Molecular Phylogenetics and Evolution 38 (2006) 416–425 www.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 b Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, 12587 Berlin, Germany 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 2005 Available online 6 October 2005 Abstract The Dalmatian cyprinid genus Phoxinellus is characterized by reductive characters most likely associated with the environmental conditions of small karstic streams, where all species of this genus occur. Based on 33 morphological traits, nuclear and mtDNA sequences Phoxinellus was found to be paraphyletic and included three not closely related monophyletic units. The scientiWc name Phoxinellus should therefore be restricted to species having plain coloration, small or absent postcleithrum, no genital papilla and an almost entirely naked body such as P. alepidotus, P. dalmaticus, and P. pseudalepidotus. Species that also have a small or absent postcleithrum and no genital papilla but display a dark stripe from the head to the caudal peduncle, and are entirely covered by distinct, not overlapping scales should be positioned closely to Telestes. Thus, we suggest inclusion of Phoxinellus croaticus, P. fontinalis and Paraphoxinus metohiensis in the genus Telestes. The Phoxinellus species that have a irregularly spotted color pattern, a large postcleithrum, an increased number of precaudal anal-Wn pterygiophores, and a large genital papilla in females represent its own evolutionary line closely related to the Balkan species of Pseudophoxinus. For this monophyletic group, we propose to introduce a new genus: Delminichthys. This genus includes the species 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 Teleost family Cyprinidae is one of the most species rich Wsh families (Nelson, 1994). The phylogenetic structure of this family is still under debate, but several subfamilies are widely accepted and seem to form strongly supported monophyletic clades (Chen et al., 1984; Howes, 1991; Liu et al., 2002). One of these is the subfamily Leuciscinae, which is widely distributed throughout northern Eurasia, North America 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 North America about 45–50 native genera including more than 250 species are recognized (Mayden, 1991). The subfamily Leuciscinae was subdivided by Bogutskaya (1988a,b, 1990a,b) into the tribes Abramidini, Alburnini, Aspinini, Leuciscini, Pseudaspinini, Elopichthyini, Pelecini, and Hypophthalmichthyini. The latter three are very specialized, monotypic or almost monotypic (Yue, 2000). The inclusion of Elopichthyini and Hypophthalmichthyini within Leuciscinae is still under debate (Liu et al., 2002). If Elopichthys and Hypophthalmichthys are excluded, Leuciscinae is clearly divided into two major groups, Leuciscini s.l. (including Aspinini + Alburnini + Abramidini + Pelecini) on one hand and Pseudaspinini on the other. These two groups coincide with the informal J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 groups “leuciscins” ( D Leuciscini) and “phoxinins” ( D Pseudaspini) proposed by Cavender and Coburn (1992). This principal dichotomy is also supported by molecular data (Pyron, 1996; Briolay et al., 1998; Gilles et al., 2001; HänXing and Brandl, 2000; Cunha et al., 2002; Liu et al., 2002) and this is the reason, why Phoxinus phoxinus was used as outgroup in this study. Pseudaspinini have a holarctic distribution, but have their highest diversity in the Nearctic. Leuciscini are mostly restricted to the palearctic with most species occurring in Europe and Western Asia. Notemigonus chrysoleucas is the only representative of Leuciscini in North America, what was already proposed by Berg (1949) and Illick (1956) based on morphological data and is well supported 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, they colonized Europe from East or Central Asia during the Oligocene reaching the Iberian Peninsular by the late Oligocene-early Miocene (Sanjur et al., 2003; see also Cavender, 1991 for review). The relationships among palearctic leuciscins is still far from being settled and many species were re-grouped during the last decades (see Kottelat, 1997 for overview). This is especially true for the genus Phoxinellus Heckel (1843) (type species: Phoxinellus alepidotus Heckel, 1843). Various small Mediterranean leuciscins were included in this genus and later re-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 were recently discovered (Zupanbib and Bogutskaya, 2000; Zupanbib and Bogutskaya, 2002; Bogutskaya and Zupanbib, 2003). In their most recent review, Bogutskaya and Zupanbib (2003) restrict Phoxinellus to 10 species all endemic to Dalmatia, sharing the following set of characters: pharyngeal teeth 5–5 or 5–4, 6 1/2 to 8 1/2 branched dorsal- and anal-Wn rays, absence of a communication between the preoperculo-mandibular and infraorbital sensory canals and small size (standard length usually smaller than 150 mm). These diagnostic characters can be interpreted as reductive, most likely associated with the environmental conditions of small karstic streams, where all 10 species occur (Zupanbib and Bogutskaya, 2002). In 2003, a Weld expedition to Bosnia-Herzegovina and Croatia gave JF and NB the unique opportunity to study all species of Phoxinellus in their natural habitat. It became obvious during this Weld work that some Phoxinellus were morphologically very similar to Telestes while others seemed to be unique. It was therefore the aim of this study, to analyze the phylogenetic structure of Phoxinellus within the Palearctic Leuciscinae and to test if Phoxinellus is monophyletic or includes several phylogenetic lineages. Similarities within Phoxinellus could be due to parallel adaptation to similar habitats (karst streams). 417 2. Materials and methods 2.1. Sample origin and morphological analysis The location of voucher specimens and the origin of samples are listed in Appendix A. Methods of morphological analysis followed Hubbs and Lagler (1958). All materials and morphological characters examined and morphological results are presented by Bogutskaya and Zupanbib (2003) and not presented again in the present study. Fish were caught by handnet or portable electroshockers and preserved in 10% formalin. One Wn of each specimen was preserved in 98% ethanol for molecular analysis. 2.2. Sequence analyses Total genomic DNA from ethanol preserved specimens was extracted using standard procedures (QIAamp DNA Blood and Tissue Kit, Qiagen). The entire mitochondrial cytochrome b gene was ampliWed in two overlapping fragments with primers GluDgL; Cb3H (Palumbi et al., 1991) and Cytb-F; Thr-R (Zardoya and Doadrio, 1998). PCR reaction mixtures contained 0.8 U AmpliTaq DNA Polymerase (Perkin–Elmer), 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200
M dNTPs, 2
M of each primer and approximately 100 ng of DNA in a Wnal volume of 25
L. Reaction mixtures were subjected to the following PCR cycling protocol on a GeneAmp PCR System 2400 (Perkin Elmer): initial denaturation (94 °C: 3 min), 35 cycles (94°C: 15 s; 50 °C: 20s; 72 °C: 1min) and Wnal extension (72 °C: 7min). All PCR products were puriWed (QIAquick PCR PuriWcation Kit, Qiagen), and directly sequenced with either one of the primers described above, using the Xuorescent Prism BigDye Terminator Cycle Sequencing kit (ABI) according to the manufacturers instructions followed by product separation on an automated 3100 Genetic Analyzer (ABI). To evaluate the molecular data from the mitochondrial cytochrome b gene we investigated the phylogenetic information content of a recently described highly variable noncoding nuclear region according to Lieckfeldt et al., 2006). Genomic DNA was extracted from tissue samples using the DNeasy Tissue Kit (Qiagen). Primers for the intron of the RAG1 gene (Venkatesh et al., 1999) were used for several species. After cloning and sequencing, speciWc primers (Cyp_unFLP1F 5⬘-A AGTGGTGCATCGTGTTGTG-3⬘; Cyp_unFLP1R 5⬘-CA GCCTGAACAATCAAAACAG-3⬘) were designed for a convenient PCR product covering a great portion of the variable region. AmpliWcation was carried out in 25
l reaction volumes containing 50–100 ng of DNA, 1.5mM MgCl2, 10 mM Tris–HCl (pH 8.3), 50mM KCl, 200
M of each dNTP, 10pmol of each primer and 0.5 units of AmpliTaq DNA polymerase (Perkin–Elmer). Reaction mixtures were subjected to the following cycling protocol: initial denaturation (94°C: 3min), 35 cycles (94 °C: 15s; 55°C: 20 s; 72°C: 45 s) and Wnal extension (72°C: 7min). PCR products were puriWed by treatment with ExoSAP-ITTM (USB), and directly sequenced. The nuclear fragment was sequenced in 26 species (Table 1). 418 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 Table 1 ScientiWc name in source Accession Nr. cyt b / nuca Reference Phoxinus phoxinus Chondrostoma vardarensis Chondrostoma prespensis Chondrostoma nasus Chondrostoma oxyrhynchum Chondrostoma soetta Chondrostoma polylepis Chondrostoma duriensis Chondrostoma willkommii Telestes pleurobipunctatus Telestes p. alWensis Telestes beoticus Leuciscus souYa Rutilus rutilus Leuciscus borysthenicus Leuciscus keadicus Leuciscus pyrenaicus Leuciscus aradensis Leuciscus cephalus vardarensis Alburnoides bipunctatus Tropidophoxinellus hellenicus Scardinius erythrophthalmus Leucaspius delineatus Alburnus alburnus Anaecypris hispanica Notemigonus crysoleucas Blicca bjoerkna Leuciscus leuciscus Pachychilon pictus Phoxinellus prespensis Pseudophoxinus stymphalicus Pseudophoxinus s. marathonicus Pseudophoxinus s. thesproticus Phoxinellus alepidotus Phoxinellus pseudalepidotus Phoxinellus dalmaticus Phoxinellus ghetaldii Phoxinellus jadovensis Phoxinellus krbavensis Phoxinellus adspersus Phoxinellus croaticus Phoxinellus fontinalis Phoxinellus metohiensis Squalius cephalus Squalius microlepis Squalius albus Telestes muticellus Telestes turskyi Telestes polylepis Alburnus mento Alburnus baliki Ladigesocypris ghigii Pelecus cutratus Pseudophoxinus minutus Y10448 AF090749 AF090747 Z75109 AF095606 AF533767 AF045982 AF045983 AF045984 AF090764 AF090765 AF090770 Y10439 Y10440 AF090759 AF090760 AF045991 AF421825 AF090754 Y10445 AF090776 Y10444/AY831422 Y10447 Y10443 AF045978 U01318 Y10442 Y10449 AF090762 AF090763 AF090767 AF090768 AF090769 AY838925/AY831414 AY838926/AY831415 AY838927 AY838929/DQ077154 AY838924/DQ077155 AY838930/DQ077156 AY838923/DQ077153 AY838932/DQ077158 AY838928/DQ077157 AY838931/DQ077159 AY549461/AY831424 AY549462/AY831425 AY549460/AY831427 AY838934/AY831416 AY549463/AY831417 AY838933/DQ077160 AY838935 AY838936 AY838937 AY838938 AY838939/AY831421 Briolay et al., 1998 Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Briolay et al., 1998 Zardoya and Doadrio (1999) Durand et al., 2003 Zardoya and Doadrio (1998) Zardoya and Doadrio (1998) Zardoya and Doadrio (1998) Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Briolay et al., 1998 Briolay et al., 1998 Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Zardoya and Doadrio (1998) Sanjur et al., 2003 Zardoya and Doadrio (1999) Briolay et al., 1998 Zardoya and Doadrio (1999) Briolay et al., 1998 Briolay et al., 1998 Briolay et al., 1998 Zardoya and Doadrio (1998) Schmidt et al., 1998 Briolay et al., 1998 Briolay et al., 1998 Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) Zardoya and Doadrio (1999) this study this study this study this study this study this study this study this study this study this study Freyhof et al., 2005 Freyhof et al., 2005 Freyhof et al., 2005 this study Freyhof et al., 2005 this study this study this study this study this study this study a New name Telestes alWensis Telestes souYa Petroleuciscus borysthenicus Squalius keadicus Squalius pyrenaicus Squalius aradensis Squalius vardarensis Pachychilon pictum Pseudophoxinus prespensis Pseudophoxinus marathonicus Pseudophoxinus thesproticus Delminichthys ghetaldii Delminichthys jadovensis Delminichthys krbavensis Delminichthys adspersus Telestes croaticus Telestes fontinalis Telestes metohiensis all nuclear sequences resulted from this study or Freyhof et al. (2005). 2.3. Phylogenetic analyses We carried out the following types of phylogenetic analyses 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 calculated applying Tamura and Nei’s (1993) method using a
shape parameter of gamma distribution calculated by maximum likelihood. Non-parametric bootstrap analyses (Felsenstein, 1985) with 1000 pseudo-replicates were J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 performed to obtain estimates of support for each node of the NJ trees. For MP analyses, we excluded constant and uninformative sites, weighted all characters and character transformations equally, and used the TBR branch swapping option. Because exhaustive and branch and bound analyses result in prohibitively long computation times, we used the heuristic search option with 1000 replicates of random sequence addition. Statistical support for recovered nodes was assessed using non-parametric bootstrap analysis with 1000 pseudo-replicates. For the ML analyses, we used likelihood ratio tests and the computer application MODELTEST 3.06 (Posada and Crandall, 1998) to determine the best-suited model of sequence evolution. The accompanying parameter values for these data (base frequency, instantaneous rates for each substitution type, shape of the distribution used to accommodate the among-site rate variation, proportion of invariant sites) were then applied to reconstruct phylogenetic trees. The best-Wt model selected by MODELTEST for the data set was the general time-reversible model (Rodrigez et al., 1990) with an allowance for invariant sites and a gamma shape for among-site rate variation under the hierarchical likelihood ratio test method. Heuristic ML searches were performed with 10 replicates of random sequence addition and TBR branch swapping. ML bootstraps employed 1000 iterations. A likelihood ratio test for rate constancy (Felsenstein, 1988) was performed using PUZZLE 4.02 (Strimmer and von Haeseler, 1996), where the likelihood of the ML tree was compared with the likelihood of the same tree with the constraint of a strict molecular clock. Because rate heterogeneity among lineages was highly signiWcant, we dated the nodes by using the nonparametric rate smoothing (NPRS) method of Sanderson (1997). This method estimates rates and divergence times by using a criterion that maximizes the autocorrelation of rates within clades. The ML tree with optimized branch lengths obtained using PAUP¤ 4.0 was transformed into an ultrametric tree by using the NPRS algorithm implemented in the software TreeEdit (version 1.0 alpha 4–61, August 2000, written by Andrew Rambaut and Mike Charleston and available at http://evolve.zoo.ox. ac.uk/software/TreeEdit/main.html). This approach does not assume a molecular clock, but assumes that rates of change tend to be similar between adjacent branches on the tree. It produces an ultrametric tree by minimizing the sum of squared changes in rate between ancestor and descendant branches across the tree. To transform relative time to absolute ages we calibrated the tree with a well-dated geological event from the late Pliocene: the opening of the strait of Korinthos, which separates the Peloponnesus from the mainland (Dermitzakis, 1990). To compute error estimates for the ages inferred from the cyt b gene, we reapplied the NPRS procedure to 100 bootstrapped matrices obtained by resampling the data using PHYLIP 3.573c (Felsenstein, 1993). 419 3. Results 3.1. Phylogenetic analysis of cytochrome b sequences The entire cytochrome b (1141 bp) was used for phylogenetic reconstructions. Topologies were nearly identical under MP (Fig. 1), ML and NJ. The parsimonious tree was 3551 steps long (CI D 0.221; RI D 0.452). All three tree constructing methods (MP, ML, NJ) generated six major lineages (clades A–F, Fig. 2) and, eight monotypic lineages: Notemigonus, Pelecus, Pachychilon, Leuciscus, Blicca, Rutilus, Alburnoides, and Tropidophoxinellus. Clade A, supported by high bootstrap values, contained 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 a separate clade C. Clade D is composed of two lineages: Pseudophoxinus and Delminichthys. This split is supported by high bootstrap values in the MP and NJ analyses but not in the ML approach. Furthermore, the newly proposed genus 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, Ladigesocypris, 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 by the 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 following species were also used for the characterisation of an intergeneric hypervariable region: Alburnoides maculatus (AY831413); Alburnus albidus (AY831418); Leuciscus danilewski (AY831419); Rutilus rubilio (AY831420); Rutilus aula (DQ088993); Squalius tenellus (AY831426); Squalius aphipsi (DQ088994); Scardinius graecus (AY831423) and Scardinius dergle (AY831428). We observed a great length variability of the PCR fragment ranging from 120 bp in Pseudophoxinus minutus up to 643 bp in Leuciscus danilewski. IntraspeciWc variability was not detected as also described previously (Lieckfeldt et al., 2006). Alignments are available upon request from the authors. While the number of species is more limited, the topologies of the diVerent former species of Phoxinellus obtained by the nuclear marker are similar to the mtDNA trees (Fig. 3). In agreement with the mtDNA trees we observed the following clades: one clade containing the species of the genera Telestes and Phoxinellus, a second clade contain the species of the genus Squalius, a third clade uniWes the Scardinius-species. Most important, in all nuclear trees we found a high bootstrap support for the phylogenetic aYnity of the new genus Delminichthys as also found in phylogenetic reconstructions based on mtDNA. In 420 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 Chondrostoma vardarensis Chondrostoma prespensis Chondrostoma nasus Chondrostoma oxyrhynchum Chondrostoma soetta Chondrostoma polylepis Chondrostoma duriensis 100/100/86 57/72/92 Chondrostoma willkommii 99/98/99 Phoxinellus pseudalepidotus 100/100/100 Phoxinellus alepidotus Phoxinellus dalmaticus 100/100/94 Telestes pleurobipunctatus 95/98/84 Telestes alfiensis alfiens Telestes beoticus Telestes souffia 64/59/74 Telestes turskyi Telestes polylepis Telestes muticellus Telestes metohiensis 100/100/99 Telestes fontinalis Telestes croaticus kroaticus Pseudophoxinus prespensis 71/63/67 100 65/75/90 Pseudophoxinus marathonicus 100 100/100/56 Pseudophoxinus stymphalicus 94 Pseudophoxinus thesproticus Pseudophoxinus minutus 76/98/91 Delminichthys Dalmatichthys adspersus 100/100/97 Dalmatichthys ghetaldii Delminichthys Delminichthys Dalmatichthys jadovensis 100/99/99 Delminichthys Dalmatichthys krbavensis Petroleuciscus borystenicus Squalius keadicus 80 80/86/89 Squalius vardarensis 68 46 Squalius cephalus 50/56/42 Squalius albus 73/64/46 Squalius pyrenaicus Squalius microlepis Squalius ardensis Ladigesocypris ghigii Tropidophoxinellus hellenicus Alburnoides bipunctatus Scardinius erythrophthalmus Leucaspius delineatus 55 51/67/44 Alburnus alburnus 97 75 94/99/82 Alburnus mento 99 41 67 Anaecypris hispanica Alburnus baliki Rutilus rutilus Blicca bjoerkna Leuciscus leuciscus Pachychilon pictum Pelecus cultratus Notemigonus crysoleucas Phoxinus phoxinus 86/95/95 80/87/91 100/100/64 52/64/52 87/98/78 89/99/86 99/100/89 92/98/65 A B C D E F Fig. 1. Most parsimonious tree calculated in PAUP¤ 4.0b10 (SwoVord, 2002) based on entire cytochrome b sequences. The MP-, ML-, and NJ-topologies were very similar. Details of calculations are discussed in the text. Bootstrap values (MP/NJ/ML) are shown on branches. general, the topology of the nuclear trees is concordant with the results of the mitochondrial data set and of the morphological characters. 3.3. Unequal rates of change and dating divergences With its interspersed long and short branches our phylogram (Fig. 2) clearly violates a molecular clock. A general clock-like behavior was also rejected because the constrained and unconstrained analyses were signiWcantly diVerent in a likelihood ratio test (without clock, ¡ln D 18466.95; with clock, ¡ln D 19122.76; P < 0.005;  D 1311.61). Because the tests of rate heterogeneity among lineages were signiWcant, we dated the nodes by using a tree-based methodology (Sanderson, 1997) that relies on geological calibration (Dermitzakis, 1990) of nucleotide substitution rates. The molecular clock was calibrated using a well-dated geological event from the late Pliocene: the opening of the strait of Korinthos (2.5 MYA), which separates the Peloponnesus from the mainland (Dermitzakis, 1990). Telestes pleuropibunctatus and J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 421 Chondrostoma vardarensis Chondrostoma prespensis Chondrostoma nasus Chondrostoma oxyrhynchum Chondrostoma soetta Chondrostoma polylepis Chondrostoma duriensis Chondrostoma willkommii Phoxinellus pseudalepidotus Phoxinellus alepidotus Phoxinellus dalmaticus Telestes pleurobipunctatus Telestes alfiensis alfiens Telestes beoticus Telestes souffia Telestes turskyi Telestes polylepis Telestes muticellus Telestes metohiensis Telestes fontinalis Telestes kroaticus croaticus Rutilus rutilus Petroleuciscus borystenicus Squalius keadicus Squalius pyrenaicus Squalius ardensis Squalius microlepis Ladigesocypris ghigii Squalius vardarensis Squalius albus Squalius cephalus Alburnoides bipunctatus Tropidophoxinellus hellenicus Scardinius erythrophthalmus Leucaspius delineatus Alburnus alburnus Alburnus mento Anaecypris hispanica Alburnus baliki Notemigonus crysoleucas Blicca bjoerkna Leuciscus leuciscus Pelecus cultratus Pachychilon pictum Pseudophoxinus prespensis Pseudophoxinus minutus Pseudophoxinus marathonicus Pseudophoxinus stymphalicus Pseudophoxinus thesproticus Dalmatichthys Delminichthysadspersus adspersus Dalmatichthys Delminichthysjadovensis jadovensis Dalmatichthys Delminichthyskrbavensis krbavensis Dalmatichthys Delminichthysghetaldii ghetaldii 25 20 15 10 5 0 Million Years Fig. 2. Clock-constrained ML tree (chronogram) showing the cladogenesis of cyprinid taxa examined. The tree was constructed under the GTR+G+I model (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 the molecular clock based on the opening of the Strait of Korinthos (see text). The corresponding split between Telestes pleurobipunctatus from the Arachthos River and T. alWensis from the Alphios River is indicated (arrow). T. alWensis were separated by the formation of the Strait of Korinthos (Zardoya and Doadrio, 1999). In the mitochondrial cytochrome b gene, genetic distance and substitution plots indicated some degree of saturation. The data set contains both deep splitting events (Telestes croaticus/T. fontinalis vs. all other Telestes species) as well as very recent separation events (genera Telestes and Squalius) (Figs. 1 and 2). Based on morphological characters, Bogutskaya and Zupanbib (2003) suspected, that Phoxinellus might be divided into tree groups. Molecular data support theses groups within Phoxinellus. Phoxinellus should therefore be restricted to species with plain coloration, small or absent postcleithrum, no genital papilla and an almost naked body such as P. alepidotus, P. dalmaticus, and P. pseudalepidotus. 422 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 Telestes polylepis Telestes muticellus 89/93/89 Telestes turskyi 70/63/69 Telestes metohiensis Telestes croaticus Telestes fontinalis 75/60/64 62/51/69 Phoxinellus alepidotus Phoxinellus pseudalepidotus Leuciscus danilewski 95/97/91 Squalius tenellus 70/67/69 Squalius microlepis 91/92/87 Squalius cephalus 79/76/95 Squalius albus Squalius aphipsii Scardinius graecus 89/91/95 62 Scardinius dergle 68/69/-71 Scardinius erythrophthalmus 81 Rutilus aula Rutilus rubilio Alburnoides maculatus Alburnus maculatus Alburnus albidus 70/76/55 93/93/89 99/99/99 Pseudophoxinus minutus Dalmatichthys Delminichthys krbavensis krbavensis Dalmatichthys jadovensis Delminichthys jadovensis 96/96/98 Delminichthys adspersus 68/72/69 Delminichthys ghetaldii 0.01 Fig. 3. Neighbour-joining tree of the nuclear intergeneric region based on Kimura-2 Parameter distance values calculated in MEGA 2.1 based on the nuclear sequences. Bootstrap percentages >50% (Neighbour-Joining/Minimum Evolution/Maximum Parsimony) are shown on branches. The monophyly of this group is supported by high bootstrap values. Similar bootstrap support is given for Phoxinellus as sister taxon of Chondrostoma, and for both as sister taxa of Telestes forming a species-rich monophyletic group within Leuciscini. Morphologically, Phoxinellus is distinguished from other genera by a set of leuciscine plesiomorphic character states in combination with autapomorphic reductive elements (reduction of scales and postcleithrum). Species with a dark stripe from the head to the caudal peduncle, small or absent postcleithrum, no genital papilla and with a body covered by distinct, not overlapping scales should be positioned into the genus Telestes. Such species are T. croaticus, T. metohiensis, and T. fontinalis. Species such as Phoxinellus adspersus, P. ghetaldii, P. jadovensis and P. krbavensis are only distantly related to Phoxinellus. These species all have an irregularly spotted color pattern, a large postcleithrum, an increased number of precaudal anal-Wn pterygiophores, and a large genital papilla in females. We propose to join these species in a new genus, named Delminichthys. Supported by high bootstrap values, Delminichthys was positioned as sister genus of Pseudophoxinus. Interestingly, these two groups may form a sister group to all other Leuciscini. 3.4. Description of the new genus 3.4.1. Delminichthys, new genus 3.4.1.1. Type species. Leucos adspersus Heckel (1843) 3.4.2. Diagnosis Lower 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 overlapping; 3 (rarely 4) anal-Wn pterygiophores in front of Wrst caudal haemal spine; postcleithrum very strong, thickened and long, its lower end reaches much below the pectoral-Wn base; entire dorsal surface and Xanks covered by numerous dark spots of irregular shape and size; spots dense on back and may be fused together forming larger irregularly shaped blotches; large genital papilla formed like a thickened triangular fold with anus at base on its ventral surface in females; in adult females (larger than 53 mm SL) genital papilla base wide, extending over lowermost part of the Wrst anal-Wn rays; pectoral Wn almost reaches or reaches the pelvic-Wn base in males; and the pelvic Wn reaches way beyond the anal-Wn origin in males. 3.4.3. Etymology Derived from Delminium, the capital of the pre-Roman Dalmatia and greek ichthys (Wsh). Gender masculine. 3.4.4. Remarks Delminichthys includes the following named species: D. adspersus (Heckel, 1843), D. ghetaldii (Steindachner, 1882), D. jadovensis (Zupanbib and Bogutskaya, 2002), D. krbavensis (Zupanbib and Bogutskaya, 2002). For description of 423 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 species see Zupanbib and Bogutskaya, 2002 and Bogutskaya and Zupanbib (2003). Delminichthys is distinguished from other European leuciscine cyprinids by the following combination of characters: scales very thin, not overlapping (vs. scales well ossiWed, overlapping in all genera, but in Phoxinellus and Telestes in part); lower jaw never with a horny sheath (vs. present in Chondrostoma); 8–12 gill rakers (vs. 84–130 in Anaecypris); no midventral keel in front of anus (vs. presence in Abramis, Alburnus [including Chalcalburnus], Alburnoides, Ballerus, Blicca, Leucaspius, Pelecus, Tropidophoxinellus; Vimba); lower lip without median lobe (vs. present in Pachychilon); 3 (rarely 4) anal-Wn pterygiophores in front of Wrst caudal haemal spine (vs. 1–2 in Leuciscus [including Aspius], Squalius, Petroleuciscus, Phoxinellus, Telestes); postcleithrum very strong, thickened and long, its lower end reaches much below the pectoral-Wn base (very thin or absent in Phoxinellus, Telestes croaticus, T. metohiensis, T. fontinalis); entire dorsal surface and Xanks covered by numerous dark spots of irregular shape and size (vs. body coloration plain in Phoxinellus, with a wide lateral stripe from the eye to the caudal Wn in Telestes); large genital papilla (vs. no genital papilla in Phoxinellus, Telestes); scales embedded in skin (vs. not embedded in Pseudophoxinus). 4. Discussion Both molecular data sets and morphological characters well agree in the grouping of species of Phoxinellus as shown by Bogutskaya and Zupanbib (2003). Ketmaier et al. (2004) already postulated a close relationship of Phoxinellus croaticus and P. metohiensis to species of the genus Telestes by mtDNA data. The position of other Phoxinellus species within Leuciscini was never truly investigated due to a mosaic of plesiomorphic leuciscine traits and apomorphic, but reductive characters. Some character states such as increased number of deeply embedded scales, characteristic for Delminichthys and Dalmatian Telestes croaticus, T. fontinalis, T. polylepis, and T. turskyi are also found in endemic Dalmatian leuciscine cyprinids such as Chondrostoma phoxinus, Squalius microlepis, and S. tenellus which are not closely related to any above mentioned species. The reduction of scales as in Phoxinellus is also found in the endemic Dalmatian barbine cyprinid Aulopyge huegelii and in the barbine cyprinid genus Sinocyclocheilus that inhabits karstic waters in China (Yue, 2000). These characters may reXect an adaptation on their speciWc environmental conditions in the karstic waters of Dalmatia. The limited resolving power of the molecular data could be caused by diVerent rates of substitutions among taxa. The detected lower rate of evolution in Delminichthys compared to other leuciscine cyprinids (Fig. 4) might be related to a very long generation time, all species of this genus inhabit cold, karstic springs and migrate to subterranean waters during winter and summer droughts (Curcic, 1913; Trgovcevic, 1905; Vukovic and Ivanovic, 1971; Vukovic, 1977). Because detailed ecological data from species of the genus Delminichthys are absent, it must remain speculative how physiological parameters (for example a lower metabolic rate) might inXuence the evolutionary rate. The limited resolving power of the molecular data could also be an eVect of long branch attraction between taxa with a long history of isolated evolution, such as Telestes croaticus, and T. fontinalis. A further cause could also be a fast radiation relative to the substitution rate of sequences analyzed. It can not be excluded that cyprinids radiated within a relatively short time frame. This rapid radiation following a long time of stasis may have resulted in the partly low resolution of the data set (Fig. 1). Banarescu and Herzig-Straschil (1998) reviewed Telestes (as a subgenus of Leuciscus) based on morphological data. Telestes as a valid genus was re-established by 0.25 y = 0.0085x DNA sequence divergence R2= 0.7391 0.20 0.15 0.10 y = 0.0052x R2 = 0.9385 0.05 0 0 5 10 15 20 25 time in million years 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 Wgure demonstrates the lower evolutionary rate of the species from the genus Delminichthys (lower line) in relation to the other Leuciscinae-species included in this study. Time values were taken from Fig. 2. 424 J. Freyhof et al. / Molecular Phylogenetics and Evolution 38 (2006) 416–425 Ketmaier et al. (1998) based on enzyme electrophoretic data. The distinctness of Telestes from Leuciscus (including Aspius) and Squalius is also strongly supported by mtDNA data (Gilles et al., 2001; Zardoya and Doadrio, 1999). Squalius and the recently described genus Petroleuciscus can be clearly diagnosed by apomorphic morphological characters (Bogutskaya, 1996, 2002), whereas Telestes and Leuciscus are diYcult to distinguish morphologically. Both show a set of plesiomorphic leuciscine characters and almost no autapomorphies. Our data suggest the ancestor of the widespread genera Chondrostoma (see Durand et al., 2003 for overview) and Telestes occurred within Dalmatian water bodies. Following the molecular data, Telestes croaticus and T. fontinalis may be interpreted as the most basic Telestes, and Phoxinellus most likely forms the sistergroup of Chondrostoma. Basic Telestes and all Phoxinellus are strictly endemic to Dalmatia. Some descendants from this group may have remained in Dalmatia and have formed geographically very restricted species (T. metohiensis, T. polylepis, T. turskyi, and unstudied T. ukliva). Following the molecular clock attempt by Zardoya and Doadrio (1999), Delminichthys forms its own lineage, separated from European Pseudophoxinus since middle Miocene (13 MY). Delminichthys is a remnant of the Wrst major radiation within leuciscines during mid-Miocene (Zardoya and Doadrio, 1999) leading to most of the generic groups recognized today. Most of these generic groups rapidly reached a widespread geographic distribution within Europe and western Asia. In contrast, Delminichthys represents one of the geographically most isolated genera of leuciscine cyprinids supporting the Miocene refuge hypothesis of Dalmatian karst habitats. The Dinaric orogenesis, as a part of the Alpine orogenesis came to its end about 10–8 MY ago. It is likely that Delminichthys were trapped in Dalmatia by the rise of mountains. This might be true for Phoxinellus and 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 following phylogenetic and biogeographic conclusions: Chondrostoma, Telestes and Phoxinellus form one monophyletic group which is likely to have arisen in Dalmatia. Delminichthys belongs to a group of genera, which is restricted to the southern Balkan and might have invaded Dalmatia coming from this area. Having a very restricted distribution area, all species of Delminichthys, Phoxinellus and Dalmatian Telestes, are highly endangered. Massive alterations are obvious in the habitats of all species (Povc et al., 1990). Water shortage due to increasing economic development and projected climate changes will further contribute to the ongoing decline of all species. Further attempts for their conservation are strongly recommend to save these old evolutionary lines. Acknowledgments NGB was sponsored by a grant of the Naturhistorisches Museum Wien (2000), a grant from the Russian Foundation for Basic Research (N 01-04-49552), and a support from Zagreb University. Authors are grateful to E. Mikschi, B. Herzig, C. Prenner, H. Wellendorf (Naturhistorisches Museum, Wien), H. Wilkens, and G. Schulze (Zoologisches Museum und Institut der Universität Hamburg), F. Krupp, K. Jentoch (Senckenberg Museum, Frankfurt/Main), M. Mrakovcic (Zagreb University), and B. Sket (Ljubljana University) for their help during the work with collections under their 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) for helpful comments and discussions. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.ympev.2005.07.024. References Almaca, C., 1977. 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