Taxonomic investigation of spined loaches from Dalmatia and Herzegovina was conducted on specimens from 14 localities. The results of the detailed morphological investigations were combined with genetic data (based on one mitochondrial and two nuclear genes) in order to resolve the taxonomic status of each Cobitis population. Among the investigated features of external morphology, the appearance of spots on the caudal fin base turned out to have the greatest diagnostic value. Furthermore, the number of branched fin rays enabled the discrimination of several species. No morphometric character alone could ensure determination of any Cobitis species. Nevertheless, groups of populations that are more similar in their body shapes correspond to mitochondrial phylogenetic lineages. Based on molecular genetic markers, Dalmatian and Herzegovinian spined loaches form independent lineages inside the Adriatic phylogenetic group. Mitochondrial DNA phylogenetic reconstruction revealed six monophyletic lineages, corresponding to six species distributed in the investigated area. The population distributed in Mostarsko blato karstic field in Bosnia and Herzegovina is described as a new species based on a unique combination of morphological characters: a single triangular Canestrini scale; usually 51/2 branched anal fin rays, 61/2 branched dorsal fin rays, 14 branched caudal fin rays; no spots in the surface pigmentation layer on the caudal fin base; scales on the body very small.

Department of Zoology Faculty of Science University of Zagreb Zagreb Croatia

Faculty of Teacher Education University of Zagreb Zagreb Croatia

National Museum Prague Czech Republic

Zobrazit více v PubMed

Chen X, Jiang K, Guo P, Huang S, Rao D, et al. (2014) Assessing species boundaries and the phylogenetic position of the rare Szechwan ratsnake, PubMed

Knowles LL, Carstens BC (2007) Delimiting Species without Monophyletic Gene Trees. Syst Biol 56: 887–895. PubMed

Sites JW, Marshall JC (2004) Empirical criteria for delimiting species. Ann Rev Ecol Evol Syst 35: 199–227.

Carstens BC, Dewey TA (2010) Species Delimitation Using a Combined Coalescent and Information-Theoretic Approach: An Example from North American PubMed PMC

Grummer JA, Bryson jr RW, Reeder TW (2014) Species Delimitation Using Bayes Factors: Simulations and Application to the PubMed

Schulter D (1996) Ecological speciation in postglacial fishes. Phil Trans R Soc Lond B 351: 807814.

Taylor EB (1999) Species pairs of north temperate freshwater fishes: Evolution, taxonomy, and conservation. Rev Fish Biol Fisher 9: 299–324.

Bernardi G (2013) Speciation in fishes. Mol Ecol 22: 54985502. PubMed

Kottelat M, Freyhof J (2007) Handbook of European freshwater fishes. Kottelat, Cornol, Switzerland and Freyhof, Berlin, Germany. 646 p.

Kottelat M (2012) Conspectus Cobitidium: An inventory of the loaches of the world (Teleostei: Cypriniformes: Cobitoidei). Raffles B Zool Suppl. 26: 1–199.

Mustafić P, Marčić Z, Duplić A, Mrakovčić M, Ćaleta M, et al. (2008) A new loach species of the genus

Vasil’eva ED (2000) Sibling species in the genus

Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, et al. (2006) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22: 148–155. PubMed

Schneider D, Mrakovčić M, Mustafić P, Kerovec M (2000) Morphological differences in some

Freyhof J, Stelbrink B (2007)

Ráb P, Rábová M, Bohlen J, Lusk S (2000) Genetic differentiation of the two hybrid diploid-polyploid complexes of loaches, genus

Boroń A (2003) Karyotypes and Cytogenetic Diversity of the Genus PubMed

Perdices A, Doadrio I (2001) The molecular systematics and biogeography of the European cobitids based on mitochondrial DNA sequences. Mol Phylogenet Evol 19: 468–478. PubMed

Janko K, Vasil’ev VP, Ráb P, Rábová M, Šlechtova V, et al. (2005) Genetic and morphological analyses of 50–chromosome spined loaches (

Bohlen J, Perdices A, Doadrio I, Economidis PS (2006) Vicariance, colonization, and fast local speciation in Asia Minor and the Balkans as revealed from the phylogeny of spined loaches (Osteichthyes; Cobitidae). Mol Phylogenet Evol 39: 552–561. PubMed

Buj I, Podnar M, Mrakovčić M, Choleva L, Šlechtova V, et al. (2008) Genetic diversity and phylogenetic relationships of spined loaches (genus

Perdices A, Bohlen J, Doadrio I (2008) The molecular diversity of Adriatic spined loaches (Teleostei, Cobitidae). Mol Phylogenet Evol 46: 382–390. PubMed

Banarescu P, Herzig-Straschil B (1998) Beitrag zur Kenntnis der

Bogutskaya NG, Zupančič P (1999) A re-description of

Mrakovčić M, Brigić A, Buj I, Ćaleta M, Mustafić P, et al.. (2006) Red book of freshwater fish of Croatia. Zagreb: Ministry of Culture and State Institute for Nature Protection, Republic of Croatia. 253 p.

Karaman S (1928) Prilozi ihtiologiji Jugoslavije. Glasnik Skopskog naučnog društva 6: 163–164 (in Serbian and in German)..

Schneider D, Mustafić P, Mrakovčić M, Mihaljević Z (2000) Some aspects of the biology of the Neretvan spined loach. Folia Zool 49: 159–165.

Šanda R, Bogut I, Doadrio I, Kohout J, Perdices A, et al. (2008) Distribution and taxonomic relationships of spined loaches (Cobitidae,

Elliot NG, Haskard K, Koslow JA (1995) Morphometric analysis of orange roughy (

Turan C (2004) Stock identification of Mediterranean horse mackerel (

Klingenberg CP, Gidaszewski NA (2010) Testing and Quantifying Phylogenetic Signals and Homoplasy in Morphometric Data. Syst Biol 59: 245–261. PubMed

Kotusz J (2000) Intra- and interpopulation morphological variability in diploid and varied-ploidy

Gambetta L (1934) Sulla variabilita del cobite fluviale (

Saitoh T, Aizawa H (1987) Local Differentiation within the Striata Spined Loach (the

Hrbek T, Stolting KN, Bardakci F, Kucuk F, Wildekamp RH, et al. (2004) Plate tectonics and biogeographical patterns of the PubMed

Perdices A, Doadrio I, Bermingham E (2005) Evolutionary history of the synbranchid eels (Teleostei: Synbranchidae) in Central America and Caribbean islands inferred from their molecular phylogeny. Mol Phylogenet Evol 37: 460–473. PubMed

Šlechtová V, Bohlen J, Tan HH (2007) Families of Cobitoidea (Teleostei; Cypriniformes) as revealed from nuclear genetic data and the position of the mysterious genera PubMed

Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882. PubMed PMC

Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68: 978–989. PubMed PMC

Stephens M, Scheet P (2005) Accounting for decay of linkage disequilibrium in haplotype inference and missing data imputation. Am J Hum Genet 76: 449–462. PubMed PMC

Swofford DL (2002) PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4 [Computer software and manual]. Sunderland, MA: Sinauer Associates.

Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics 133: 693–709. PubMed PMC

Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585–595. PubMed PMC

Librado P, Rozas J (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451–1452. PubMed

Hudson RR (1987) Estimating the recombination parameter of a finite population model without selection. Genet Res 50: 245–250. PubMed

Hudson RR, Kaplan NL (1985) Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111: 147–164. PubMed PMC

Tamura K, Dudley J, Nei M, Kumar S (2007) PubMed

Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755. PubMed

Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818. PubMed

Templeton AR, Crandall KA, Sing CF (1992) A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132: 619–633. PubMed PMC

Clement M, Posada D, Crandall KA (2000) TCS: a computer program to estimate gene genealogies. Mol Ecol 9: 1657–1659. PubMed

Xie WG, Lewis PO, Fan Y, Kuo L, Chen MH (2011) Improving marginal likelihood estimation for Bayesian phylogenetic model selection. Syst Biol 60: 150?160. PubMed PMC

Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7: 214. PubMed PMC

Rambaut A, Drummond AJ (2007) Tracer v1.4. Available from: URL http://beast.bio.ed.ac.uk/Tracer.

Lartillot N, Philippe H (2006) Computing Bayes factors using thermodynamic integration. Syst Biol 55: 195–207. PubMed

Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29: 1969–1973. PubMed PMC

Hey J, Wakeley J (1997) A coalescent estimator of the population recombination rate. Genetics 145: 833–846. PubMed PMC

Li WLS, Drummond AJ (2012) Model Averaging and Bayes Factor Calculation of Relaxed Molecular Clocks in Bayesian Phylogenetics. Mol Biol Evol 29: 751–761. PubMed PMC

Perea S, Bohme M, Zupancic P, Freyhof J, Sanda R, et al.. (2010) Phylogenetic relationships and biogeographical patterns in Circum-Mediterranean Subfamily Leuciscinae (Teleostei, Cyprinidae) inferred from both mitochondrial and nuclear data. BMC Evol Biol, doi:10.1186/1471–2148–10–265. PubMed PMC

Carstens BC, Knowles LL (2007) Shifting distributions and speciation: species divergence during rapid climate change. Mol Ecol 16: 619–627. PubMed

Gamble T, Berendzen PB, Shaffer HB, Starkey DE, Simons AM (2008) Species limits and phylogeography of North American cricket frogs (Acris: Hylidae). Mol Phylogenet Evol 48: 112–125. PubMed

Marková S, Šanda R, Crivelli A, Shumka S, Wilson IF, et al. (2010) Nuclear and mitochondrial DNA sequence data reveal the evolutionary history of PubMed

Buj I, Podnar M, Mrakovčić M, Ćaleta M, Mustafić P, et al. (2008) Morphological and genetic diversity of

Doadrio I, Perdices A (2005) Phylogenetic relationships among the Ibero-African cobitids ( PubMed

Šanda R, Vukić J, Choleva L, Křížek J, Šedivá A, et al. (2008) Distribution of loach fishes (Cobitidae, Nemacheilidae) in Albania, with genetic analysis of population of

Karyotype and chromosomal characteristics of rDNA of Cobitisstrumicae Karaman, 1955 (Teleostei, Cobitidae) from Lake Volvi, Greece

Different Histories, Different Destinies‒Impact of Evolutionary History and Population Genetic Structure on Extinction Risk of the Adriatic Spined Loaches (Genus Cobitis; Cypriniformes, Actinopterygii)