrio, 1999) leading to most of the genericgroups recognized today. Most of these generic groups rap-idly reached a widespread geographic distribution withinEurope and western Asia. In contrast, Delminichthys repre-sents one of the geographically most isolated genera of leu-ciscine cyprinids supporting the Miocene refuge hypothesisof Dalmatian karst habitats. The Dinaric orogenesis, as apart of the Alpine orogenesis came to its end about 10–8MYago. It is likely that Delminichthys were trapped in Dalmatiaby the rise of mountains. This might be true for Phoxinellusand Dalmatian species of Telestes (T. croaticus, T. fontinalis,T. metohiensis, T. polylepis, T. ukliva, and T. turskyi) as well.

Considering the outcome of our studies, we draw the fol-lowing phylogenetic and biogeographic conclusions:Chondrostoma, Telestes and Phoxinellus form one mono-phyletic group which is likely to have arisen in Dalmatia.Delminichthys belongs to a group of genera, which isrestricted to the southern Balkan and might have invadedDalmatia coming from this area. Having a very restricteddistribution area, all species of Delminichthys, Phoxinellusand Dalmatian Telestes, are highly endangered. Massivealterations are obvious in the habitats of all species (Povcet al., 1990). Water shortage due to increasing economicdevelopment and projected climate changes will furthercontribute to the ongoing decline of all species. Furtherattempts for their conservation are strongly recommend tosave these old evolutionary lines.

Acknowledgments

NGB was sponsored by a grant of the NaturhistorischesMuseum Wien (2000), a grant from the Russian Foundation

for Basic Research (N 01-04-49552), and a support fromZagreb University. Authors are grateful to E. Mikschi, B.Herzig, C. Prenner, H. Wellendorf (NaturhistorischesMuseum, Wien), H. Wilkens, and G. Schulze (ZoologischesMuseum und Institut der Universität Hamburg), F. Krupp,K. Jentoch (Senckenberg Museum, Frankfurt/Main), M.Mrakovcic (Zagreb University), and B. Sket (Ljubljana Uni-versity) for their help during the work with collections undertheir care. We are thankful to Primoc Zupancic (Ljubljana)for his help during Weld work. The authors thank J. Fickel(Leibniz-Institute for Zoo and Wildlife Research Berlin) forhelpful comments and discussions.

Appendix A. Supplementary material

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ympev.2005.07.024.

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