Spatial Heterogeneity in the Mediterranean Biodiversity Hotspot Affects Barcoding Accuracy of Its Freshwater ﬁshes

Molecular Ecology Resources (2014) doi: 10.1111/1755-0998.12257

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater ﬁshes

M. F. GEIGER,1 F. HERDER,1 M. T. MONAGHAN,2 V. ALMADA,3 R. BARBIERI,4 M. BARICHE,5 P. BERREBI,6 J. BOHLEN,7 M. CASAL-LOPEZ,8 G. B. DELMASTRO,9 G. P. J. DENYS,10 A. DETTAI,10 € I. DOADRIO,8 E. KALOGIANNI,4 H. KARST,11 M. KOTTELAT,12 M. KOVACIC,13 M. LAPORTE,6,14 € M. LORENZONI,15 Z. MARCIC,16 M. OZULUG,17 A. PERDICES,8 S. PEREA,8 H. PERSAT,18 S. PORCELOTTI,19 C. PUZZI,20 J. ROBALO,3 R. SANDA,21 M. SCHNEIDER,11 V. SLECHTOVA,7 M. STOUMBOUDI,4 S. WALTER1 and J. FREYHOF 1,2 1Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute for Animal Biodiversity, Adenauerallee 160, 53113 Bonn, Germany, 2Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Muggelseedamm€ 301 & 310, 12587 Berlin, Germany, 3Eco-Ethology Research Unit, ISPA-Instituto Universitario, Rua Jardim do Tabaco, 34, 1149-041, Lisbon, Portugal, 4Hellenic Centre for Marine Research, 46.7 km Athens Sounio ave., P.C. 19013 Anavyssos, Greece, 5American University of Beirut, Riad El 6 Solh, 1107 2020 Beirut, Lebanon, Institut des Sciences de l’Evolution de Montpellier, Place Eugene Bataillon – Universite Montpellier 2, 34095 Montpellier Cedex 05, France, 7Institute of Animal Physiology and Genetics, Libechov Rumburska 89, 277 21 Libechov, Czech Republic, 8National Museum of Natural History, Jose Gutierrez Abascal 2, 28006 Madrid, Spain, 9Natural History Museum, Cascina Vigna via San Francesco di Sales 188, 10022 Carmagnola, Italy, 10Museum national d’Histoire naturelle, 57 Rue Cuvier, 75005 Paris, France, 11Deutsche Killiﬁschgemeinschaft e.V., 65183 Wiesbaden, Germany, 12Route de la Baroche, 2952, Cornol, Switzerland, 13Natural History Museum Rijeka, Lorenzov prolaz 1, 51000 Rijeka, Croatia, 14Centre de Recherche sur les 15 Interactions Bassins Versants – Ecosystemes Aquatiques, C.P. 500, Trois-Rivieres, QC G9A 5H7, Canada, University of Perugia, Via Elce di Sotto, 06123 Perugia, Italy, 16University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia, 17Istanbul University, 34134 Vezneciler, Istanbul, Turkey, 18Universite Claude Bernard, Lyon 1, 69622 Villeurbanne Cedex, France, 19Associazione Ichthyos Italia, Via. A Cecchi 12, 52100 Arezzo, Italy, 20Gestione e Ricerca Ambientale Ittica Acque, 21020 Varano Borghi, Italy, 21National Museum, Vinohradska 1, 110 00 Prague, Czech Republic

Abstract

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endan- gered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

Keywords: DNA barcoding, Evolutionarily Distinct and Globally Endangered score, ﬁsh, freshwater diversity, Mediter- ranean Biodiversity Hotspot, molecular identiﬁcation Received 14 January 2014; revision received 15 March 2014; accepted 19 March 2014

Correspondence: Matthias F. Geiger, Fax: +0049 228 9122 252; E-mail: [email protected]

unique evolutionary history (Isaac et al. 2007; Safi et al. Introduction 2013). The Biodiversity Hotspot (BH) concept serves to prior- The EDGE approach appears compelling, especially itize geographical regions of high conservation value considering the growing availability of DNA barcode (Myers 1988; Mittermeier et al. 1999), and the Mediter- data (Hebert et al. 2003, 2004; Hebert & Gregory 2005; La- ranean area was included in the first list of 25 globally haye et al. 2008; May 2011; Mora et al. 2011; April et al. important BHs (Myers et al. 2000). The area is geo- 2013); however, its meaningful application depends on graphically highly structured and includes 23 ecore- correctly identified material underlying barcode refer- gions in 20 countries (Abell et al. 2008). While the ence libraries. Moreover, a recent study in aquatic insects initial Hotspot classification was based on plant diver- concluded that the reliability of species determination by sity, the freshwater fauna of the region is also consid- DNA barcoding may be scale dependent (Bergsten et al. erably rich (Mittermeier et al. 1999; De Figueroa et al. 2012). This means that large-scale databases may be 2013). Despite the disproportionately small global sur- problematic for species identification, because evolution- face cover of freshwater habitats (‘paradox of freshwa- arily close relatives may occur in distant localities. Thus, ter biodiversity’, Martens 2009), the importance of as the geographical and taxonomic size of the database freshwater biodiversity for humans is increasingly rec- increases, the resolution may decrease because of the ognized (Dudgeon et al. 2006). Hotspots of freshwater inclusion of ever more closely related species. This may diversity are not necessarily congruent with terrestrial be particularly problematic in geographically highly hotspots (Abell et al. 2008), nonetheless, the species structured areas, where allopatric speciation likely numbers reported by Mittermeier et al. (2004) were accounts for a considerable proportion of the species surprisingly low: only 216 native freshwater fishes for diversity. the Mediterranean BH with 63 endemic species. That Here, we present a DNA barcode database of fresh- these numbers were a gross underestimation became water fishes of the Mediterranean BH that covers 98% of clear when the IUCN Red List for endemic Mediterra- the species known from the area. This comprehensive nean freshwater fish species, published in 2006, database is verified by taxonomic experts and allows us included 253 species (Smith & Darwall 2006). Recent to address the following questions: How closely do descriptions of new freshwater fish species demon- sequence-based diversity estimates of this highly geo- strate that the Mediterranean BH remains only graphically structured area mirror the estimates based partially explored (Kottelat & Freyhof 2007; Fontaine on morphology? How do analytical, intrinsic biological et al. 2012; Essl et al. 2013), which is a stumbling block (e.g. introgressions) and extrinsic geographical (scale) for conservation planning (Freyhof & Brooks 2011). To factors affect accuracy and practical implementation of overcome this, molecular information is increasingly DNA barcoding? Can phylogenetic information from used to explore biodiversity and improve biodiversity DNA barcode data be used to assist selection of EDGE knowledge, including large-scale barcoding approaches species for conservation? for molecular identification systems that serve various purposes including conservation (Hebert et al. 2003, Materials and methods 2004; Hebert & Gregory 2005; Lahaye et al. 2008; May 2011; Mora et al. 2011). The Mediterranean BH of Mittermeier et al. (1999) and Molecular information is also increasingly used to Myers et al. (2000) includes all areas of the Mediterra- evaluate conservation priorities, complementing the nean floral zone, therefore including Portugal and the IUCN classification (Rolland et al. 2012; Abellan et al. Atlantic parts of Spain and Morocco, as well as the 2013). Such data are of special importance for freshwater Macaronesian islands. The geographical area consid- fishes in the Mediterranean BH, where a large number of ered for this study (Fig. 1) is the same used for the threatened species occur (56% of 253 evaluated species, IUCN Red List assessment (Smith & Darwall 2006). Smith & Darwall 2006; 17 endemics already extinct Frey- The Macaronesian islands and Libya were not consid- hof & Brooks 2011). One approach to assess priority spe- ered because of the absence of endemic fish species. A cies from a given list of threatened species is the total of three endemic species from the Egyptian Medi- Evolutionarily Distinct and Globally Endangered terranean area were included, and all others belong to (EDGE) score, which combines estimators for unique the largely afro-tropical fish fauna (Roberts 1975) and evolutionary history with the formal IUCN conservation are excluded. status (Isaac et al. 2007). In this approach, globally threa- The analysed material was collected with the aid of tened species that represent isolated and phylogeneti- numerous colleagues, or stems from available collec- cally old lineages receive highest conservation priority, tions. When possible, multiple individuals (2–38) from because their loss would mean a disproportionate loss of single species from different drainages were included

Fig. 1 Sample locations (n = 657) for materials obtained from within the Mediterranean Biodiversity Hotspot with major rivers and country borders.

to estimate intraspecific genetic variation (Appendix S1, Molecular data analysis Table S1, Supporting information). As the barcode (cytochrome c oxidase subunit 1: COI) data published Details for DNA extraction, PCR and standard barcode here will serve as a reference database, we applied a data preparation are listed in the Supporting informa- strict policy to ensure data quality and considered only tion (Appendix S2). Clustering with Kimura 2-cor- material reliably determined by taxonomic specialists. rected distances (K2P) in SpeciesIdentifier (Meier et al. With a few exceptions (see Results section), all 2006) was used to cluster sequences at 2%, 1% and an sequences were taken from individual voucher speci- optimized, data-derived threshold. Taking into account mens deposited in publicly available collections (Table the number of true and false positives, and true and S1). Data associated with each specimen (taxonomy, false-negative identifications at a given threshold with collection sites and voucher catalogue numbers) are cumulative error (false negative + false positive), the available via the respective GenBank accession numbers optimized threshold was derived from each data parti- (see Data accessibility below, Table S1) and will also be tion with the SPIDER package (Brown et al. 2012) in R v made available via the BOLD data portal. As reference 2.15.1 (R Development Core Team 2011). We then to which we compare our molecular-based diversity counted the number of clusters in agreement with estimates, we use the species listed as valid in Esch- existing taxonomy, that is, containing only sequences meyer’s Catalogue of Fishes (Eschmeyer & Fong 2013). of one morphological species (‘perfect match’), as well We evaluated the performance of different analytical as the total number of clusters (Hendrich et al. 2010). approaches (distance- versus tree-based species delimi- We treated clusters containing all COI sequences of tation) in terms of congruence to traditional taxonomy one species together with one or more sequences that and compare the standard barcoding metrics of the were determined to genus level only as a ‘perfect Mediterranean Hotspot to other large-scale DNA bar- match’. As the simplest, but most widely used dis- coding studies. Our sampling scheme allowed us to tance-based approach has been criticized for being assess the recently demonstrated impact of geographi- arbitrarily chosen without sound biological back- cal scale on DNA barcoding accuracy (Bergsten et al. ground (Meier et al. 2006; Srivathsan & Meier 2012; 2012; Lou & Golding 2012). We did so by separately Collins & Cruickshank 2013), and the threshold varies analysing the fish faunas from three Mediterranean between species from different groups (Hebert et al. drainages in a focal approach, which constitutes a prac- 2003), we applied additional methods to analyse the tical and realistic monitoring scenario on a local scale. data. First, the species criterion of monophyly was For this, we chose the Po in Italy (48 species, 229 indi- applied to test for congruence, requiring the grouping viduals), the Vardar in Greece (36 species, 159 individ- of all COI haplotypes of a given species as a mono- uals) and the Orontes in Turkey and Syria (34 species, phyletic unit in a given phylogram; here, we screened 126 individuals). ML trees for those groups generated under the model

© 2014 John Wiley & Sons Ltd 4 M. F. GEIGER ET AL. assumptions as derived below. Singletons were consid- (www.redlist.org) or local experts (Appendix S1, ered as match, if they did not cluster unresolved Supporting information). We analysed DNA barcode within another species, and we counted every mono- data (mitochondrial COI) of 98% (498 species) of the phyletic clade without defining a certain a priori sup- remaining 509 extant species, from a total of 20 port. Second, the general mixed Yule-coalescent countries (Fig. 1, Table 1). We newly sequenced DNA (GMYC) approach was used, which identifies clusters barcodes of 2899 individuals from 487 species, added by fitting models that predict the inter- and intraspe- 89 sequences from 21 species from GenBank and 183 cific divergence rates and threshold times differentiat- sequences from 58 species from other DNA barcoding ing these processes to multispecies coalescent trees studies (Appendix S1, Supporting information). Eleven (Monaghan et al. 2009; Powell 2012). The model species were represented with material from outside assumes that branching patterns within genetic clus- the Mediterranean due to their rarity, or because they ters reflect neutral coalescent processes and occur are thought to be extirpated in the BH (Appendix S1, within species (Kingman 1982), whereas branching Supporting information). Altogether, 2809 COI between clusters can reflect the timing of a speciation sequences belonged to material unambiguously identi- event (Yule 1924). Thus, it identifies a species bound- fied to species level, and an additional 318 sequences ary by identifying independently evolving lineages from 30 genera where species membership was uncer- and the transition from coalescent to speciation tain a priori (e.g. juveniles or unclear species bound- branching patterns on a phylogenetic tree. Although aries). After re-examination of the vouchers from not uncontroversial (Lohse 2009; Esselstyn et al. 2012), which DNA was extracted, literature research, and the theory behind the model is very attractive and it taking into account their haplotype clustering (Fig. S1, has proven to deliver reliable and biological sound Supporting information), 115 of the 318 unidentified species number estimates (Fujita et al. 2012; Powell specimens could be assigned to 18 species currently 2012; Puillandre et al. 2012; Talavera et al. 2013). recognized as synonyms (Eschmeyer & Fong 2013) Details for the generation of ultrametric trees for the (Table S2, Supporting information). GMYC analysis are given in the Supporting information (Appendix S2). Finally, we applied an extension Introgression and hybrids to the GMYC approach (Powell 2012) that takes into account additional models with slightly lower likeli- Hybridization and introgression are common in freshwa- hoods and uses an Akaike information criterion (AIC) ter fishes, in particular in the Cyprinidae (Scribner et al. of all single- and multiple-threshold models (Powell 2000; Freyhof et al. 2005), which comprised 56% of 2012). The probabilities that two haplotypes belong to species in our data set. Seven hybrids identified in the the same entity are then based on the weights associ- field were excluded from subsequent analyses; all but ated with each model, and the variance is estimated one of these belonged to the Cyprinidae, including two from model averaging. Thus, uncertainty in species cases of intergeneric crosses (Table S2, Supporting infor- boundaries can directly be incorporated into diversity mation). In addition, 44 individuals (1.4%) from 26 spe- estimates. Based on the probabilities associated with cies had COI haplotypes not matching expectations from each genetic cluster, we derived the number of morphological species supported by the GMYC approach, Table 1 Summary of basic barcode statistics with ranges or sub- analogous to the ‘perfect match’ above, also including sets analysed in parentheses singletons. Basic statistic Evolutionary distinctiveness (ED) was calculated from an ultrametric tree with all endemic species only Total individuals 3165 with the CAPER package in R v 2.15.1 (R Development Species total number (analysed) 526 (498) Core Team 2011). The ED estimates where then used to Number of genera 113 calculate the EDGE scores as outlined in Isaac et al. Species assessed as extinct 18 Extant endemics (barcoded) 382 (372) (2007). Alien species 37 Mean number of individuals per species 5.4 (1–38) Results Number of singletons 33 Mean sampling events per species 2 (1–18) Mean sequence length in base pairs 646 (454–652) DNA barcode library Mean intraspecific distance 0.59% (0.02–12.5%) – Of the 526 species of freshwaters fishes currently Mean smallest interspecific distance 4.10% (0.1 16.8%) 95% intraspecific variance ≤ 1.98% recognized for the Mediterranean BH (Eschmeyer & 95% smallest interspecific distance ≥ 0.21% Fong 2013), 17 are considered to be extinct by IUCN

© 2014 John Wiley & Sons Ltd DNA BARCODING MEDITERRANEAN FRESHWATER FISHES 5 morphological identification (Table S2, Supporting infor- those species showed above 1% COI divergence. A mation). Fourteen of these were in the Cyprinidae, refined barcode gap [difference between the maximum mostly in species for which introgression has been intraspecific and minimum interspecific divergence reported elsewhere, but to our knowledge, some cases (Meyer & Paulay 2005; Meier et al. 2008)] was present in are reported here for the first time. One case of presumed 74.8% of all species. The distribution of intra- and inter- introgression was observed in Oxynoemacheilus seyhanico- specific congeneric K2P-corrected distances displayed la with two distinct COI haplotype groups (>12% K2P), considerable overlap (Fig. 2). Among all individual which can however not be attributed to a recent intro- intraspecific comparisons, 91.5% were below 2% gression event, because neither haplotype occurred in sequence divergence. Interspecific distances were below any other species. 2% sequence divergence in 11.3% of all pairwise comparisons (Table 1). Cytochrome c oxidase subunit 1 divergence in the full data set DNA barcoding accuracy Mean K2P nucleotide divergence increased from species When applying the 2% K2P divergence clustering (m = 0.59%, SE = 0.183) to genera (m = 2.89%, approach to the complete set of 498 freshwater fish spe- SE = 0.300) to families (m = 6.44%, SE = 1.000). Mean cies, a total of 391 clusters were recovered; 230 of these distance to the closest congener across all genera was were in perfect match with the species entities deter- 4.10% (SE = 0.500), seven times higher than the mean mined by morphology. This translates to 44.8% of the intraspecific divergence. A lack of variation (i.e. identical expected species recovered using the simple distance- COI sequences) was observed in 28% of all species with based approach. For the Cyprinidae (data set IV), the multiple sequences (2–12). For intraspecific distances, match increased from 34.8% to 44.7% when using a 1% 4.9% of the 390 species with multiple individuals and threshold (Table 2). Highest accuracy, with 82% of the nonzero variation had values over 2%, while 11.0% of species resolved as perfect barcode cluster applying a 1%

35 Mediterranean BH 35 Orontes River 498 species 34 species 30 3165 individuals 30 126 individuals 25 25

20 20

15 15 Percentage Percentage 10 intra 10 intra inter inter 5 5

0 0 01234567891011121314151617181920 012 3 4 5 6 7 8 9 101112131415161718 K2P distance (%) K2P distance (%)

35 Po River 35 Vardar River 48 species 36 species 30 229 individuals 30 159 individuals 25 25

20 20

15 15 Percentage Percentage 10 intra 10 intra inter inter 5 5

0 0 012345678910111213141516171819 0123456789101112 K2P distance (%) K2P distance (%)

Fig. 2 Frequency distribution of the intra- and interspeciﬁc pairwise Kimura 2-corrected (K2P) distances for all 498 species (upper left), and each native ﬁsh fauna for three selected drainages of the Mediterranean Biodiversity Hotspot.

Table 2 Number of clusters obtained with different distance thresholds and identiﬁcation success percentage for different analytical methods; seq: number of cytochrome c oxidase subunit 1 sequences analysed; spp: number of species; optimum: threshold values from cumulative error estimation in parenthesis; perfect cluster: percentage of clusters in congruence with taxonomy; general mixed Yule- coalescent (GMYC): single- and multiple-threshold entity estimation with conﬁdence intervals (CI)

Distance cluster % perfect cluster GMYC entities

Group Seq Spp 2% 1% Optimum 2% 1% Optimum Mono GMYC Single CI Multi CI

I 289 62 69 77 69 (2.04) 67.7 67.7 67.7 88.7 86.9 79 73–85 77 71–91 II 331 44 53 60 57 (1.19) 80.0 82.0 80.0 94.0 93.2 62 56–70 46 42–83 III 247 34 34 37 31 (2.29) 62.9 62.9 57.1 77.1 73.5 36 32–42 38 30–51 IV 1914 289 196 268 247 (1.24) 34.8 44.7 39.4 71.7 81.9 322 305–349 347 346–363 V 215 34 31 33 31 (1.78) 69.0 71.9 69.0 90.6 90.6 7 1–88 37 13–47 VI 175 35 11 13 10 (5.5) 10.3 10.3 10.3 46.2 78.1 9 8–31 20 9–33 All 3165 498 391 490 433 (1.45) 44.8 51.2 49.2 – 85.2* 463.54 (variance 6.6)*

Group composition: (I) Barbatula, Cobitis, Misgurnus, Oxynoemacheilus, Paramisgurnus, Sabanejewia, Seminemacheilus; (II) Acipenser, Ameiu- rus, Anguilla, Aphanius, Chelon, Clarias, Esox, Fundulus, Gambusia, Huso, Lota, Mugil, Mystus, Poecilia, Pterygoplichthys, Silurus, Valencia, Xiphophorus; (III) Atherina, Cottus, Dicentrarchus, Economidichthys, Gasterosteus, Knipowitschia, Neogobius, Ninnigobius, Orsinigobius, Padogo- bius, Platichthys, Pomatoschistus, Proterorhinus, Pungitius, Syngnathus; (IV) Abramis, Acanthobrama, Achondrostoma, Alburnoides, Alburnus, Anaecypris, Aulopyge, Barbus, Blicca, Capoeta, Carasobarbus, Carassius, Chondrostoma, Clupeonella, Ctenopharyngodon, Cyprinus, Delminichthys, Garra, Gobio, Hypophthalmichthys, Iberochondrostoma, Iberocypris, Ladigesocypris, Leucaspius, Leuciscus, Luciobarbus, Mirogrex, Pachychilon, Parachondrostoma, Pelasgus, Petroleuciscus, Phoxinellus, Phoxinus, Protochondrostoma, Pseudochondrostoma, Pseudophoxinus, Pseudorasbora, Pterocapoeta, Rhodeus, Romanogobio, Rutilus, Scardinius, Squalius, Telestes, Tinca, Tropidophoxinellus, Vimba; (V) Amatitlania, Australoheros, Caspiomyzon, Coptodon, Eudontomyzon, Gymnocephalus, Haplochromis, Hemichromis, Lampetra, Lepomis, Micropterus, Morone, Odontesthes, Oreochromis, Perca, Petromyzon, Pseudocrenilabrus, Salaria, Sander, Sarotherodon, Tristramella, Zingel; (VI) Alosa, Clupeonella, Coregonus, Hucho, Ictalurus, Oncorhynchus, Salmo, Salvelinus, Thymallus; (All) all sequences including unidentified specimens. *Based on the average numbers over all models, calculated with the modified GMYC approach (Powell 2012). divergence criterion, was achieved in group II, a hetero- (SD = 10.7), and 83% of these clusters had support val- geneous group composed of 14 families with 44 species ues above 90%. As for the GMYC analysis, we did not only. Lowest congruence (10.3%) between morphological observe a relationship between number of individuals species and barcode clusters detected is present in group per species and bootstrap support (GLM, P = 0.8201). VI (salmonids and herrings), irrespective of the distance threshold chosen. In summary, distance-based methods Cytochrome c oxidase subunit 1 divergence within appear suitable to detect families and species groups, but selected drainages did not support all of the species expected in the full data set. We used our comprehensive database to test whether The tree-based analysis outperformed the distance- barcoding accuracy changes under conservation- and based approach in recovering patterns congruent operator-oriented conditions on a smaller scale, using between a priori species identifications and molecular- the species sets from three selected large Mediterranean based units (Table 2). In total, 83% of all species were drainages (Po in Italy, 48 species; Vardar in Greece, 36 recovered as GMYC entities, with probabilities above species; Orontes in Turkey and Syria, 34 species). Here, 0.95 containing only conspecifics, and including single- the mean distance to the closest congeneric species was tons. In four of the six data partitions, a multiple-thresh- similar for all three drainages (6.50–6.93%) and was, on old model provided the best fit to the data, but the even average, 19 times higher than the mean intraspecific distribution of the weights given to each model suggests divergence (0.26–0.55%). Compared with the ratio that no single model best represented species boundaries derived from the complete data set, this is a threefold in a given subset of the data (Appendix S2, Supporting increase. We found intraspecific divergences exceeding information). We did not observe a relationship between 2% in one species in the Orontes (Aphanius mento), number of haplotypes per species and assignment proba- where a cryptic species might be involved, in two cases bilities (GLM, P = 0.2372), thus we did not find an effect in the Po (Thymallus aeliani and Barbus plebejus) where of sample size bias on GMYC accuracy. Of all species introgression by alien, congeneric species is likely, and represented by multiple individuals (n = 465), 74.4% car- in two individuals of Orsinigobius punctatissimus. In all ried COI haplotypes that formed monophyla in the species occurring in the three drainages, the maximum screened ML trees. Mean bootstrap support derived from intraspecific divergence was smaller than the minimum 500 pseudoreplicates for those species was 95.4% interspecific divergence, indicating the presence of the

© 2014 John Wiley & Sons Ltd DNA BARCODING MEDITERRANEAN FRESHWATER FISHES 7 refined barcode gap (Meyer & Paulay 2005; Meier et al. Discussion 2008) (Fig. 2). Barcoding accuracy under the 2% distance-based approach was between 96% (Po) and 100% The present data (with 98% species coverage) comprise (Orontes and Vardar). For the Po sample, the two Salmo the first comprehensive molecular study on the freshwa- and Alosa species occurring within the drainage show ter fish diversity of a complete BH and will serve as ref- COI sequence divergences below 2% and thus erence for future studies of this large fauna. Our finding decreased overall accuracy. that model-based clustering of sequences outperformed the simplest, but most widely used version of distance- based clustering is consistent with theoretical and previ- Candidate species ous empirical work (Papadopoulou et al. 2008; April Divergence levels between haplotypes exceeding 2% et al. 2011; Bergsten et al. 2012). Low levels of introgres- K2P remained in ten of the species analysed (Table S2, sion and the focal analyses of three drainages demon- Supporting information). Examples for allopatric subc- strated that DNA barcoding is a powerful tool for lades with geographically structured haplotype pools specimen identification at the drainage scale, even with included A. mento (intraspecific distance ≤8%) and rapid distance-based methods. With drainage-specific Telestes pleurobipunctatus (intraspecific distance ≤3%). species lists and barcoding data available, species can be The first species is challenging, given that material identified with high accuracy. Our findings support the from the type locality (Northern Iraq) is currently not notion that challenges of barcode identification may available; the type series of T. pleurobipunctatus is com- increase with the complexity of the reference database posed of populations found to belong to different subc- and corroborate a recent empirical test of the effects of lades and, awaiting nomenclatural clarification, cannot spatial scale on DNA barcoding (Bergsten et al. 2012). be applied to a particular one. Here, we define candi- We identified several candidate populations that may be date species as (i) intraspecific clusters exceeding 2% new species and require focused taxonomic research, K2P distance, (ii) a priori to genus level only identified potentially raising diversity by 12%. Phylogenetic infor- populations with over 2% K2P distance to a described mation from COI barcode data delivered estimates of ED species, and (iii) the 18 species which were treated as for the calculation of EDGE scores and allowed the iden- synonym before, and which are proposed to be revali- tification of species for conservation prioritization. This dated. All candidate species fulfilled the monophyly constitutes a promising approach to practically benefit criterion and were detected as GMYC entities. This from growing DNA barcode libraries to assist conserva- indicates the existence of cryptic diversity representing tion planning, or rapidly pre-assess data-deficient up to 64 candidate species (Table S2 and Fig. S1, species. Supporting information). Divergence levels in Mediterranean freshwater fishes Evolutionarily Distinct and Globally Endangered score The large geographical scale of our study may explain and IUCN status why divergence levels within species largely met expec- We used the COI sequence data to calculate EDGE scores tations from other studies of freshwater fishes (Ward for 311 IUCN Red List fish species endemic to the Medi- et al. 2005; April et al. 2011; De Carvalho et al. 2011; Pere- terranean Hotspot. We found no correlation between ED ira et al. 2011, 2013), but divergence within genera did and IUCN ranking (Spearman’s rank-order r = 0.0965, not (April et al. 2011; Pereira et al. 2013). The only other P = 0.08), but a clear correlation between EDGE scores study of freshwater fishes of comparable scale and scope and IUCN ranks (r = 0.963, P < 0.0001, Fig. S2, Support- (i.e. including several ecoregions) (April et al. 2011) ing information). This correlation was expected given reported a mean divergence within species of 0.73% that the IUCN status contributes to the EDGE score (see (0.59% this study), within genera of 13.67% (2.89% this Materials and methods). Consequently, it was not sur- study) and within families of 15.91% (6.44% this study) prising that the top 10 EDGE score species are also listed in North America. Comparisons with studies of marine as critically endangered; yet, a number of ‘outlier’ spe- fishes (Ward et al. 2005; Lakra et al. 2011) are of limited cies with higher than expected EDGE scores for their value because dispersal of pelagic larvae should lead to IUCN category were identified (Fig. S2, Supporting lower levels of allopatric divergence through gene flow information). In addition, the top three species, ranked (Helfman et al. 2009). It has also been proposed that dif- according to their ED, include two listed by IUCN as ferences in vagility, as in the marine environment as data deficient (DD), the other as least concern (LC), fol- opposed to the freshwater realm, can be responsible for lowed by a set of species which is different from the set different divergence levels as well (Bergsten et al. 2012; ranked by EDGE scores. Young et al. 2013).

The low divergence within genera and families that genetic distance to the closest heterospecific decreases we observed suggests that species in Mediterranean gen- with increasing geographical scale of sampling, linked to era and families are more closely related than in North the larger intraspecific variance when incorporating America and has implications for the use of DNA bar- more populations. A positive correlation between intra- codes for identification. In the BH, relatively recent (2.5– specific genetic variation and geographical scale is pre- 0.01 Ma) dispersal and vicariance events are thought to dicted by concepts like isolation by distance (Wright have had the greatest impact on species formation and 1943), where more distantly occurring subpopulations contemporary diversity (Banarescu 1989; Zardoya & Do- show higher divergence. Assuming that vicariance adrio 1999). The low differentiation in COI between sev- events were important in shaping the Mediterranean eral Mediterranean freshwater fish species might also be freshwater fish diversity, we do not expect the closest related to a combination of renewed species concepts in relatives to occur in the same drainage. Without doubt, ichthyology (Kottelat 1997) and increased importance of this is reflected in the increased accuracy of DNA bar- molecular tools for species discoveries and descriptions coding for species identification within the three river (Doadrio & Perdices 1997). A review of the European catchments analysed separately, and in general, only few freshwater fish fauna in 2007 (Kottelat & Freyhof 2007) cases of low interspecific divergence levels in sympatry recognized 546 native species, compared with only 215 were detected (e.g. Lampetra planeri and Lampetra fluviatil- species recognized in 1977 (Maitland 1977). From the is, Alosa alosa and Alosa fallax or Alosa algeriensis, or vari- Mediterranean freshwaters alone, 99 species have been ous Salmo species from Lake Ohrid and Salmo farioides, described since the year 2000 (22% of the fauna recog- Salmo marmoratus and Salmo obtusirostris in some rivers nized before), while only 76 and 25 have been described in the Adriatic). Congeneric species occurring in allopa- from North America and Australia, respectively, (9% try frequently possessed low or very low levels of molec- and 16% of fauna recognized before) during the same ular divergences, which hampered their detection. A period (Froese & Pauly 2013). This could also explain the practical solution to overcome this is to combine genetic difference in the proportion of candidate species sug- and distributional data to cope with spatial scale effects gested, which – if all would be formally described – in order to obtain similar identification rates at global would lead to a 28% increase in North American species and regional barcoding campaigns (Papadopoulou et al. diversity (April et al. 2011) versus up to 12% for the 2008; Bergsten et al. 2012), or to construct and implement Mediterranean fauna presented herein. barcode databases on local or regional scales. Most of the species that were not genetically recognized with GMYC or distance approaches (Table S2, Sup- Conservation prioritization porting information) exhibit diagnosable morphological differences, which led to their formal recognition. We fol- Evolutionarily Distinct and Globally Endangered species low Padial et al. (2010) in that congruence between mor- are threatened species with few or no close relatives on phology and DNA barcode unit is preferred for the the tree of life (Isaac et al. 2007). To date, the EDGE recognition of species entities, but emphasize that this approach has only been applied to globally complete criterion has clear limits, for example in cases of recent phylogenies of three major taxonomic groups: mammals speciation. Neglecting the morphological differences to (Isaac et al. 2007), amphibians (Safi et al. 2013) and reef adapt strictly DNA barcode-based species leads away corals (Huang 2012). The approach has not been used for from a concept of species as evolving, natural, diagnos- regional data sets, such as single BHs, because excluding able units (Simpson 1961; Wiley 1978). On the other close relatives occurring outside the study area can lead hand, some morphological species that were nested to falsely elevated EDGE scores. Given that there are within larger COI haplotype clusters may not be recog- approximately 29 000 species of fishes (Levêque et al. nized as species if taxonomic revisions were to be carried 2007), but only 5500 species of mammals (Wilson & Ree- out. It is not our aim to review all these cases, but the der 2005) and 7044 species of amphibians (Frost 2013), a data made available will aid future taxonomic studies complete global fish phylogeny remains a larger task. At (Table S2, Supporting information). present, this precludes fishes and other highly diverse groups from a global EDGE assessment. We see no rea- son not to apply the methodology to a regional fauna, if Sampling strategy species with close relatives outside the studied area are The high species coverage achieved might have influ- excluded, here based on published comprehensive fau- enced the performance of the nontree-based species nal assessments covering the Mediterranean BH delimitation approach. This is a general problem for (Levêque & Daget 1984; Geldiay & Balık 2007; Kottelat & large-scale barcoding studies, as demonstrated empiri- Freyhof 2007). As for amphibians (Safi et al. 2013) and cally by Bergsten et al. (2012), who concluded that the mammals (Isaac et al. 2007), we found no correlation

© 2014 John Wiley & Sons Ltd DNA BARCODING MEDITERRANEAN FRESHWATER FISHES 9 between the IUCN threat category of a species and its these new developments and calls for attention when ED. Therefore, a ranking according to EDGE scores analysing large DNA barcode data sets. allows prioritizing conservation efforts for species within one IUCN threat category. In the Mediterranean BH, the Acknowledgements two highest ranked EDGE species (Valencia spp.) are the only members of the endemic family Valenciidae. Spe- We thank numerous colleagues for providing additional sam- cies with higher EDGE score than expected from their ples, including N. Alwan, S. Brosse, A. Dunz, M. Englund, T. € IUCN category (outliers in Fig. S2, Supporting informa- Eriksson, F. Kronke, S. Kullander, N. Mesquita, D. Neumann, A. Nolte, L. Ruber,€ K. Rylkova, A. Scharbert, U. Schliewen, S. Saba- tion) could be candidates for a re-evaluation of their tino, S. Valdesalici and P. Vonlanthen. Sebastian Hullen,€ F.W. IUCN status. A conservation prioritization based on ED Miesen and J. Huylebrouck are thanked for clarifying some leads to a different set, the three most distinct species are unclear determinations. Claudia Etzbauer, J. Schmidt, I. Stoeger, not even present in the top 10 EDGE list. It seems prom- S. Guse€ and K. Gritzalis are acknowledged for help with logis- ising to use molecular information (also obtained from tics and wet-laboratory routines. We are grateful to S. Rutsch- DNA barcode data) to either complement a species’ mann, J. Powell and J. Pfaender for fruitful discussions on IUCN assessment, or to estimate ED for species that are methodological issues. not assessed. The latter approach would particularly benefit from large-scale barcode libraries. Funding The project FREDIE (Freshwater Diversity Identification Conclusions for Europe, www.fredie.eu) is funded by the Joint Initiative for Research and Innovation (PAKT) program This is the first large-scale study that uses DNA barcode data to estimate conservation priorities from a nearly of the Leibniz Association. RS acknowledges support complete freshwater fish fauna of a globally important from the Czech Ministry of Culture (DKRVO2012 and BH. The barcodes and associated vouchers, from 3165 DKRVO2013/14, National Museum, 00023272). This study is a product of the FREDIE project, financed in the individuals from 498 Mediterranean freshwater fish species, will serve as baseline for all forthcoming related SAW programme by the Leibniz Association. studies in the region. The freshwater fish diversity in the Mediterranean BH with its 23 different ecoregions (Abell et al. 2008) is indeed remarkable, but remains References underestimated. 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Appendix S1 (A) List of species barcoded, including those Wright S (1943) Isolation by distance. Genetics, 28, 114–138. where we suggest a taxonomic update, and also including to Young MK, McKelvey KS, Pilgrim KL, Schwartz MK (2013) DNA barcod- genus level only identified specimens. In parentheses number ing at riverscape scales: assessing biodiversity among fishes of the of individuals, an ‘e’ marks species for which individuals genus Cottus (Teleostei) in northern Rocky Mountain streams. Molecu- where used from outside the Mediterranean region. Taxa with lar Ecology Resources, 13, 583–595. potential candidate species or recovered synonyms are marked Yule GU (1924) A mathematical theory of evolution, based on the conclu- with an ’*’. Alien taxa in the Mediterranean BH are marked sions of Dr. J. C. Willis, F.R.S.. Philosophical Transactions of the Royal with an ‘$’; (B) list of Mediterranean freshwater fish species Society of London. Series B, 213,21–87. for which no material could be acquired; (Ex) indicates that Zardoya R, Doadrio I (1999) Molecular evidence on the evolutionary and biogeographical patterns of European cyprinids. Journal of Molecular the species is probably extinct; (F) indicates the failure or poor Evolution, 49, 227–237. quality of COI PCR amplification; (C) list of Mediterranean freshwater fish species for which COI sequences were obtained from NCBI GenBank with their respective accession M.F.G., F.H., M.T.M. and J.F. designed and performed numbers; (D) DNA barcoding studies that contributed materials to the study. the research. M.F.G., J.F., V.A., R.B., M.B., P.B., J.B., M.C.L., G.B.D., G.P.J.D., I.D., E.K., H.K., M.K., M.K., Appendix S2 (A) Methodological details for DNA extraction, € M.La., M.Lo, Z.M., M.O., A.P., S.P., H.P., S.P., C.P., J.R., PCR and molecular data analysis; (B) best-fit and other GMYC = R.S., M.S., V.S., M.S., and S.W. collected, provided or models within delta AICc 0.99 ranked by increasing delta AICc; weights: Akaike weight given to the model in the aver- determined specimens and helped with logistics in the aged parameter estimates below; method: single- or multiple- field. M.F.G. performed all analyses. M.F.G. and A.D. threshold GMYC model. obtained and analysed sequence data. M.F.G., M.T.M., F.H. and J.F. wrote the article. All authors contributed to Fig. S1 Collection of the maximum-likelihood trees obtained from the different data partitions (see Methods), with details for the final version of the text. calculation given for each tree.

Fig. S2 (A) For each IUCN category (LC, least concern; NT, near threatened; VU, vulnerable; EN, endangered; CR, critically Data Accessibility endangered; number of species in parentheses), the 25–75 per cent quartiles of the EDGE scores are drawn as box, the medians Newly generated DNA sequences: GenBank Accession given as horizontal line. Whiskers show largest and smallest nos. KJ552094-KJ554964; KC354979-KC354984; KC3550 data points <1.5 times the box height from the box. Outliers 19, KC355024, KC355025; details in Table S1. (stars) are values further than three times the box height from BOLD public project-ID: FFMBH. the box, values outside the inner fences are shown as circles; (B)

© 2014 John Wiley & Sons Ltd 12 M. F. GEIGER ET AL. top 10 species listed according to decreasing EDGE score and Table S2 (A) List of proposed updated taxonomy and, where according to decreasing ED (evolutionary distinctiveness) with applicable, mean K2P distances to former synonyms, and to their respective IUCN classification. Outliers with respect to nearest neighbour species if different; (B) commented lists of IUCN category combined with EDGE score are marked with an individuals identified as hybrids in the field with their respec- (*). Only endemic species without closely related congeneric tive putative parental species and number of individuals given species outside the Mediterranean are considered. in parentheses; (C) species or populations with haplotype-shar- ing indicating the need for more systematic research; (D) diver- Table S1 Excel spreadsheet with the name and sampling details gent lineages indicating introgressions and/or the need for for all newly generated individuals included in this study, more systematic research. museum or field accession numbers for specimens, and GenBank accession number for their DNA sequence.

© 2014 John Wiley & Sons Ltd Appendix S1 A) List of species barcoded, including those where we suggest a taxonomic update, and also including to genus level only identified specimens. In parentheses number of individuals, an ‘e’ marks species for which individuals where used from outside the Mediterranean region. Taxa with potential candidate species or recovered synonyms are marked with an’*’. Alien taxa in the Mediterranean BH are marked with an ‘$’; B) List of Mediterranean freshwater fish species for which no material could be acquired; (Ex) indicates that the species is probably extinct; (F) indicates the failure or poor quality of COI PCR amplification; C) List of Mediterranean freshwater fish species for which COI sequences were obtained from NCBI GenBank with their respective accession numbers; D) DNA barcoding studies that contributed materials to the study.

A) Abramis brama (10), Acanthobrama lissneri (3), Acanthobrama orontis* (11), Acanthobrama telavivensis (1), Achondrostoma arcasii (3), Achondrostoma occidentale (2), Achondrostoma oligolepis (6), Achondrostoma salmantinum (2), Acipenser baerii (5)e$, Acipenser naccarii (3), Acipenser stellatus (5)e, Acipenser sturio (2)e, Alburnoides bipunctatus (3), Alburnoides devolli (7), Alburnoides fangfangae (6), Alburnoides ohridanus (6), Alburnoides prespensis (6), Alburnoides sp. (1), Alburnoides strymonicus* (5), Alburnoides thessalicus* (18), Alburnus adanensis (3), Alburnus albidus (7), Alburnus alburnus (16), Alburnus arborella (20), Alburnus attalus (5), Alburnus baliki (3), Alburnus battalgilae (3), Alburnus belvica (6), Alburnus carinatus (3), Alburnus cf. maximus (2), Alburnus demiri (3), Alburnus escherichii (6), Alburnus istanbulensis (6), Alburnus kotschyi (3), Alburnus macedonicus (3), Alburnus nasreddini (4), Alburnus neretvae (2), Alburnus orontis (2), Alburnus qalilus (5), Alburnus scoranza (6), Alburnus sp. (9), Alburnus thessalicus (6), Alburnus vistonicus (1), Alburnus volviticus (4), Alosa agone (5), Alosa algeriensis (2), Alosa alosa (7), Alosa fallax (2), Alosa immaculata (1), Alosa macedonica (3), Alosa sp. (2), Alosa vistonica (1), Amatitlania nigrofasciata (3)$, Ameiurus melas (7$), Ameiurus nebulosus (2)e$, Anaecypris hispanica (3), Anaecypris punica (3), Anguilla anguilla (8), Aphanius almiriensis (7), Aphanius anatoliae* (9), Aphanius apodus (3), Aphanius baeticus (4), Aphanius danfordii (1), Aphanius dispar (2)e, Aphanius fasciatus (21), Aphanius fontinalis* (5), Aphanius iberus (7), Aphanius iconii* (2), Aphanius maeandricus* (5), Aphanius mento* (32), Aphanius meridionalis* (7), Aphanius richardsoni* (5), Aphanius saldae* (2), Aphanius saourensis (5), Aphanius sirhani (2), Aphanius sureyanus (2), Aphanius transgrediens (8), Aphanius villwocki (2), Atherina boyeri (8), Atherina pontica (1), Atherina sp.* (7), Aulopyge huegelii (4), Australoheros facetus (2)$, Barbatula quignardi (11), Barbatula sturanyi (9), Barbatula vardarensis (5), Barbatula zetensis (6), Barbus balcanicus (7), Barbus barbus (6), Barbus caninus (8), Barbus cyclolepis (9), Barbus euboicus (4), Barbus haasi (3), Barbus macedonicus (6), Barbus meridionalis (8), Barbus niluferensis (3), Barbus oligolepis (6), Barbus peloponnesius (7), Barbus pergamonensis (13), Barbus plebejus (6), Barbus prespensis (5), Barbus rebeli (7), Barbus sp.* (11), Barbus sperchiensis (4), Barbus strumicae (13), Barbus tyberinus (17), Blicca bjoerkna (1), Capoeta angorae (2), Capoeta antalyensis (2), Capoeta barroisi (4), Capoeta bergamae (4), Capoeta caelestis (4), Capoeta damascina (16), Capoeta erhani (4), Capoeta mauricii (1), Capoeta pestai (5), Capoeta sp. (6), Capoeta tinca (2), Capoeta turani (1), Carasobarbus canis (3), Carasobarbus chantrei (6), Carasobarbus fritschii* (11), Carasobarbus harterti (6), Carassius auratus (6)$, Carassius gibelio (15)$, Carassius langsdorfii (1)$, Caspiomyzon graecus (2), Caspiomyzon hellenicus (7), Chelon abu (5)$, Chelon auratus (15), Chelon carinatus (2)$, Chelon haematocheilus (10)$, Chelon labrosus (10), Chelon ramada (9), Chelon saliens (6), Chelon sp. (1), Chondrostoma beysehirense (2), Chondrostoma fahirae (8), Chondrostoma holmwoodii (3), Chondrostoma kinzelbachi (3), Chondrostoma knerii (5), Chondrostoma meandrense (3), Chondrostoma nasus (6), Chondrostoma ohridanus* (7), Chondrostoma phoxinus (7), Chondrostoma prespense (10), Chondrostoma soetta (18), Chondrostoma sp. (12), Chondrostoma vardarense (22), Clarias gariepinus (1), Clupeonella cultriventris (1), Cobitis arachthosensis (5), Cobitis battalgili (7), Cobitis bilineata (11), Cobitis bilseli (3), Cobitis calderoni (4), Cobitis dalmatina (2), Cobitis evreni (2), Cobitis fahireae (2), Cobitis hellenica (4), Cobitis illyrica (6), Cobitis jadovaensis (2), Cobitis levantina (2), Cobitis maroccana (3), Cobitis meridionalis (2), Cobitis narentana (4), Cobitis ohridana (3), Cobitis paludica (3), Cobitis phrygica (5), Cobitis puncticulata (2), Cobitis punctilineata (6), Cobitis sp.* (24), Cobitis splendens (2), Cobitis strumicae (2), Cobitis trichonica (1), Cobitis turcica (5), Cobitis vardarensis (5), Cobitis vettonica (4), Cobitis zanandreai (6), Coptodon zillii (7), Coregonus sp. (4)$, Cottus gobio (10), Cottus petiti (3), Cottus rondeleti (6), Cottus scaturigo (4), Ctenopharyngodon idella (3)$, Cyprinus carpio (13), Delminichthys adspersus (2), Delminichthys ghetaldii (5), Delminichthys jadovensis (3), Delminichthys krbavensis (2), Dicentrarchus labrax (2), Economidichthys pygmaeus (6), Economidichthys trichonis (5), Esox cisalpinus (23), Esox lucius (11), Eudontomyzon sp.* (2), Eudontomyzon stankokaramani (7), Fundulus heteroclitus (4)$, Gambusia holbrooki (8)$, Garra caudomaculata (3), Garra culiciphaga (5), Garra festai (7), Garra ghorensis (5), Garra kemali (2), Garra klatti (6), Garra nana (5), Garra rufa (5), Garra sauvagei (2), Garra variabilis (5), Gasterosteus sp. (43), Gobio balcanicus (6), Gobio battalgilae (3), Gobio bulgaricus (7), Gobio feraeensis (7), Gobio gobio (18), Gobio gymnostethus (4), Gobio hettitorum (4), Gobio insuyanus (4), Gobio intermedius (4), Gobio lozanoi (4), Gobio maeandricus (3), Gobio microlepidotus (7), Gobio occitaniae (18), Gobio ohridanus (7), Gobio skadarensis (5), Gobio sp. (5), Gymnocephalus cernua (1)$, Haplochromis desfontainii (6), Haplochromis flaviijosephi (3), Haplochromis sp. (6), Hemichromis guttatus (2), Hemichromis letourneuxi (3), Hemichromis saharae* (3), Hucho hucho (1)$, Huso huso (5), Hypophthalmichthys molitrix (4)$, Hypophthalmichthys nobilis (4)$, Iberochondrostoma almacai (2), Iberochondrostoma lemmingii (2), Iberochondrostoma lusitanicum (5), Iberochondrostoma oretanum (1), Iberocypris alburnoides (7), Ictalurus punctatus (1)$, Knipowitschia byblisia (9), Knipowitschia caucasica (12), Knipowitschia ephesi (3), Knipowitschia mermere (7), Knipowitschia milleri (12), Knipowitschia montenegrina (5), Knipowitschia mrakovcici (8), Knipowitschia panizzae (17), Knipowitschia radovici (6), Knipowitschia thessala (3), Ladigesocypris ghigii (3), Ladigesocypris irideus (2), Ladigesocypris mermere (2), Lampetra alavariensis (1), Lampetra auremensis (1), Lampetra fluviatilis (2)e, Lampetra lanceolata (2), Lampetra lusitanica (6), Lampetra planeri (20), Lampetra sp. (7), Lampetra zanandreai (6), Lepomis gibbosus (12)$, Lepomis macrochirus (1)$, Leucaspius delineatus* (2), Leuciscus aspius (3), Leuciscus burdigalensis (4), Leuciscus idus (2)$, Leuciscus leuciscus (3), Leuciscus vorax (2), Lota lota (6), Luciobarbus albanicus (22), Luciobarbus biscariensis (5), Luciobarbus bocagei (11), Luciobarbus callensis (6), Luciobarbus comizo (4), Luciobarbus figuigensis (2), Luciobarbus graecus (11), Luciobarbus graellsii (6), Luciobarbus guiraonis (6), Luciobarbus issenensis (3), Luciobarbus kottelati (1), Luciobarbus ksibi (8), Luciobarbus labiosa (10), Luciobarbus lepineyi (5), Luciobarbus leptopogon* (2), Luciobarbus longiceps (2), Luciobarbus lorteti (1), Luciobarbus lydianus (3), Luciobarbus magniatlantis (5), Luciobarbus massaensis (3), Luciobarbus microcephalus (3), Luciobarbus moulouyensis (7), Luciobarbus nasus (4), Luciobarbus pallaryi (12), Luciobarbus pectoralis (7), Luciobarbus sclateri (3), Luciobarbus setivimensis (6), Luciobarbus sp.* (15), Luciobarbus steindachneri (7), Micropterus salmoides (2)$, Mirogrex terrasanctae (5), Misgurnus anguillicaudatus (3)$, Morone hybrid (3)$, Mugil cephalus (13), Mystus pelusius (2), Neogobius fluviatilis (3), Neogobius melanostomus (1), Neogobius nigricans (8), Ninnigobius canestrinii (3), Ninnigobius montenegrensis (5), Ninnigobius sp. (5), Odontesthes bonariensis (3)$, Oncorhynchus mykiss (3)$, Oreochromis aureus (2), Oreochromis niloticus (2), Orsinigobius croaticus (7), Orsinigobius punctatissimus* (2), Oxynoemacheilus anatolicus (2), Oxynoemacheilus angorae (2), Oxynoemacheilus atili(3), Oxynoemacheilus bureschi (10), Oxynoemacheilus ceyhanensis (2), Oxynoemacheilus cinicus (3), Oxynoemacheilus eregliensis (3), Oxynoemacheilus evreni (2), Oxynoemacheilus galilaeus (2), Oxynoemacheilus germencicus* (4), Oxynoemacheilus hamwii (6), Oxynoemacheilus insignis (2), Oxynoemacheilus leontinae (4), Oxynoemacheilus mediterraneus (2), Oxynoemacheilus mesudae (2), Oxynoemacheilus namiri (4), Oxynoemacheilus panthera (2), Oxynoemacheilus phoxinoides (4), Oxynoemacheilus pindus (8), Oxynoemacheilus samanticus (2), Oxynoemacheilus seyhanensis (7), Oxynoemacheilus seyhanicola (4), Oxynoemacheilus simavicus (2), Oxynoemacheilus sp.* (19), Oxynoemacheilus theophilii (4), Pachychilon macedonicum (14), Pachychilon pictum (5), Padogobius bonelli (12), Parachondrostoma arrigonis (2), Parachondrostoma miegii (5), Parachondrostoma toxostoma (1), Parachondrostoma turiensis (3), Paramisgurnus dabryanus (4)$, Pelasgus laconicus (7), Pelasgus marathonicus (7), Pelasgus minutus (4), Pelasgus prespensis (3), Pelasgus sp.* (12), Pelasgus stymphalicus (5), Pelasgus thesproticus (6), Perca fluviatilis (23), Petroleuciscus borysthenicus (2), Petroleuciscus smyrnaeus (2), Petromyzon marinus (4), Phoxinellus alepidotus (4), Phoxinellus dalmaticus (8), Phoxinellus pseudalepidotus (3), Phoxinus bigerri (3), Phoxinus lumaireul* (10), Phoxinus septimaniae (23), Phoxinus sp.* (1), Phoxinus strandjae (4), Phoxinus strymonicus (5), Platichthys flesus (1), Poecilia reticulata (4)$, Pomatoschistus marmoratus (6), Pomatoschistus microps (4), Proterorhinus semilunaris (3), Protochondrostoma genei (13), Pseudochondrostoma duriense (10), Pseudochondrostoma polylepis (13), Pseudochondrostoma willkommii (2), Pseudocrenilabrus multicolor (2), Pseudophoxinus alii (2), Pseudophoxinus anatolicus (2), Pseudophoxinus antalyae (3), Pseudophoxinus battalgilae* (6), Pseudophoxinus burduricus (6), Pseudophoxinus caralis (2), Pseudophoxinus crassus (4), Pseudophoxinus drusensis (5), Pseudophoxinus egridiri (10), Pseudophoxinus elizavetae (3), Pseudophoxinus evliyae (2), Pseudophoxinus fahrettini (2), Pseudophoxinus hasani (2), Pseudophoxinus hittitorum (3), Pseudophoxinus kervillei (10), Pseudophoxinus libani (7), Pseudophoxinus maeandricus (4), Pseudophoxinus meandri (4), Pseudophoxinus ninae (4), Pseudophoxinus sp.* (13), Pseudophoxinus syriacus (2), Pseudophoxinus zekayi (4), Pseudophoxinus zeregi (5), Pseudorasbora parva (11)$, Pterocapoeta maroccana (2), Pterygoplichthys cf. disjunctivus (2)$, Pungitius hellenicus (3), Pungitius laevis (2), Pungitius platygaster (3)e, Rhodeus amarus (6), Rhodeus meridionalis (12), Romanogobio banarescui* (6), Romanogobio benacensis (1), Romanogobio elimeius (7), Rutilus albus (6), Rutilus aula (9), Rutilus basak (5), Rutilus frisii (1), Rutilus heckelii (4), Rutilus ohridanus (8), Rutilus panosi (12), Rutilus pigus (4), Rutilus prespensis (15), Rutilus rubilio* (20), Rutilus rutilus (16), Rutilus sp. (2), Rutilus ylikiensis (6), Sabanejewia balcanica (6), Sabanejewia larvata (3), Salaria atlantica (3), Salaria economidisi (8), Salaria fluviatilis (38), Salaria sp.* (3), Salmo akairos (1), Salmo aphelios (2), Salmo carpio (4), Salmo cettii (3), Salmo chilo (2), Salmo farioides (9), Salmo fibreni (3), Salmo labecula (3), Salmo letnica (7), Salmo lourosensis (7), Salmo macedonicus (2), Salmo marmoratus (7), Salmo obtusirostris (2), Salmo ohridanus (7), Salmo opimus (4), Salmo peristericus (5), Salmo pellegrini (9), Salmo platycephalus (2), Salmo rhodanensis (2), Salmo sp. (18), Salmo trutta (7)e, Salmo visovacensis (7), Salmo zrmanjaensis (4), Salvelinus fontinalis (3)$, Salvelinus umbla (3)e$, Sander lucioperca (2)$, Sander volgensis (1)e$, Sarotherodon galilaeus (1), Scardinius acarnanicus (19), Scardinius dergle (5), Scardinius elmaliensis (2), Scardinius erythrophthalmus (11), Scardinius graecus (8), Scardinius hesperidicus (5), Scardinius knezevici (3), Scardinius plotizza (3), Scardinius scardafa (2), Scardinius sp. (1), Seminemacheilus ispartaensis (5), Seminemacheilus lendlii (2), Seminemacheilus sp.* (3), Silurus aristotelis (10), Silurus glanis (3), Squalius adaenensis (2), Squalius albus (2), Squalius anatolicus (2), Squalius aradensis (4), Squalius aristotelis (2), Squalius cappadocicus (4), Squalius carinus (4), Squalius carolitertii (10), Squalius castellanus (1), Squalius cephaloides (2), Squalius cephalus (10), Squalius cii (4), Squalius fellowesii (22), Squalius illyricus (5), Squalius janae (8), Squalius keadicus (3), Squalius kosswigi (5), Squalius kottelati (3), Squalius laietanus (7), Squalius lucumonis (6), Squalius malacitanus (3), Squalius microlepis (3), Squalius moreoticus (6), Squalius orpheus (4), Squalius pamvoticus (5), Squalius peloponensis (2), Squalius platyceps (10), Squalius prespensis (6), Squalius pyrenaicus (5), Squalius recurvirostris (4), Squalius seyhanensis (3), Squalius sp.* (72), Squalius squalus (23), Squalius svallize (3), Squalius tenellus (2), Squalius torgalensis (6), Squalius valentinus (3), Squalius vardarensis (14), Squalius zrmanjae (5), Syngnathus abaster (1), Syngnathus sp. (1), Telestes beoticus (3), Telestes croaticus (2), Telestes dabar (7), Telestes fontinalis (4), Telestes karsticus (2), Telestes metohiensis (3), Telestes montenigrinus (3), Telestes muticellus* (17), Telestes pleurobipunctatus* (26), Telestes polylepis (5), Telestes souffia* (7), Telestes turskyi (2), Telestes ukliva (2), Thymallus aeliani* (5), Thymallus thymallus (6), Tinca tinca (8), Tristramella simonis (4), Tropidophoxinellus callensis (10), Tropidophoxinellus chaignoni (2), Tropidophoxinellus hellenicus (10), Tropidophoxinellus spartiaticus (3), Valencia hispanica (5), Valencia letourneuxi (6), Valencia sp.* (5), Vimba melanops (12), Vimba mirabilis (1), Vimba vimba (5), Xiphophorus helleri (1)e$, Zingel asper (2)

B) List of Mediterranean freshwater fish species for which no material could be acquired Acanthobrama centisquama (Ex), Acanthobrama tricolor (Ex, F), Alburnoides manyasensis, Alburnus akili (Ex), Alburnus nicaeensis (Ex), Aphanius splendens (Ex), Chondrostoma scodrense (Ex), Clupeonella muhlisi (F), Cobitis kurui, Cobitis stephanidisi (Ex), Iberocypris palaciosi (Ex), Knipowitschia caunosi, Knipowitschia goerneri, Labeobarbus reinii (Ex), Mirogrex hulensis (Ex), Pelasgus epiroticus (Ex), Pseudophoxinus handlirschi (Ex), Salmo balcanicus (Ex), Salmo dentex, Salmo lumi, Salmo macrostigma (Ex), Salmo montenigrinus, Salmo pallaryi (Ex), Salmo pelagonicus, Salmo taleri, Telestes miloradi (Ex), Tristramella sacra (Ex), Zingel balcanicus

C) COI sequences used from Genbank Genus species accession numbers Acipenser baerii GQ328793,GQ328795,GQ328797,GQ328799,GQ328801 Acipenser stellatus HQ960585-HQ960589 Chelon abu JQ060447 Chelon aurata JQ060456,JQ060457,JQ060459,JQ060460,HM208835,HM208836 Chelon carinata FN600159, JQ623947 Chelon haematocheila EU392236-EU392238,JN242615-JN242620,HM180700 Chelon ramada JQ060479-JQ060480,HM208839,HM208840 Chelon saliens HM208833,JQ060483 Chondrostoma phoxinus HM560257 Esox cisalpinus HM563688,HM563691,HM563692,HM563694-HM563698,HM563700,HM563703- HM563707 Esox lucius HM563689,HM563690,HM563693,HM563699,HM563701,HM563702 Fundulus heteroclitus EU524627-EU524630 Huso huso FJ809715-FJ809719 Hypophthalmichthys molitrix FJ459499-FJ459502 Hypophthalmichthys nobilis HQ682696-HQ682699 Iberochondrostoma lemmingii HM560267 Iberochondrostoma oretanum HM560268 Mugil cephalus JQ060529,JQ060532-JQ060534,JQ060536 Parachondrostoma miegii HM989722 Paramisgurnus dabryanus HM446339,HM446340,HM446342,HM446343 Tropidophoxinellus chaignoni HM560301 D) Project description and number of COI barcodes used (published) FBPIS Fauna Bavarica Pisces (BOLD), 1 (no) FFMDR Fishes of the French Mediterranean Drainage (BOLD), 55(yes) NGLF Barcoding analysis of fish species from four North Greek Lakes (BOLD), 125(yes) NRM Sweden (“A DNA key to all Swedish vertebrates.", funded by the Swedish Research Council (FORMAS 2006-714), 2(no)

Appendix S2 A) Methodological details for DNA extraction, PCR and molecular data analysis; B) Best-fit and other GMYC models within delta AICc = 0.99 ranked by increasing delta AICc; weights: Akaike weight given to the model in the averaged parameter estimates below; method: single or multiple threshold GMYC model.

Genomic DNA was extracted using Machery & Nagel NucleoSpin® Tissue kits following the manufacturer’s protocol on an Eppendorf EpMotion® pipetting-roboter with vacuum manifold. The standard vertebrate DNA barcode region of COI was PCR-amplified using different primer combinations (Folmer et al. 1994; Ivanova et al. 2007). Sequencing of the ExoSAP-IT (USB) purified PCR product in both directions was conducted at Macrogen Europe Laboratories with either sequencing primer M13F (5’ GTAAAACGACGGCCAGT) and M13R-pUC (5’ CAGGAAACAGCTATGAC), HCO2198 and LCO1490, or at Genoscope (Centre national de séquencage) with primers TelF1 and TelR1.

Data processing and sequence assembly was done in Geneious (Geneious created by Biomatters 2013). The Muscle algorithm (Edgar 2004) was used to create DNA sequence alignments containing groups of sequences belonging to various taxonomic ranks (e.g. all Perciformes, all Cyprinidae) in order to restrict computational demand, and to take into account the heterogeneous substitution rates expected (e.g. Brown et al. 1979). Taxonomic composition (including outgroup choice) of data partitions were aided by an approximately- maximum-likelihood guide tree composed of all 3165 COI sequences, calculated in FastTree with default settings (Price et al. 2009). Clustering with Kimura2-corrected distances (K2P) in SpeciesIdentifier (Meier et al. 2006a) was used to cluster sequences at 2%, 1% and an optimized, data-derived threshold. Taking into account the number of true & false positives, and true & false negative identifications at a given threshold with cumulative error (false negative + false positive), the optimized threshold was derived from each data partition with the Spider package (Brown et al. 2012) in R v 2.15.1 (R Development Core Team 2011). We then counted the number of clusters in agreement with existing taxonomy, i.e. containing only sequences of one morphological species (‘perfect match’), as well as the total number of clusters (e.g. (Hendrich et al. 2010)). We treated clusters containing all COI sequences of one species together with one or more sequences that were determined to genus level only as a ‘perfect match’. The number of genetically differentiated clusters found with the ‘traditional’ barcoding approach, i.e. distance-based with a priori defined thresholds for cluster delimitation at 2% and 1% sequence divergence (Hebert et al. 2003a; April et al. 2011) was chosen to enable comparison to results of other comprehensive barcode studies. For the complete fish faunas of three single drainage systems (Po, Italy; Vardar, Greece; Orontes, Turkey) we calculated standard barcoding statistics to test for barcoding accuracy under more realistic ‘operator-oriented’ (e.g. environment agencies) conditions. Species that are known to occur in these drainages, but could not be acquired from it directly were substituted by allochthonous representatives.

As the distance-based approach has repeatedly been criticized for being arbitrarily chosen without sound biological background (Meier et al. 2006b; Srivathsan & Meier 2012), and the threshold varies considerably between species from different organism groups (Hebert et al. 2003b), we applied additional methods to analyze the COI sequences. First, the species criterion of monophyly was applied to test for congruence, requiring the grouping of all COI haplotypes of a given species as a monophyletic unit in a given phylogram; here, we screened ML trees for those groups generated under the model assumptions as derived below. Singletons were considered as match, if they did not cluster unresolved within another species, and we counted every monophyletic clade without defining a certain a priori support (e.g. bootstrap), since this would be an arbitrary choice. Second, the general mixed Yule-coalescent (GMYC) approach was used, which identifies clusters by fitting models that predict the inter- and intraspecific divergence rates and threshold times differentiating these processes to multispecies coalescent trees (Monaghan et al. 2009; Powell 2012). The model assumes that branching patterns within genetic clusters reflect neutral coalescent processes and occur within species (Kingman 1982) whereas branching between clusters can reflect the timing of a speciation event (Yule 1924). Thus, it identifies a species boundary by identifying independently evolving lineages and the transition from coalescent to speciation branching patterns on a phylogenetic tree. Although not uncontroversial (Lohse 2009; Esselstyn et al. 2012), the theory behind the model is very attractive and it has proven to deliver reliable and biological sound species number estimates (Monaghan et al. 2009; Fujita et al. 2012; Puillandre et al. 2012).

GMYC analysis requires an ultrametric gene tree of unique haplotypes, since zero-length branches of identical sequences would imply infinite branching rates (Williams et al. 2012). Alignments were collapsed to contain only unique haplotypes using FaBox 1.40 (http://users-birc.au.dk/biopv/php/fabox/). Modeltest in Mega 5 (Tamura et al. 2011) was used to determine the most likely substitution model for each alignment, and the one with the lowest BIC score (Bayesian Information Criterion) was chosen to generate input trees for the GMYC. The ultrametric trees were obtained by the Bayesian Evolutionary Analysis Utility (BEAUti) v 1.7.4 and Bayesian Evolutionary Analysis by Sampling Trees (BEAST; v 1.7.4) (Drummond et al. 2012) using the substitution models determined before under a relaxed (uncorrelated lognormal) molecular clock model with a coalescent tree prior. Depending on the size of the matrix, three to six parallel chains were run between 30,000,000 and 100,000,000 generations each. Trees were sampled every 1,000 to 10,000 generations. Resulting log files were combined in LogCombiner v 1.7.4 and parameters checked for their ESS values, aiming at ESS > 300 for the prior and likelihood parameter to achieve stable parameter estimations. After combining the tree files in LogCombiner v 1.7.4 and discarding the first 20% of each run as burn-in, we used TreeAnnotator v.1.5.4 to produce a consensus tree from the tree samples with a posterior probability of 0.5, targeting the maximum clade credibility tree and target node heights. We performed all GMYC analyses with the single and multiple threshold methods (Monaghan et al. 2009) using the SPLITS package (T. Ezard; r-forge.rproject.org/projects/splits) for R (R Development Core Team 2011). We report the number of GMYC entities as the sum of groups and singletons as proxy for species number estimation. Finally, we applied an extension to the GMYC approach (Powell 2012) that takes into account additional models with slightly lower likelihoods and uses an Akaike Information Criterion (AIC) of all single- and multiple- threshold models (Powell 2012). The probabilities that two haplotypes belong to the same entity are then based on the weights associated with each model, and the variance is estimated from model averaging. Thus, uncertainty in species boundaries can directly be incorporated into diversity estimates. Based on the probabilities associated with each genetic cluster, we derived the number of morphological species supported by the GMYC approach, analogous to the ‘perfect match’ above, also including singletons.

combinatio n primer 5' - 3' sequence remarks FishF2_t TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGC Ivanova 1 AC FishR2_t CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAG Ivanova 1 AA most universal TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGC combination Ivanova VF2_t1 AC CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCAR Ivanova FR1d_t1 AA samples from Dettai TelF1 TCGACTAATCAYAAAGAYATYGGCAC MNHN Dettai TelR1 ACTTCTGGGTGNCCAAARAATCARAA TGTAAAACGACGGCCAGTATTCAACCAATCATAAAGATATTG Mammal LepF1_tl G TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATT Mammal VF1_tl GG TGTAAAACGACGGCCAGTTCTCAACCAACCACAARGAYATY for some Mammal VF1d_tl GG Alburnoides, Mammal VFli_tl TGTAAAACGACGGCCAGTTCTCAACCAACCAIAAIGAIATIGG Aphanius, CAGGAAACAGCTATGACTAAACTTCTGGATGTCCAAAAAATC Acanthobrama, Mammal LepR1_tl A Cobitis, CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAY Proterorhinus Mammal VR1d_tl CA CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAAT Mammal VR1_tl CA Mammal VRli_tl CAGGAAACAGCTATGACTAGACTTCTGGGTGICCIAAIAAICA Folmer LCO1490 GGTCAACAAATCATAAAGATATTGG for some Cobitis & Folmer HC02198 TAAACTTCAGGGTGACCAAAAAATCA Garra

PCR conditions with Qiagen® multiplex mix 1) 15m @ 95°C 2) 35s @ 94°C 3) 90s @ 52°C to 49°C touch down 10 cycles (+25 cycles @ 55°C) 4) 90s @ 72°C 5) 10m @ 72°C 6) forever @ 10°C

dataset I method likelihood AICc clusters entities ∆AICc weights 1 multiple 1588.97 -3165.52 61 73 0.000 0.0625 2 single 1587.63 -3164.96 64 79 0.562 0.0472 3 single 1587.55 -3164.8 63 78 0.723 0.0435 4 single 1587.53 -3164.77 65 80 0.753 0.0429 5 multiple 1589.65 -3164.74 63 77 0.787 0.0422 6 multiple 1588.58 -3164.73 60 72 0.788 0.0421 7 multiple 1589.63 -3164.70 63 76 0.825 0.0414 8 multiple 1589.63 -3164.69 62 76 0.828 0.0413

Summary of models (averaged): estimate variance clusters 62.08 1.71 entities 77.12 3.99

dataset II method likelihood AICc clusters entities ∆AICc weights 1 multiple 1313.15 -2611.71 37 51 0.000 0.063 2 multiple 1313.06 -2611.52 37 52 0.191 0.057 3 multiple 1312.82 -2611.04 37 50 0.669 0.045 4 multiple 1312.81 -2611.02 37 51 0.692 0.044 5 multiple 1312.68 -2610.76 36 50 0.948 0.039

Summary of models (averaged) estimate variance clusters 41.36 5.48 entities 61.76 12.10

dataset III method likelihood AICc clusters entities ∆AICc weights 1 multiple 1009.21 -2003.61 31 38 0.000 0.069 2 multiple 1009.20 -2003.59 32 39 0.018 0.068 3 multiple 1008.03 -2003.47 32 38 0.139 0.064 4 multiple 1009.13 -2003.44 32 38 0.162 0.063 5 multiple 1008.97 -2003.14 32 37 0.468 0.054 6 multiple 1007.69 -2002.77 33 38 0.834 0.045 7 multiple 1008.78 -2002.75 32 38 0.851 0.045

Summary of models (averaged) estimate variance clusters 31.79 1.36 entities 37.31 2.31

dataset IV method likelihood AICc clusters entities ∆AICc weights 1 multiple 8784.21 -17550.23 229 346 0.000 0.024 2 multiple 8784.20 -17550.21 230 347 0.020 0.024 3 multiple 8784.20 -17550.21 229 347 0.023 0.024 4 multiple 8784.20 -17550.2 229 347 0.031 0.024 5 multiple 8784.17 -17550.15 230 348 0.081 0.023 6 multiple 8784.17 -17550.14 229 348 0.097 0.023 7 multiple 8784.17 -17550.14 229 348 0.097 0.023 8 multiple 8784.13 -17550.07 230 349 0.166 0.022 9 multiple 8784.13 -17550.07 231 349 0.168 0.022 10 multiple 8784.12 -17550.05 230 349 0.179 0.022 11 multiple 8784.08 -17549.97 231 350 0.267 0.021 12 multiple 8784.07 -17549.95 230 350 0.285 0.021 13 multiple 8784.01 -17549.82 231 351 0.410 0.020 14 multiple 8784.00 -17549.81 231 351 0.423 0.020 15 multiple 8784.00 -17549.8 230 351 0.436 0.020 16 multiple 8783.92 -17549.65 231 352 0.581 0.018 17 multiple 8783.9 -17549.61 232 352 0.622 0.018 18 multiple 8783.9 -17549.6 232 352 0.631 0.018 19 multiple 8783.89 -17549.59 231 352 0.643 0.018 20 multiple 8783.84 -17549.48 231 352 0.753 0.017 21 multiple 8783.83 -17549.47 232 352 0.763 0.017 22 multiple 8783.82 -17549.45 232 353 0.788 0.016 23 multiple 8783.81 -17549.43 232 353 0.805 0.016 24 multiple 8783.81 -17549.42 231 353 0.813 0.016 25 multiple 8783.74 -17549.29 231 353 0.940 0.015 26 multiple 8783.73 -17549.27 232 353 0.959 0.015

Summary of models (averaged): estimate variance clusters 231.23 1.10 entities 353.47 4.49

dataset V method likelihood AICc clusters entities ∆AICc weights 1 single 461.08 -911.41 14 22 0.000 0.049 2 multiple 462.12 -911.19 13 20 0.214 0.044 3 single 460.90 -911.05 15 23 0.355 0.041 4 multiple 462.03 -911.01 21 37 0.397 0.040 5 multiple 462.02 -911.00 21 38 0.413 0.040 6 multiple 461.99 -910.92 21 38 0.488 0.038 7 multiple 461.92 -910.80 12 19 0.612 0.036 8 single 460.68 -910.62 13 21 0.785 0.033 9 multiple 461.81 -910.56 12 16 0.844 0.032 10 single 460.63 -910.52 13 20 0.888 0.031 11 multiple 461.75 -910.46 12 19 0.952 0.030

Summary of models (averaged): estimate variance clusters 14.46 3.19 entities 23.57 7.52

dataset VI method likelihood AICc clusters entities ∆AICc weights 1 single 5343.59 -1057.88 6 9 0.000 0.0534 2 single 5342.68 -1057.7 9 13 0.183 0.0487 3 multiple 5353.50 -1057.52 9 12 0.368 0.0444 4 multiple 5353.50 -1057.52 9 12 0.368 0.0444 5 multiple 5353.06 -1057.43 8 11 0.456 0.0425 6 single 5339.93 -1057.15 6 10 0.731 0.0370 7 single 5339.17 -1057. 10 14 0.885 0.0343

Summary of models (averaged): estimate variance clusters 13.69 6.27 entities 18.57 7.90

All method likelihood AICc clusters entities ∆AICc weights 1 multiple 16618.74 -33219.37 366 468 0 0.01525 2 multiple 16618.73 -33219.35 367 469 0.0196232 0.0151 3 multiple 16618.7 -33219.3 366 467 0.0667598 0.01475 4 multiple 16618.7 -33219.29 367 468 0.0828841 0.01463 5 multiple 16618.68 -33219.26 367 468 0.1102221 0.01443 6 multiple 16618.68 -33219.25 367 468 0.1171038 0.01438 7 multiple 16618.66 -33219.22 368 468 0.1464015 0.01417 8 multiple 16618.66 -33219.21 368 469 0.1623253 0.01406 9 multiple 16618.65 -33219.19 367 469 0.1820813 0.01392 10 multiple 16618.65 -33219.18 366 467 0.1844626 0.0139 11 multiple 16618.64 -33219.17 367 467 0.1946338 0.01383 12 multiple 16618.63 -33219.15 366 467 0.219051 0.01366 13 multiple 16618.61 -33219.11 367 467 0.2560431 0.01341 14 multiple 16618.61 -33219.11 367 468 0.2582147 0.0134 15 multiple 16618.61 -33219.1 367 468 0.2649349 0.01335 16 multiple 16618.59 -33219.08 366 468 0.2882941 0.0132 17 multiple 16618.59 -33219.07 365 466 0.3015253 0.01311 18 multiple 16618.58 -33219.06 366 466 0.3113541 0.01305 19 multiple 16618.57 -33219.03 365 466 0.3369965 0.01288 20 multiple 16618.55 -33218.99 366 467 0.3753263 0.01264 21 multiple 16618.52 -33218.94 366 467 0.4302676 0.0123 22 multiple 16618.49 -33218.88 367 467 0.490291 0.01193 23 multiple 16618.48 -33218.86 369 461 0.5116825 0.0118 24 multiple 16618.48 -33218.86 367 468 0.5127439 0.0118 25 multiple 16618.48 -33218.85 366 466 0.5153482 0.01178 26 multiple 16618.45 -33218.8 366 466 0.5654785 0.01149 27 multiple 16618.43 -33218.76 369 460 0.6094319 0.01124 28 multiple 16618.42 -33218.74 367 466 0.6331505 0.01111 29 multiple 16618.42 -33218.73 367 467 0.6417651 0.01106 30 multiple 16618.4 -33218.69 365 465 0.6776312 0.01086 31 multiple 16618.4 -33218.69 368 459 0.6810092 0.01085 32 multiple 16618.39 -33218.68 368 459 0.6861074 0.01082 33 multiple 16618.38 -33218.65 365 465 0.7230621 0.01062 34 multiple 16618.36 -33218.61 367 458 0.7572932 0.01044 35 multiple 16618.34 -33218.57 366 465 0.7986665 0.01023 36 multiple 16618.28 -33218.46 371 461 0.9062066 0.00969 37 multiple 16618.26 -33218.42 370 460 0.9502652 0.00948 38 multiple 16618.24 -33218.38 371 460 0.9862114 0.00931

Summary of models (averaged): estimate variance clusters 366.48 2.22 entities 463.54 6.59

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Cobitis battalgili Beysehir Turkey WH13 F5 1108 Cobitis battalgili Beysehir Turkey WH13 F7 1108 98 Cobitis battalgili Beysehir Turkey WH13 E5 1082 Cobitis battalgili Manavgat Turkey Ex60div F1 1657 Cobitis battalgili Beysehir Turkey WH13 E3 1082 100 Cobitis battalgili Beysehir Turkey WH13 E6 1082 Cobitis battalgili Manavgat Turkey Ex60div F3 1657 Cobitis turcica Tuz Turkey Ex52 E2 958 Cobitis turcica Tuz Turkey Ex52 D12 958 99 Cobitis turcica Tuz Turkey Ex52 E8 1086 Cobitis turcica Tuz Turkey Ex52 E7 1086 Cobitis turcica Tuz Turkey Ex52 E9 1086 97 Cobitis sp. Egirdir Turkey Ex15 E2 1103 78 Cobitis sp. Egirdir Turkey Ex15 E3 1103 Cobitis bilseli Beysehir Turkey Ex13 A12 55 Cobitis bilseli Beysehir Turkey Ex13 A10 55 98 Cobitis bilseli Beysehir Turkey Ex13 A11 55 87 Cobitis sp. Kirgz Turkey Ex19 E2 1539 100 Cobitis sp. Kirgz Turkey Ex19 E3 1539 Cobitis sp. Soysalli Turkey Ex52 D11 965 100 Cobitis sp. Dalaman Turkey Ex14 B11 333 Cobitis sp. Dalaman Turkey Ex14 B12 333 99 Cobitis phrygica Esen Turkey Ex13 B6 59 97 Cobitis phrygica Esen Turkey Ex13 B7 59 Cobitis phrygica Aci Turkey Ex18 D10 979 99 Cobitis phrygica Aci Turkey Ex18 D11 979 99 Cobitis phrygica Aci Turkey Ex18 D12 979 100 Cobitis meridionalis Prespa Greece WH11 D7 55809 Cobitis meridionalis Prespa Greece WH11 D8 55810 Cobitis arachthosensis Arachthos Greece Ex10 C12 55798 75 Cobitis arachthosensis Arachthos Greece Ex10 D4 55802 Cobitis arachthosensis Arachthos Greece Ex10 D1 55799 100 Cobitis arachthosensis Arachthos Greece Ex10 D2 55800 Cobitis arachthosensis Arachthos Greece Ex10 D3 55801 95 Cobitis hellenica Louros Greece Ex10 D11 55804 Cobitis hellenica Louros Greece Ex10 E1 55806 Cobitis hellenica Louros Greece Ex10 E2 55807 Cobitis hellenica Louros Greece Ex10 D12 55805 Cobitis sp. Seyhan Turkey Ex39 E2 289 100 Cobitis sp. Seyhan Turkey Ex47 B7 289 79 Cobitis sp. Seyhan Turkey Ex47 B8 289 Cobitis sp. Glbasi Turkey Ex18 E2 982 Cobitis sp. Glbasi Turkey Ex18 E3 982 100 Cobitis sp. Glbasi Turkey Ex18 E4 982 Cobitis levantina Orontes Syria Ex52 F8 1214 100 Cobitis levantina Orontes Syria Ex52 F9 1214 Cobitis evreni Ceyhan Turkey Ex75 A2 300TK 100 Cobitis evreni Ceyhan Turkey Ex75 A1 299TK Cobitis trichonica Trichonis Greece DNADIV A4 D2162 Cobitis vardarensis Vardar Greece boldgr AUTH06-116 Cobitis vardarensis Pinios Greece Ex10 H2 55827 100 Cobitis vardarensis Vardar Greece boldgr AUTH07-117 Cobitis vardarensis Pinios Greece Ex10 H3 55822 Cobitis vardarensis Pinios Greece Ex10 H5 55824 100 Cobitis fahireae Madra Turkey Ex52 H11 308 95 Cobitis fahireae Madra Turkey Ex61WH21 C8 308 100 Cobitis sp. Sapanca Turkey WH07 F2 173 Cobitis sp. Sapanca Turkey WH07 F3 173 88 100 Cobitis puncticulata Apolyont Turkey Ex13 G4 180 Cobitis puncticulata Apolyont Turkey Ex45 D12 180 100 Cobitis splendens Kavukkavla Turkey Ex29 C1 882 Cobitis splendens Kavukkavla Turkey Ex29 C2 882 Cobitis bilineata Rhone France Ex61WH21 G8 53571 Cobitis sp. Skadar Montenegro Ex20 E1 G325-331 Cobitis sp. Buna Albania WH04 E12 Co26 100 Cobitis sp. Skadar Montenegro Ex20 D12 G325-331 Cobitis sp. Karadirek Turkey Ex19 C9 1520 Cobitis sp. Karadirek Turkey Ex19 C10 1520 100 Cobitis sp. BykMenderes Turkey Ex52 F12 1513 Cobitis sp. BykMenderes Turkey Ex52 F10 1513 Cobitis sp. BykMenderes Turkey Ex52 F11 1513 Cobitis sp. BykMenderes Turkey Ex31 H8 977 98 100 Cobitis strumicae Strymon Greece WH07 D11 55817 Cobitis strumicae Strymon Greece WH07 D12 55818 Cobitis punctilineata Strymon Greece Ex11 C5 55815 89 Cobitis punctilineata Strymon Greece Ex11 C6 55816 Cobitis punctilineata Strymon Greece Ex11 C3 55813 100 Cobitis punctilineata Strymon Greece Ex08 A9 50730 Cobitis punctilineata Strymon Greece Ex11 C4 55814 Cobitis punctilineata Strymon Greece Ex08 A10 50731 Cobitis calderoni Ebro Spain WH08 A3 AT13774 100 Cobitis calderoni Ebro Spain WH08 A5 AT13775 Cobitis calderoni Ebro Spain WH08 A2 AT13774 Cobitis calderoni Ebro Spain WH08 A4 AT13775 97 100 Cobitis maroccana Loukkos Morocco Ex20 H9 1906 Cobitis maroccana Loukkos Morocco Ex20 H10 1906 Cobitis maroccana Loukkos Morocco Ex20 H8 1906 100 Cobitis vettonica Tagus Spain Ex39 A1 AT2372 99 Cobitis vettonica Tagus Spain Ex39 A3 AT2374 99 Cobitis paludica Tagus Spain Ex32 F6 AT9859 Cobitis paludica Tagus Spain WH08 A6 AT9858 Cobitis paludica Tagus Spain Ex17 C9 AT8838 98 Cobitis vettonica Tagus Spain Ex32 F7 AT15824 Cobitis vettonica Tagus Spain Ex32 F8 AT15825 100 Misgurnus anguillicaudatus Po Italy Ex11 H9 51022 Misgurnus anguillicaudatus Po Italy Ex11 H10 51023 Misgurnus anguillicaudatus Po Italy Ex11 H7 51020 Cobitis zanandreai Fondi Italy WH07 H9 36G Cobitis zanandreai Fondi Italy WH07 H10 38G 100 Cobitis zanandreai Fondi Italy WH07 H8 12G Cobitis zanandreai Fondi Italy WH07 H7 11G 99 Cobitis zanandreai Fondi Italy WH07 H5 59157 Cobitis zanandreai Fondi Italy Ex32 E5 11G Cobitis ohridana Shushica Albania Ex34 H4 CO128 Cobitis ohridana Devolli Albania WH04 E9 A3014 99 78 Cobitis ohridana Shkumbini Albania Ex34 H5 CO129 Cobitis illyrica Imotzki BiH WH04 E8 A1991 Cobitis illyrica Imotzki BiH WH08 H9 A1988 91 100 Cobitis illyrica Imotzki BiH WH08 H11 A1990 Cobitis illyrica MostBlato BiH RS B2 A1975 84 Cobitis illyrica MostBlato BiH RS A9 A1960 Cobitis illyrica Imotzki BiH WH08 H10 A1989 Cobitis narentana Neretva BiH RS A1 A1699 92 Cobitis narentana Neretva BiH RS A7 A1928 98 98 Cobitis narentana Neretva BiH RS A2 A1700 Cobitis narentana Neretva BiH Ex21 B8 213 Cobitis dalmatina Cetina Croatia WH08 E9 G316-321 97 Cobitis dalmatina Cetina Croatia WH08 E10 G316-321 100 Cobitis jadovaensis Jadova Croatia Ex50 B7 VP2729 Cobitis jadovaensis Jadova Croatia Ex50 B8 VP2729 Cobitis bilineata Arno Italy WH08 F3 50341 Cobitis bilineata Po Italy WH07 C2 16 97 Cobitis bilineata Po Italy WH07 C3 16 Cobitis bilineata Rhone France Ex61WH21 G9 53571 Cobitis bilineata Arno Italy WH08 F2 50340 99 Cobitis bilineata Po Italy WH07 C7 16 Cobitis bilineata Po Italy WH07 C8 16 Cobitis bilineata Po Italy WH07 C4 16 79 Cobitis bilineata Po Italy WH07 C5 16 Cobitis bilineata Po Italy WH07 C6 16 Paramisgurnus dabryanus HM446343 Paramisgurnus dabryanus HM446342 100 Paramisgurnus dabryanus HM446339 90 Paramisgurnus dabryanus HM446340 100 Sabanejewia larvata Po Italy Ex03 F2 45874 Sabanejewia larvata Po Italy Ex03 F3 45875 Sabanejewia larvata Po Italy Ex03 F1 45873 Sabanejewia balcanica Vardar Greece Ex12 A12 55964 100 Sabanejewia balcanica Vardar Greece Ex12 A11 55962 Sabanejewia balcanica Vardar Greece Ex11 C7 55961 100 Sabanejewia balcanica Vardar Greece Ex11 C8 55963 Sabanejewia balcanica Vardar Greece Ex11 C9 55965 Sabanejewia balcanica Vardar Greece Ex11 C10 55966 Oxynoemacheilus bureschi Vardar Greece Ex12 B8 55910 Oxynoemacheilus bureschi Kossynthos Greece Ex12 C7 55906 Oxynoemacheilus bureschi Strymon Greece Ex09 H4 55730 Oxynoemacheilus bureschi Kossynthos Greece Ex12 C6 55905 Oxynoemacheilus bureschi Vardar Macedonia Ex32 D9 17832 100 Oxynoemacheilus bureschi Vardar Macedonia Ex32 D10 17839 Oxynoemacheilus bureschi Strymon Greece Ex09 H5 55731 Oxynoemacheilus bureschi Strymon Greece Ex11 B10 55907 99 Oxynoemacheilus bureschi Strymon Greece Ex11 B11 55908 83 Oxynoemacheilus bureschi Strymon Greece Ex11 B12 55909 Oxynoemacheilus pindus Devolli Albania Ex35 G11 A578 Oxynoemacheilus pindus Shkumbini Albania Ex34 H7 OX3 Oxynoemacheilus pindus Aoos Greece Ex11 B2 55911 100 Oxynoemacheilus pindus Aoos Greece Ex11 B3 55912 Oxynoemacheilus pindus Aoos Greece Ex11 B4 55913 Oxynoemacheilus pindus Aoos Greece Ex11 B5 55914 Oxynoemacheilus pindus Aoos Greece Ex11 B6 55915 Oxynoemacheilus pindus Gjanices Albania Ex34 H6 OX2 Seminemacheilus ispartaensis Egirdir Turkey Ex19 D8 1536 100 Seminemacheilus ispartaensis Egirdir Turkey Ex19 D9 1536 Seminemacheilus ispartaensis Egirdir Turkey Ex19 D7 1536 96 Seminemacheilus ispartaensis Aksehir Turkey Ex16 D8 1665 Seminemacheilus ispartaensis Aksehir Turkey Ex16 D7 1665 Seminemacheilus sp. Kirgz Turkey Ex14 C7 336 100 Seminemacheilus sp. Kirgz Turkey Ex14 C5 336 100 Seminemacheilus sp. Kirgz Turkey Ex14 C6 336 100 Seminemacheilus lendlii Seyhan Turkey Ex15 B3 956 100 Seminemacheilus lendlii Seyhan Turkey Ex15 B4 956 Oxynoemacheilus namiri Orontes Syria Ex45 F1 1144 Oxynoemacheilus leontinae Litani Lebanon Ex25 E5 2148 Oxynoemacheilus namiri Orontes Syria Ex15 F7 1144 Oxynoemacheilus leontinae Litani Lebanon Ex25 F2 2152 100 Oxynoemacheilus leontinae Litani Lebanon Ex25 E4 2148 Oxynoemacheilus namiri Orontes Syria Ex45 E12 1144 Oxynoemacheilus leontinae Litani Lebanon Ex25 E6 2148 Oxynoemacheilus namiri Orontes Syria Ex45 E11 1144 Oxynoemacheilus panthera Barada Syria Ex15 H8 1177 100 Oxynoemacheilus panthera Barada Syria Ex15 H9 1177 100 Oxynoemacheilus sp. Orontes Turkey Ex52 D6 391 Oxynoemacheilus sp. Orontes Turkey Ex52 D7 391 Oxynoemacheilus sp. Orontes Turkey Ex52 D5 391 Oxynoemacheilus sp. Seyhan Turkey Ex27 B6 369 88 Oxynoemacheilus sp. Ceyhan Turkey Ex29 C11 943 94 Oxynoemacheilus sp. Seyhan Turkey Ex27 B5 369 Oxynoemacheilus sp. Ceyhan Turkey Ex29 C12 943 100 Oxynoemacheilus ceyhanensis Ceyhan Turkey Ex19 B2 1395 Oxynoemacheilus ceyhanensis Ceyhan Turkey Ex19 B3 1395 Oxynoemacheilus galilaeus Muzarib Syria Ex18 G8 1240 100 Oxynoemacheilus galilaeus Muzarib Syria Ex18 G9 1240 99 Oxynoemacheilus insignis AlTammasiyyar Syria WH01 C1 1180 100 Oxynoemacheilus insignis AlTammasiyyar Syria WH07 F1 1180 100 Oxynoemacheilus sp. Orontes Syria Ex18 F2 1227 Oxynoemacheilus sp. Orontes Syria Ex18 F3 1227 98 Oxynoemacheilus seyhanicola Seyhan Turkey Ex16 C10 1492 Oxynoemacheilus sp. Orontes Syria Ex18 F3 1227 98 Oxynoemacheilus seyhanicola Seyhan Turkey Ex16 C10 1492 100 Oxynoemacheilus seyhanicola Seyhan Turkey Ex47 A1 318 100 Oxynoemacheilus sp. Soysalli Turkey Ex18 D1 970 Oxynoemacheilus sp. Soysalli Turkey Ex18 D2 970 Oxynoemacheilus evreni Ceyhan Turkey Ex16 C6 1486 100 Oxynoemacheilus evreni Ceyhan Turkey Ex16 C7 1486 Oxynoemacheilus seyhanicola Seyhan Turkey Ex47 A2 318 92 100 Oxynoemacheilus seyhanicola Seyhan Turkey Ex47 A4 318 Oxynoemacheilus hamwii Orontes Turkey Ex19 B6 1499 Oxynoemacheilus hamwii Orontes Turkey Ex47 A12 339 100 Oxynoemacheilus hamwii Orontes Turkey Ex47 A11 339 95 Oxynoemacheilus hamwii Orontes Turkey Ex47 B1 339 Oxynoemacheilus hamwii Orontes Turkey Ex47 B3 339 81 Oxynoemacheilus hamwii Orontes Turkey Ex47 B2 339 100 Oxynoemacheilus eregliensis Melendiz Turkey Ex31 D8 34 Oxynoemacheilus eregliensis Melendiz Turkey Ex31 D9 34 100 Oxynoemacheilus eregliensis Melendiz Turkey Ex31 D7 34 94 Oxynoemacheilus atili Beysehir Turkey Ex27 H8 43 94 Oxynoemacheilus atili Beysehir Turkey Ex27 H9 43 98 Oxynoemacheilus atili Beysehir Turkey Ex45 H8 1531 Oxynoemacheilus sp. Beysehir Turkey Ex40 G4 1573 Oxynoemacheilus sp. Beysehir Turkey Ex40 G5 1573 100 Oxynoemacheilus sp. Beysehir Turkey Ex40 G3 1573 81 Oxynoemacheilus sp. Beysehir Turkey Ex45 H7 1573 Oxynoemacheilus seyhanensis Seyhan Turkey Ex55 A5 1399 Oxynoemacheilus seyhanensis Seyhan Turkey Ex55 A1 1399 100 Oxynoemacheilus seyhanensis Seyhan Turkey Ex55 A4 1399 Oxynoemacheilus seyhanensis Seyhan Turkey Ex55 A3 1399 Oxynoemacheilus seyhanensis Seyhan Turkey Ex55 A2 1399 76 Oxynoemacheilus seyhanensis Seyhan Turkey Ex19 B5 1399 Oxynoemacheilus seyhanensis Seyhan Turkey Ex19 B4 1399 100 Oxynoemacheilus simavicus Simav Turkey Ex57 F10 51 Oxynoemacheilus simavicus Simav Turkey Ex57 F9 51 97 Oxynoemacheilus samanticus Seyhan Turkey Ex16 B4 1402 100 Oxynoemacheilus samanticus Seyhan Turkey Ex16 B5 1402 100 Oxynoemacheilus sp. Gediz Turkey Ex55 E2 273 Oxynoemacheilus sp. Gediz Turkey WH03 E12 273 Oxynoemacheilus phoxinoides Iznik Turkey Ex51 A3 379 Oxynoemacheilus phoxinoides Iznik Turkey Ex51 A4 379 Oxynoemacheilus phoxinoides Iznik Turkey Ex51 A2 379 100 Oxynoemacheilus phoxinoides Iznik Turkey Ex51 A1 379 Oxynoemacheilus angorae Ilgin Turkey Ex51 H2 1527 97 Oxynoemacheilus angorae Ilgin Turkey WH07 F9 1527 Oxynoemacheilus theophilii Bakir Turkey Ex19 B9 1511 100 Oxynoemacheilus theophilii Bakir Turkey Ex19 B10 1511 Oxynoemacheilus theophilii Evergetoulas Greece Ex09 B4 50832 Oxynoemacheilus theophilii Evergetoulas Greece Ex09 B5 50833 99 Oxynoemacheilus mediterraneus Kpr Turkey Ex19 F2 1561 Oxynoemacheilus mediterraneus Kpr Turkey Ex19 F3 1561 98 Oxynoemacheilus sp. Eber Turkey Ex19 C5 1518 99 Oxynoemacheilus sp. Eber Turkey Ex19 C6 1518 99 Oxynoemacheilus anatolicus Burdur Turkey Ex15 E10 1110 Oxynoemacheilus anatolicus Burdur Turkey Ex15 E11 1110 88 Oxynoemacheilus germencicus Gediz Turkey Ex27 B3 372 Oxynoemacheilus germencicus Gediz Turkey Ex27 B4 372 96 88 Oxynoemacheilus germencicus BykMenderes Turkey Ex14 D9 351 Oxynoemacheilus germencicus BykMenderes Turkey Ex14 D10 351 Oxynoemacheilus cinicus BykMenderes Turkey Ex19 B12 1514 98 Oxynoemacheilus cinicus BykMenderes Turkey Ex19 C1 1514 Oxynoemacheilus cinicus BykMenderes Turkey Ex19 B11 1514 Oxynoemacheilus mesudae Isikli Turkey Ex67 A12 38 Oxynoemacheilus mesudae Isikli Turkey Ex70 H10 1077 Barbatula quignardi Lez France boldfr FFFtag4261 Barbatula quignardi Lez France boldfr FFFtag4262 Barbatula quignardi Lez France boldfr FFFtag4260 Barbatula quignardi Rhone France Ex56 G11 34 93 Barbatula quignardi Rhone France Ex26 B6 55594 Barbatula quignardi Rhone France Ex56 G12 34 100 Barbatula quignardi Rhone France Ex26 B4 55592 Barbatula quignardi Rhone France Ex26 B5 55593 Barbatula quignardi Rhone France Ex26 B8 55596 Barbatula quignardi Agly France Ex32 D5 17608 99 Barbatula quignardi Rhone France Ex26 H10 55668 Barbatula sturanyi Ohrid Macedonia Ex41 A7 A2946 Barbatula sturanyi Ohrid Macedonia Ex41 A2 A2941 Barbatula sturanyi Ohrid Macedonia Ex41 A5 A2944 100 100 Barbatula sturanyi Ohrid Macedonia Ex41 A4 A2943 Barbatula sturanyi Ohrid Macedonia Ex41 A3 A2942 Barbatula sturanyi Ohrid Albania Ex36 H7 Bast1 Barbatula sturanyi Ohrid Macedonia Ex32 E3 17830 Barbatula sturanyi Ohrid Macedonia Ex32 E4 17830 Barbatula sturanyi Ohrid Macedonia Ex41 A6 A2945 Barbatula vardarensis Vardar Greece Ex33 C9 D2490 94 100 Barbatula vardarensis Vardar Macedonia Ex32 D8 17840 Barbatula vardarensis Vardar Greece Ex33 C8 D2489 Barbatula vardarensis Vardar Greece Ex33 C7 D2488 Barbatula vardarensis Vardar Macedonia Ex32 D7 17840 Barbatula zetensis Skadar Montenegro 3Skadar FSJF1044 81 Barbatula zetensis Skadar Montenegro 4Skadar FSJF1044 Barbatula zetensis Skadar Montenegro 6Skadar FSJF1044 100 Barbatula zetensis Skadar Montenegro Zeta1 FSJF1108 Barbatula zetensis Skadar Montenegro Zeta2 FSJF1108 Barbatula zetensis Skadar Montenegro 5Skadar FSJF1044

0.05 92 Aphanius fontinalis Yarisli Turkey Ex25 D6 MS4a Aphanius fontinalis Yarisli Turkey Ex25 D7 MS4b Aphanius fontinalis Aci Turkey Ex25 D8 MS5 95 Aphanius sureyanus Burdur Turkey Ex25 D4 MS3 87 Aphanius sureyanus Burdur Turkey Ex25 D5 MS3 99 Aphanius fontinalis Salda Turkey Ex39 H8 Ms9 Aphanius saldae Salda Turkey Ex25 D1 MS1 Aphanius fontinalis Salda Turkey Ex39 H7 Ms9 95 98 Aphanius saldae Salda Turkey Ex25 C12 MS1 Aphanius transgrediens Aci Turkey Ex25 D11 MS6 Aphanius transgrediens Aci Turkey Ex25 D10 MS6 Aphanius transgrediens Aci Turkey Ex25 D9 MS5 Aphanius transgrediens Aci Turkey Ex21 B10 1704 85 Aphanius transgrediens Aci Turkey Ex37 A1 59166 100 Aphanius transgrediens Aci Turkey Ex37 A2 59167 Aphanius transgrediens Aci Turkey Ex37 A3 59168 Aphanius transgrediens Aci Turkey Ex37 A4 59169 Aphanius iconii Egirdir Turkey Ex15 D5 1087 100 Aphanius iconii Egirdir Turkey Ex15 D6 1087 99 Aphanius maeandricus Isikli Turkey Ex19 D10 1537 Aphanius maeandricus Isikli Turkey Ex19 D11 1537 Aphanius maeandricus BykMenderes Turkey Ex61WH21 H9 54764 100 Aphanius maeandricus BykMenderes Turkey Ex61WH21 H10 54765 99 Aphanius maeandricus BykMenderes Turkey Ex61WH21 H11 54767 91 Aphanius meridionalis Sgt Turkey Ex51 A7 1102 Aphanius meridionalis Sgt Turkey Ex52 F2 1102 Aphanius meridionalis Sgt Turkey Ex51 A5 1102 100 Aphanius meridionalis Sgt Turkey Ex51 A6 1102 Aphanius meridionalis Sgt Turkey Ex52 F3 1102 91 Aphanius meridionalis Sgt Turkey Ex52 F4 1102 Aphanius meridionalis Sgt Turkey Ex52 F1 1102 100 Aphanius anatoliae Beysehir Turkey Ex14 G8 393 91 Aphanius anatoliae Beysehir Turkey Ex14 G9 393 Aphanius anatoliae Tuz Turkey Ex18 C6 964 100 Aphanius anatoliae Tuz Turkey Ex18 B9 957 Aphanius anatoliae Tuz Turkey Ex18 B12 961 96 Aphanius anatoliae Tuz Turkey Ex17 G8 167 93 Aphanius anatoliae Tuz Turkey Ex15 D1 1080 Aphanius anatoliae Tuz Turkey Ex15 C4 1064 Aphanius anatoliae Tuz Turkey Ex15 C3 1064 Aphanius villwocki Ilgin Turkey Ex19 C12 1526 100 Aphanius villwocki Ilgin Turkey Ex19 D1 1526 Aphanius danfordii Soysalli Turkey Ex18 C2 962 Aphanius almiriensis Meligou Greece Ex49 E12 1706 89 Aphanius almiriensis Meligou Greece Ex49 F1 1706 Aphanius almiriensis Meligou Greece Ex21 B11 1706 100 Aphanius almiriensis Meligou Greece Ex53 H5 53110 Aphanius almiriensis Kos Greece Ex60div H9 53564 Aphanius almiriensis Meligou Greece Ex53 H6 53111 Aphanius almiriensis Meligou Greece Ex21 B12 1706 Aphanius fasciatus Ston Croatia Ex49 F9 199 100 Aphanius fasciatus Ston Croatia Ex49 F11 199 Aphanius fasciatus Ston Croatia Ex49 F10 199 100 Aphanius fasciatus Ston Croatia Ex49 F12 199 Aphanius fasciatus Zahzah Tunisia Ex49 H1 1843 Aphanius fasciatus Zahzah Tunisia Ex49 H2 1843 Aphanius fasciatus Zahzah Tunisia Ex49 G12 1843 78 Aphanius fasciatus Zahzah Tunisia Ex49 G11 1843 99 Aphanius fasciatus Zahzah Tunisia Ex49 G10 1843 Aphanius fasciatus Zeroud Tunisia Ex21 E2 1842 Aphanius fasciatus Touggourt Algeria Ex21 F2 1853 Aphanius fasciatus Touggourt Algeria Ex21 F3 1853 Aphanius fasciatus Veneto Italy Ex49 G7 713 Aphanius fasciatus Comacchio Italy Ex49 G1 707 Aphanius fasciatus Comacchio Italy Ex49 G2 707 Aphanius fasciatus Comacchio Italy Ex49 G3 707 Aphanius fasciatus Comacchio Italy Ex49 G4 707 96 Aphanius fasciatus Comacchio Italy Ex49 G5 707 Aphanius fasciatus Veneto Italy Ex49 G6 713 Aphanius fasciatus Veneto Italy Ex49 G8 713 Aphanius fasciatus Veneto Italy Ex49 G9 713 Aphanius saourensis Mazzer Algeria Ex53 G6 53088 100 Aphanius saourensis Mazzer Algeria Ex53 G8 53090 Aphanius saourensis Mazzer Algeria Ex21 A9 2090 Aphanius saourensis Mazzer Algeria Ex21 A8 2090 Aphanius saourensis Mazzer Algeria Ex21 A7 2090 Aphanius baeticus Vega Spain Ex16 H1 AT1326 100 100 Aphanius baeticus Vega Spain Ex16 H2 AT1327 Aphanius baeticus Vega Spain Ex16 F8 AT1328 Aphanius baeticus Guadalquivir Spain Ex19 G6 HK04 Aphanius iberus MenorSea Spain Ex32 F9 AT5077 Aphanius iberus MenorSea Spain Ex32 F10 AT5078 Aphanius iberus Estartit Spain Ex19 H4 HK10 100 Aphanius iberus Estartit Spain Ex53 H8 53113 Aphanius iberus Muga Spain Ex19 G9 HK06 75 Aphanius iberus Muga Spain Ex19 G10 HK06 Aphanius iberus Muga Spain Ex19 H3 HK09 100 Aphanius apodus AinLila Algeria Ex19 F9 1700 Aphanius apodus AinLila Algeria Ex19 H5 HK11 Aphanius apodus AinLila Algeria Ex19 F8 1684 Aphanius richardsoni EinFeshka Israel Ex49 F5 Ms8 100 Aphanius richardsoni EinFeshka Israel Ex49 F6 Ms8 Aphanius richardsoni EinFeshka Israel Ex49 F4 MsAr2 100 Aphanius richardsoni EinFeshka Israel Ex49 F3 MsAr2 Aphanius richardsoni EinFeshka Israel Ex49 F2 MsAr2 100 Aphanius dispar AlQuam UnitedArabEmirates Ex49 H6 HK01 100 Aphanius dispar AlQuam United Arab Emirates Ex49 H5 HK01 Aphanius sirhani Azraq Jordan Ex39 H2 Ms7b 100 Aphanius sirhani Azraq Jordan Ex39 H3 Ms7c Aphanius mento Sanawbar Syria Ex47 F6 1153 Aphanius mento Sanawbar Syria Ex47 F8 1153 Aphanius mento Sanawbar Syria Ex47 F5 1153 Aphanius mento Orontes Syria Ex47 B6 1142 99 Aphanius mento Orontes Syria Ex47 B5 1142 Aphanius mento Orontes Syria Ex47 B4 1142 97 Aphanius mento Hawaiz Syria Ex18 E10 1153 96 Aphanius mento Sanawbar Syria Ex47 F7 1153 Aphanius mento Orontes Turkey Ex47 E8 300 100 Aphanius mento Orontes Turkey Ex47 E9 300 Aphanius mento Kirgz Turkey Ex13 H7 294 Aphanius mento Kirgz Turkey Ex13 H8 294 Aphanius mento Kirgz Turkey Ex47 A8 294 100 Aphanius mento Kirgz Turkey Ex47 A9 294 Aphanius mento Kirgz Turkey Ex47 A10 294 Aphanius mento Seyhan Turkey Ex14 E10 366 100 100 Aphanius mento Seyhan Turkey Ex14 E11 366 98 Aphanius mento Eregli Turkey Ex18 E1 980 Aphanius mento Eregli Turkey Ex19 G8 HK05 Aphanius mento Ceyhan Turkey Ex15 A4 942 Aphanius mento Arsuz Turkey Ex14 A3 302 93 99 Aphanius mento Arsuz Turkey Ex14 A4 302 77 Aphanius mento AlAshaAri Syria Ex49 F7 1223 Aphanius mento AlTammasiyyar Syria Ex18 G12 1244 97 Aphanius mento Barada Syria Ex18 H7 1318 95 Aphanius mento AlAshaAri Syria Ex49 F8 1223 95 Aphanius mento Litani Lebanon Ex25 E7 2150 88 Aphanius mento Litani Lebanon Ex25 E8 2150 Aphanius mento Orontes Syria Ex18 H11 1322 89 Aphanius mento Orontes Syria Ex47 G1 1322 Aphanius mento Orontes Syria Ex47 F12 1322 Aphanius mento Orontes Syria Ex47 G2 1322 Poecilia reticulata Rhine Germany Ex51 H5 53010 100 Poecilia reticulata Rhine Germany Ex51 H3 53008 84 Poecilia reticulata Rhine Germany Ex51 H6 53011 Poecilia reticulata Rhine Germany Ex51 H4 53009 Xiphophorus helleri aquaculture Germany Ex56 H11 53338 Gambusia holbrooki Acheron Greece Ex12 H6 55867 Gambusia holbrooki Acheron Greece Ex12 H7 55868 Gambusia holbrooki Bullent Spain Ex17 B9 AT12731 Gambusia holbrooki Bullent Spain Ex17 B12 AT12733 100 Gambusia holbrooki Jordan Syria Ex16 A12 1208 Gambusia holbrooki Vardar Greece boldgr AUTH06-79 Gambusia holbrooki Vardar Greece boldgr AUTH06-77 Gambusia holbrooki Vardar Greece boldgr AUTH06-78 99 Valencia hispanica Bullent Spain Ex45 C9 AT19409 Valencia hispanica Bullent Spain Ex45 C10 AT19413 100 Valencia hispanica Bullent Spain Ex17 C6 AT19409 Valencia hispanica Ebro Spain Ex53 F6 53076 Valencia hispanica Ebro Spain Ex82mx C9 53086 Valencia hispanica Ebro Spain Ex82mx C10 53087 Valencia sp. Pinios Greece Ex53 F9 53079 86 Valencia sp. Pinios Greece Ex53 F8 53078 99 100 Valencia sp. Pinios Greece Ex53 F7 53077 Valencia sp. Pinios Greece Ex49 H3 HK03 Valencia sp. Pinios Greece Ex49 H4 HK03 Valencia letourneuxi Acheron Greece Ex10 F1 59009 99 Valencia letourneuxi Korfu Greece Ex53 F11 53081 100 Valencia letourneuxi Korfu Greece Ex53 F12 53082 Valencia letourneuxi Korfu Greece Ex53 G1 53083 Valencia letourneuxi Albania SExS02 G7 2459 Valencia letourneuxi Korfu Greece Ex53 G2 53084 Fundulus heteroclitus EU524628 100 Fundulus heteroclitus EU524627 Fundulus heteroclitus EU524630 Fundulus heteroclitus EU524629 Esox lucius Rhone France Ex61WH21 F5 51604 Esox lucius HM563693 Esox lucius Brenta Italy HM563699 94 Esox lucius HM563702 Esox lucius Volvi Greece boldgr AUTH07-145 86 Esox lucius Volvi Greece boldgr AUTH07-146 Esox lucius HM563689 Esox lucius HM563701 Esox lucius Po Italy Ex40 E12 50984 77 Esox lucius Po Italy Ex40 F1 50985 Esox lucius HM563690 Esox cisalpinus HM563705 Esox cisalpinus HM563696 Esox cisalpinus HM563704 100 Esox cisalpinus HM563706 Esox cisalpinus Garda Italy Ex56 H7 2323 Esox cisalpinus HM563700 Esox cisalpinus Garda Italy Ex56 H6 2321 Esox cisalpinus Garda Italy Ex56 H5 2320 Esox cisalpinus HM563703 Esox cisalpinus HM563695 Esox cisalpinus HM563698 Esox cisalpinus HM563694 82 Esox cisalpinus HM563707 Esox cisalpinus HM563692 Esox cisalpinus HM563688 Esox cisalpinus HM563691 Esox cisalpinus HM563697 Esox cisalpinus Garda Italy Ex56 H4 2322 Esox cisalpinus Po Italy Ex17 E8 59045 Esox cisalpinus Po Italy Ex17 E9 59047 Esox cisalpinus Po Italy Ex17 E10 47310 Esox cisalpinus Po Italy Ex17 E9 59047 Esox cisalpinus Po Italy Ex17 E10 47310 Esox cisalpinus Po Italy Ex17 E11 47311 Esox cisalpinus Po Italy Ex40 F2 50986 Acipenser baerii Yenisei Russia GQ328801 97 Acipenser baerii Lena Russia GQ328793 Acipenser baerii Lena Russia GQ328795 100 Acipenser baerii Lena Russia GQ328797 Acipenser baerii Selenga Russia GQ328799 Acipenser naccarii Po Italy SCEx02 H3 51488 Acipenser naccarii Po Italy SCEx02 G11 51484 83 Acipenser naccarii Po Italy SCEx02 G12 51485 Huso huso FJ809715 86 Huso huso FJ809716 Huso huso FJ809717 100 Huso huso FJ809718 Huso huso FJ809719 99 Acipenser stellatus Danube Czech Republic HQ960585 Acipenser stellatus Danube Czech Republic HQ960586 Acipenser stellatus Danube Czech Republic HQ960587 100 Acipenser stellatus Danube Czech Republic HQ960588 Acipenser stellatus Danube Czech Republic HQ960589 Acipenser sturio Gironde France Ex47 C11 50000 100 Acipenser sturio Gironde France Ex47 C12 50001 Anguilla anguilla Fossa Calda Italy Ex12 H8 59040 Anguilla anguilla Mediterranean Italy Ex39 H9 59038 Anguilla anguilla Volvi Greece boldgr 247 Anguilla anguilla Volvi Greece boldgr 232 Anguilla anguilla Mediterranean Italy WH01 B4 59037 100 Anguilla anguilla Acheron Greece Ex12 H4 55758 Anguilla anguilla Acheron Greece Ex12 H5 55759 Anguilla anguilla Volvi Greece boldgr 272 100 Pterygoplichthys disjunctivus Fossa Calda Italy Ex12 H11 59120 Pterygoplichthys disjunctivus Fossa Calda Italy Ex37 C6 50925 Ameiurus melas Rhone France Ex56 G5 24 Ameiurus melas Rhone France Ex56 G6 24 99 Ameiurus melas Po Italy Ex03 A5 45877 Ameiurus melas Rhone France Ex26 B3 55591 Ameiurus melas Po Italy Ex03 A4 45876 100 Ameiurus melas Arno Italy Ex04 B3 50241 Ameiurus melas Mediterranean Italy Ex12 H10 59036 Ameiurus nebulosus Vistula Poland Ex42 A2 P271 100 Ameiurus nebulosus Vistula Poland Ex42 A3 P272 Mystus pelusius Tigris Turkey WH07 G8 1446 100 Mystus pelusius Tigris Turkey WH07 G9 1446 Clarias gariepinus Jordan Jordan Ex41 E6 SYR08-40 100 Silurus glanis Arno Italy Ex39 A10 50243 Silurus glanis Arno Italy Ex39 A11 50244 Silurus glanis Arno Italy Ex39 A9 50237 Silurus aristotelis Acheloos Greece Ex06 G2 50605 Silurus aristotelis Kifissos Greece Ex08 H3 50808 100 Silurus aristotelis Kifissos Greece Ex08 H4 50809 Silurus aristotelis Kifissos Greece Ex08 H5 50810 Silurus aristotelis Kifissos Greece Ex08 H6 50811 100 Silurus aristotelis Kifissos Greece Ex08 H7 50812 Silurus aristotelis Kifissos Greece Ex08 H8 50813 Silurus aristotelis Volvi Greece boldgr AUTH07-173 Silurus aristotelis Volvi Greece boldgr AUTH07-174 Silurus aristotelis Volvi Greece boldgr AUTH07-175 Mugil cephalus JQ060534 Mugil cephalus JQ060536 Mugil cephalus JQ060533 Mugil cephalus JQ060532 Mugil cephalus Mediterranean Morocco Ex22 E4 1939 100 Mugil cephalus Mediterranean Greece Ex06 B6 50573 Mugil cephalus Mediterranean Greece Ex06 B5 50572 Mugil cephalus Mediterranean Greece Ex06 B4 50571 Mugil cephalus Kpr Turkey Ex19 E5 1540 Mugil cephalus Iqem Morocco Ex22 E7 1943 Mugil cephalus Kpr Turkey Ex19 E4 1540 Mugil cephalus Iqem Morocco Ex22 E8 1943 Mugil cephalus JQ060529 Lota lota Po Italy Ex40 E11 50966 Lota lota Rhone France Ex26 E9 55633 100 Lota lota Po Italy Ex40 E10 50965 Lota lota Po Italy Ex40 E9 50964 Lota lota Po Italy Ex40 E8 50963 Lota lota Po Italy Ex40 E7 50962 Chelon haematocheilus China Sea China JN242619 Chelon haematocheilus China Sea China JN242620 94 Chelon haematocheilus China Sea China JN242618 Chelon haematocheilus China Sea China JN242617 Chelon haematocheilus China Sea China JN242616 100 Chelon haematocheilus China Sea China JN242615 Chelon haematocheilus Korea HM180700 Chelon haematocheilus Russia EU392238 Chelon haematocheilus Russia EU392236 Chelon haematocheilus Russia EU392237 Chelon abu Orontes Turkey Ex51 D3 2012-1060 100 Chelon abu Orontes Turkey Ex51 D2 2012-1060 Chelon abu Orontes Turkey Ex51 C12 2012-1060 99 Chelon abu Orontes Turkey Ex51 D1 2012-1060 89 Chelon abu Tigris Turkey JQ060447 98 Chelon sp. Mediterranean Lebanon Ex60div C12 LC3 Chelon carinatus Red Sea Egypt FN600159 100 Chelon carinatus Turkey JQ623947 Chelon ramada HM208840 Chelon ramada Zerga Morocco JQ060480 87 Chelon ramada Iqem Morocco Ex22 D11 1935 Chelon ramada Kpr Turkey Ex19 E6 1540 Chelon ramada Istanbul Turkey Ex51 B10 2012-1058 100 Chelon ramada Iqem Morocco Ex22 D10 1935 92 Chelon ramada Iqem Morocco Ex22 D6 1931 Chelon ramada Goulette Tunisia JQ060479 88 Chelon ramada HM208839 Chelon auratus Goulette Tunisia JQ060456 Chelon auratus Agadir Morocco JQ060457 Chelon auratus HM208835 Chelon auratus Mediterranean Greece Ex06 A7 50562 Chelon auratus Mediterranean Greece Ex06 A6 50561 Chelon auratus Mediterranean Greece Ex06 A3 50558 99 99 Chelon auratus Istanbul Turkey Ex51 D7 2012-1044 Chelon auratus HM208836 Chelon auratus Mediterranean Greece Ex06 A9 50564 Chelon auratus Mediterranean Greece Ex06 A8 50563 Chelon auratus Istanbul Turkey Ex51 A8 2012-1059 Chelon auratus Mediterranean Greece Ex06 A5 50560 Chelon auratus Khnifiss Morocco JQ060459 Chelon auratus Istanbul Turkey Ex51 A9 2012-1059 86 Chelon auratus Khnifiss Morocco JQ060460 90 Chelon saliens Goulette Tunisia JQ060483 Chelon saliens HM208833 Chelon saliens Istanbul Turkey Ex51 C7 2012-1057 100 Chelon saliens Mediterranean Greece Ex06 A10 50565 Chelon saliens Mediterranean Greece Ex06 A1 50556 Chelon saliens Mediterranean Greece Ex06 A2 50557 Chelon labrosus Iqem Morocco Ex22 A7 1892 Chelon labrosus Mediterranean Greece Ex06 A11 50566 Chelon labrosus Mediterranean Greece Ex06 B3 50570 Chelon labrosus Iqem Morocco Ex22 A8 1892 100 Chelon labrosus Iqem Morocco Ex22 A9 1892 Chelon labrosus Mediterranean Greece Ex06 A12 50567 Chelon labrosus Mediterranean Greece Ex06 B1 50568 Chelon labrosus Mediterranean Greece Ex06 B2 50569 Chelon labrosus Mediterranean Greece Ex06 B10 50577 Chelon labrosus Mediterranean Greece Ex37 E4 50575

0.1 Knipowitschia radovici Neretva Croatia Ex52 C10 210 Knipowitschia mrakovcici Krka Croatia Ex49 E2 734 Knipowitschia radovici Neretva Croatia Ex52 C7 210 Knipowitschia panizzae Morinjska Montenegro Ex05 E9 731 Knipowitschia panizzae Krka Croatia Ex01 G9 727 Knipowitschia mrakovcici Krka Croatia WH07 C1 734 Knipowitschia panizzae Bacina Croatia Ex01 C5 207 Knipowitschia radovici Neretva Croatia Ex52 C9 210 Knipowitschia panizzae Morinjska Montenegro Ex05 E6 731 Knipowitschia mrakovcici Krka Croatia WH07 B12 734 Knipowitschia radovici Neretva Croatia Ex52 C8 210 Knipowitschia mrakovcici Krka Croatia Ex49 D12 734 Knipowitschia mrakovcici Krka Croatia Ex49 D10 734 Knipowitschia panizzae Neretva Croatia Ex01 H2 736 97 Knipowitschia panizzae Vransko Croatia Ex01 G5 721 Knipowitschia panizzae Morinjska Montenegro Ex05 E7 731 Knipowitschia panizzae Cetina Croatia Ex01 H4 740 Knipowitschia mrakovcici Krka Croatia Ex49 D11 734 Knipowitschia panizzae Morinjska Montenegro Ex05 E8 731 Knipowitschia panizzae Zrmanja Croatia Ex01 G3 718 Knipowitschia mrakovcici Krka Croatia Ex49 E1 734 Knipowitschia radovici Neretva Croatia Ex52 C11 210 Knipowitschia panizzae Neretva Croatia Ex01 G11 732 90 Knipowitschia mrakovcici Krka Croatia Ex49 E3 734 Knipowitschia radovici Neretva Croatia Ex52 C12 210 Knipowitschia panizzae Bolsena Italy Ex47 G9 59095 Knipowitschia panizzae Bolsena Italy Ex47 G10 59096 Knipowitschia panizzae Bolsena Italy Ex47 G6 59092 Knipowitschia panizzae Bolsena Italy Ex47 G11 59097 Knipowitschia panizzae Bolsena Italy Ex47 G7 59093 Knipowitschia panizzae Bolsena Italy Ex47 G8 59094 Knipowitschia montenegrina Skadar Montenegro Ex05 D8 725 84 Knipowitschia montenegrina Skadar Montenegro Ex05 D9 725 Knipowitschia montenegrina Skadar Montenegro Ex05 D11 725 99 Knipowitschia montenegrina Skadar Montenegro Ex05 D12 725 Knipowitschia montenegrina Skadar Montenegro Ex05 D10 725 Knipowitschia caucasica Evros Greece Ex63 B6 C1919 93 Knipowitschia caucasica Evros Greece Ex63 B7 C1918 99 Knipowitschia caucasica Beysehir Turkey Ex18 E5 983 Knipowitschia caucasica Euboia Greece Ex63 A12 D3180 Knipowitschia caucasica Euboia Greece Ex63 A11 D3181 93 Knipowitschia caucasica Euboia Greece Ex63 B1 D3179 Knipowitschia caucasica Apolyont Turkey Ex13 F5 164 99 Knipowitschia caucasica Apolyont Turkey Ex13 F6 164 Knipowitschia ephesi Kck Menderes Turkey Ex13 E12 111 Knipowitschia caucasica Apolyont Turkey Ex39 D12 179 85 Knipowitschia caucasica Volvi Greece boldgr AUTH07-155 Knipowitschia caucasica Volvi Greece Ex05 B10 669 99 Knipowitschia caucasica Apolyont Turkey Ex13 G2 179 Knipowitschia ephesi KckMenderes Turkey Ex13 F1 111 Knipowitschia ephesi KckMenderes Turkey Ex13 F2 111 Knipowitschia milleri Acheron Greece Ex45 G11 55847 Knipowitschia milleri Acheron Greece Ex45 G12 55848 Knipowitschia milleri Acheron Greece Ex45 G10 55846 Knipowitschia milleri Acheron Greece Ex47 D10 55744 99 Knipowitschia milleri Acheron Greece Ex47 D9 55743 Knipowitschia milleri Acheron Greece Ex47 D8 55742 Knipowitschia milleri Acheron Greece Ex47 D11 55745 97 Knipowitschia milleri Acheron Greece Ex45 H1 55849 Knipowitschia milleri Acheron Greece Ex47 D12 55746 Knipowitschia milleri Trichonis Greece Ex06 E2 50586 Knipowitschia milleri Trichonis Greece Ex06 E3 50586 99 Knipowitschia milleri Trichonis Greece Ex06 E1 50586 Knipowitschia byblisia Dalaman Turkey Ex47 F3 409 Knipowitschia byblisia Dalaman Turkey Ex47 F4 409 83 Knipowitschia byblisia Dalaman Turkey Ex47 F2 409 Knipowitschia byblisia Dalaman Turkey Ex47 E12 409 99 Knipowitschia byblisia Dalaman Turkey Ex18 B3 409 86 Knipowitschia byblisia Dalaman Turkey Ex14 B4 326 Knipowitschia byblisia Dalaman Turkey Ex14 B5 326 Knipowitschia byblisia Dalaman Turkey Ex47 E11 409 78 Knipowitschia byblisia Dalaman Turkey Ex47 F1 409 85 88 Knipowitschia thessala Pinios Greece Ex11 C11 55900 94 99 Knipowitschia thessala Pinios Greece Ex11 C12 55901 Knipowitschia thessala Pinios Greece Ex11 D1 55902 Knipowitschia mermere Madra Turkey Ex18 B2 402 Knipowitschia mermere Gediz Turkey Ex13 F7 175 Knipowitschia mermere Gediz Turkey Ex13 F8 175 Knipowitschia mermere Gediz Turkey Ex13 F9 175 99 Knipowitschia mermere Gediz Turkey Ex17 H4 275 Knipowitschia mermere Gediz Turkey Ex17 H5 275 Knipowitschia mermere Gediz Turkey Ex17 H6 275 Economidichthys trichonis Trichonis Greece DNADIV A2 D2146 Economidichthys trichonis Trichonis Greece DNADIV A3 D2147 99 Economidichthys trichonis Trichonis Greece DNADIV A8 Etri Economidichthys trichonis Trichonis Greece WH08 E8 55844 Economidichthys trichonis Trichonis Greece WH08 E7 55840 Economidichthys pygmaeus Louros Greece Ex10 E4 55829 98 Economidichthys pygmaeus Louros Greece Ex10 E6 55831 Economidichthys pygmaeus Kifissos Greece Ex10 G9 55833 99 Economidichthys pygmaeus Kifissos Greece Ex10 G11 55835 Economidichthys pygmaeus Louros Greece Ex10 E5 55830 Economidichthys pygmaeus Louros Greece Ex10 E7 55832 99 Orsinigobius punctatissimus Stella Italy WH07 F6 705 Orsinigobius punctatissimus Stella Italy WH08 A7 59019 87 Orsinigobius croaticus Neretva Croatia WH07 B6 210 96 Orsinigobius croaticus Neretva Croatia WH07 B9 210 99 Orsinigobius croaticus Neretva Croatia WH07 B11 702 Orsinigobius croaticus Neretva BiH Ex47 E4 212 99 Orsinigobius croaticus Neretva BiH Ex01 C10 212 Orsinigobius croaticus Neretva BiH Ex47 E6 212 Orsinigobius croaticus Neretva BiH WH07 B8 212 91 Pomatoschistus marmoratus Po Italy Ex05 H4 730 Pomatoschistus marmoratus Po Italy Ex47 A7 730 88 Pomatoschistus marmoratus Po Italy Ex47 A6 730 99 Pomatoschistus marmoratus Po Italy Ex47 A5 730 Pomatoschistus marmoratus Cetina Croatia Ex01 H5 741 99 Pomatoschistus marmoratus Po Italy Ex05 H2 697 Pomatoschistus88 microps Montpellier France Ex25 E1 55679 Pomatoschistus microps Montpellier France Ex25 E2 55680 Pomatoschistus microps Montpellier France Ex25 D12 55678 99 Pomatoschistus microps Montpellier France Ex25 E3 55681 Ninnigobius canestrinii Tiber Italy Ex04 G12 59062 96 Ninnigobius canestrinii Tiber Italy Ex04 H1 59071 Ninnigobius canestrinii Ricica Croatia Ex01 H7 745 Ninnigobius montenegrensis Skadar Montenegro Ex05 E1 726 Ninnigobius montenegrensis Skadar Montenegro Ex05 E4 726 99 Ninnigobius montenegrensis Skadar Montenegro Ex05 E2 726 99 Ninnigobius montenegrensis Skadar Montenegro Ex05 E5 726 Ninnigobius montenegrensis Skadar Montenegro Ex05 E3 726 Ninnigobius sp. Krka Croatia Ex01 G10 728 81 Ninnigobius sp. Bacina Croatia Ex01 C7 209 Ninnigobius sp. Neretva BiH Ex47 E7 212 99 Ninnigobius sp. Neretva Croatia Ex01 G12 733 Ninnigobius sp. Neretva Croatia Ex01 H3 737 Cottus scaturigo Timavo Italy WH04 A3 59081 Cottus scaturigo Timavo Italy WH04 A4 59073 Cottus scaturigo Timavo Italy Ex04 H10 59072 Cottus scaturigo Timavo Italy Ex04 H8 59080 Cottus gobio Rhone France boldfr FFFtag4420 Cottus gobio Rhone France WH03 D4 55609 87 Cottus gobio Zrmanja Croatia Ex39 D3 1771 Cottus gobio Zrmanja Croatia Ex39 D4 1771 Cottus gobio Po Italy Ex03 C1 46077 Cottus gobio Po Italy Ex39 A12 50444 Cottus cf. rondeleti Rhone France boldfr FFFtag10713 99 Cottus cf. rondeleti Rhone France boldfr FFFtag10824 Cottus petiti Lez France boldfr FFFtag4342 Cottus petiti Lez France boldfr FFFtag4266 Cottus petiti Lez France boldfr FFFtag4337 Cottus cf. rondeleti Rhone France Ex26 C7 55608 Cottus cf. rondeleti Rhone France boldfr FFFtag4185 Cottus rondeleti Herault France boldfr FFFtag4268 Cottus rondeleti Herault France boldfr FFFtag4270 Cottus gobio Stella Italy Ex39 F2 716 Cottus gobio Stella Italy Ex39 F3 716 99 Cottus gobio Stella Italy Ex39 F4 716 Cottus gobio Roya France Ex75 E5 CMK17623 99 Pungitius hellenicus Sperchios Greece Ex12 B7 55960 Pungitius hellenicus Sperchios Greece Ex12 B6 55959 86 Pungitius hellenicus Sperchios Greece Ex12 B5 55958 Pungitius platygaster SouthBug Ukraine Ex73 G9 781 99 Pungitius platygaster SouthBug Ukraine Ex73 G10 782 99 Pungitius platygaster SouthBug Ukraine Ex73 G11 783 Pungitius laevis France Ex26 F11 55646 99 Pungitius laevis France Ex26 G1 55648 Gasterosteus sp. Liri Italy Ex43 H5 59104 Gasterosteus sp. Rhone France Ex56 G9 32 Gasterosteus sp. Rhone France Ex26 D3 55616 Gasterosteus sp. Rhone France Ex56 G8 32 Gasterosteus sp. Rhone France Ex56 G7 32 Gasterosteus sp. Brague France Ex26 C10 55611 75 Gasterosteus sp. France Ex26 D2 55615 Gasterosteus sp. Liri Italy Ex43 H2 59101 Gasterosteus sp. Liri Italy Ex43 H3 59102 Gasterosteus sp. France Ex26 D6 55619 Gasterosteus sp. Fondi Italy Ex12 E3 59082 Gasterosteus sp. Liri Italy Ex43 H3 59102 Gasterosteus sp. France Ex26 D6 55619 Gasterosteus sp. Fondi Italy Ex12 E3 59082 Gasterosteus sp. Fondi Italy Ex12 E5 59084 Gasterosteus sp. Liri Italy Ex43 H7 59106 Gasterosteus sp. Fondi Italy Ex12 E4 59083 Gasterosteus sp. Liri Italy Ex43 H6 59105 99 Gasterosteus sp. Liri Italy Ex43 H4 59103 Gasterosteus sp. Dalaman Turkey Ex18 D5 974 Gasterosteus sp. Dalaman Turkey Ex18 D6 974 93 Gasterosteus sp. Dalaman Turkey WH01 F5 974 Gasterosteus sp. Aris Greece Ex09 G11 55740 Gasterosteus sp. Aris Greece Ex09 G12 55741 Gasterosteus sp. Aris Greece Ex09 G10 55739 76 Gasterosteus sp. Aris Greece Ex09 G9 55738 Gasterosteus sp. Aris Greece Ex09 G8 55737 Gasterosteus sp. Mirna Croatia Ex01 G6 722 Gasterosteus sp. Pamisos Greece Ex12 G12 55869 Gasterosteus sp. Sperchios Greece Ex11 D2 55880 Gasterosteus sp. Sperchios Greece Ex11 D3 55881 Gasterosteus sp. Sperchios Greece Ex11 D4 55882 85 Gasterosteus sp. Neretva Croatia Ex01 B12 202 Gasterosteus sp. Neretva Croatia Ex01 C3 205 Gasterosteus sp. Neretva BiH Ex01 C9 211 Gasterosteus sp. Louros Greece Ex10 D8 55873 Gasterosteus sp. Louros Greece Ex10 D9 55874 Gasterosteus sp. Louros Greece Ex10 D7 55872 Gasterosteus sp. Louros Greece Ex10 D6 55871 Gasterosteus sp. Louros Greece Ex10 D5 55870 Gasterosteus sp. Buna Albania Ex34 H1 A596 Gasterosteus sp. Acheron Greece Ex10 F2 55875 Gasterosteus sp. Acheron Greece Ex10 F3 55876 Gasterosteus sp. Acheron Greece Ex10 F4 55877 Gasterosteus sp. Acheron Greece Ex10 F5 55878 Gasterosteus sp. Acheron Greece Ex10 F6 55879 Platichthys flesus Rhone France WH07 G3 HP25 Padogobius bonelli Stella Italy Ex04 H2 59074 Padogobius bonelli Stella Italy Ex04 H4 59076 Padogobius bonelli Stella Italy Ex04 H6 59078 Padogobius bonelli Tiber Italy Ex04 D6 50405 Padogobius bonelli Tiber Italy Ex04 F8 59057 Padogobius bonelli Arno Italy Ex04 C4 50326 Padogobius bonelli Arno Italy Ex04 C2 50324 99 Padogobius bonelli Arno Italy Ex36 A8 50261 Padogobius bonelli Ricica Croatia Ex01 H6 744 Padogobius bonelli Zrmanja Croatia Ex20 D3 G301 Padogobius bonelli Zrmanja Croatia Ex20 D4 G302-307 Padogobius bonelli Zrmanja Croatia Ex20 D6 G302-307 99 Proterorhinus semilunaris Evros Greece WH12 A3 FSJF1762 Proterorhinus semilunaris Strymon Greece WH12 A9 FSJF1678 Proterorhinus semilunaris Evros Greece WH12 A2 FSJF1762 99 Neogobius fluviatilis Simav Turkey Ex16 E4 1678 77 Neogobius fluviatilis Simav Turkey Ex16 E5 1678 Neogobius fluviatilis Apolyont Turkey Ex13 G1 179 Neogobius melanostomus Istanbul Turkey Ex51 C10 2012-1061 Neogobius nigricans Arno Italy Ex36 A11 50274 Neogobius nigricans Arno Italy Ex36 A12 50274 99 Neogobius nigricans Tiber Italy Ex36 B1 59010 Neogobius nigricans Tiber Italy Ex36 B2 59011 99 Neogobius nigricans Tiber Italy Ex36 B3 59012 Neogobius nigricans Tiber Italy Ex36 B4 59013 Neogobius nigricans Arno Italy Ex36 A10 50274 Neogobius nigricans Tiber Italy Ex36 B5 59014 99 Dicentrachus labrax Atlantic Sweden NRM49173 Dicentrachus labrax Atlantic Sweden NRM56985 99 Syngnathus abaster Po Italy Ex18 B8 729 Syngnathus sp. Mediterranean Algeria Ex21 F1 1851 99 Atherina sp. Orontes Turkey Ex14 E5 361 Atherina sp. Orontes Turkey Ex14 E6 361 Atherina sp. Trichonis Greece Ex10 B12 55763 Atherina sp. Trichonis Greece Ex10 B10 55761 99 Atherina sp. Trichonis Greece Ex10 B9 55760 99 99 Atherina sp. Trichonis Greece Ex10 B11 55762 Atherina sp. Trichonis Greece Ex10 C1 55764 Atherina pontica Sapanca Turkey Ex13 E7 106 Atherina boyeri Mediterranean Algeria Ex20 E8 1852 Atherina boyeri Sebou Morocco Ex20 H11 1907 Atherina boyeri Bolsena Italy Ex47 G12 59098 99 Atherina boyeri Bolsena Italy Ex47 H1 59099 Atherina boyeri Bolsena Italy Ex47 H2 59100 Atherina boyeri Tiber Italy Ex47 D5 59107 Atherina boyeri Tiber Italy Ex47 D6 59108 Atherina boyeri Tiber Italy Ex47 D7 59109

0.05 Scardinius_acarnanicus_Trichonis_Greece_Ex06_F11_50602 Scardinius_acarnanicus_Trichonis_Greece_Ex06_G1_50604 Scardinius_acarnanicus_Trichonis_Greece_Ex06_F8_50599 Scardinius_acarnanicus_Trichonis_Greece_Ex06_E8_50587 Scardinius_acarnanicus_Trichonis_Greece_Ex06_E7_50587 Scardinius_acarnanicus_Trichonis_Greece_Ex06_E6_50587 Scardinius_acarnanicus_Trichonis_Greece_Ex06_E5_50587 Scardinius_acarnanicus_Trichonis_Greece_Ex06_E4_50587 83 Scardinius_acarnanicus_Trichonis_Greece_Ex06_C9_50581 Scardinius_acarnanicus_Trichonis_Greece_Ex06_C8_50581 Scardinius_acarnanicus_Trichonis_Greece_Ex06_C7_50581 Scardinius_acarnanicus_Trichonis_Greece_Ex06_C6_50581 Scardinius_acarnanicus_Louros_Greece_Ex09_F10_50886 Scardinius_acarnanicus_Louros_Greece_Ex09_F8_50884 Scardinius_acarnanicus_Trichonis_Greece_Ex06_C10_50581 Scardinius_acarnanicus_Trichonis_Greece_Ex06_F7_50598 Scardinius_acarnanicus_Trichonis_Greece_Ex06_F9_50600 Scardinius_acarnanicus_Trichonis_Greece_Ex06_F12_50603 Scardinius_acarnanicus_Trichonis_Greece_Ex06_F10_50601 Scardinius_sp._Ohrid_Albania_Ex34_C2_D3805 Scardinius_erythrophthalmus_Sapanca_Turkey_Ex13_E4_100 90 Scardinius_erythrophthalmus_Apolyont_Turkey_Ex13_C1_64 Scardinius_erythrophthalmus_Sapanca_Turkey_Ex13_C2_66 Scardinius_erythrophthalmus_Sapanca_Turkey_Ex13_B11_61 Scardinius_erythrophthalmus_Apolyont_Turkey_Ex13_B12_64 99 Scardinius_erythrophthalmus_Volvi_Greece_boldgr_AUTH07-134 Scardinius_erythrophthalmus_Volvi_Greece_boldgr_AUTH07-135 Scardinius_erythrophthalmus_Volvi_Greece_boldgr_AUTH07-133 Scardinius_erythrophthalmus_Volvi_Greece_Ex05_B4_663 Scardinius_erythrophthalmus_Strymon_Greece_Ex05_B9_668 Scardinius_erythrophthalmus_Volvi_Greece_Ex05_B6_663 Scardinius_graecus_Yliki_Greece_Ex05_A3_457 Scardinius_graecus_Yliki_Greece_Ex08_G4_50797 Scardinius_graecus_Yliki_Greece_Ex08_G5_50798 Scardinius_graecus_Yliki_Greece_Ex08_G6_50799 99 Scardinius_graecus_Yliki_Greece_Ex08_H9_50814 Scardinius_graecus_Yliki_Greece_Ex08_H10_50815 Scardinius_graecus_Yliki_Greece_Ex08_H11_50816 99 Scardinius_graecus_Yliki_Greece_Ex08_G3_50796 99 Scardinius_knezevici_Skadar_Montenegro_Ex05_D6_698 Scardinius_knezevici_Skadar_Montenegro_Ex05_D7_698 Scardinius_knezevici_Skadar_Montenegro_Ex05_D4_698 Scardinius_scardafa_Scanno_Italy_Ex05_G10_675 Scardinius_plotizza_Neretva_BiH_Ex34_C3_C1058 Scardinius_plotizza_Neretva_BiH_Ex34_C4_BH50 75 Scardinius_hesperidicus_Po_Italy_Ex03_F11_46000 Scardinius_hesperidicus_Po_Italy_Ex03_F12_46001 Scardinius_hesperidicus_Po_Italy_Ex03_G1_46002 86 Scardinius_hesperidicus_Po_Italy_Ex03_G3_46004 Scardinius_plotizza_Neretva_Croatia_Ex01_G4_720 Scardinius_scardafa_Scanno_Italy_Ex05_G12_675 Scardinius_hesperidicus_Po_Italy_Ex03_G2_46003 Scardinius_elmaliensis_Akcay_Turkey_Ex14_G10_394 99 Scardinius_elmaliensis_Akcay_Turkey_Ex14_G11_394 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_C11_378 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_H4_1535 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_C8_378 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex14_D2_342 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex14_D3_342 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_C9_378 86 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_H5_1535 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_H6_1535 83 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_H7_1535 99 Pseudophoxinus_egridiri_Egirdir_Turkey_Ex48_C10_378 Pseudophoxinus_sp._Beysehir_Turkey_Ex45_F7_1119 Pseudophoxinus_sp._Beysehir_Turkey_Ex15_F5_1119 99 Pseudophoxinus_sp._Beysehir_Turkey_Ex45_F6_1119 Pseudophoxinus_sp._Beysehir_Turkey_Ex45_F8_1119 Tropidophoxinellus_callensis_Soumam_Algeria_WH02_E3_1863 Tropidophoxinellus_callensis_Soumam_Algeria_Ex21_G6_1863 89 Tropidophoxinellus_callensis_Soumam_Algeria_Ex21_G7_1863 Tropidophoxinellus_callensis_Kebir_Tunisia_Ex21_E12_1850 Tropidophoxinellus_callensis_Kebir_Tunisia_Ex21_E11_1850 Tropidophoxinellus_callensis_Kebir_Tunisia_Ex21_D6_1838 Tropidophoxinellus_callensis_Soumam_Algeria_Ex21_A3_1874 99 Tropidophoxinellus_callensis_Soumam_Algeria_Ex21_F4_1854 Tropidophoxinellus_callensis_Soumam_Algeria_Ex21_F5_1854 Tropidophoxinellus_callensis_Kebir_Tunisia_Ex21_D11_1841 Tropidophoxinellus_chaignoni_Abid_Tunisia_Ex21_E10_1849 99 Tropidophoxinellus_chaignoni_Joumine_Tunisia_HM560301 99 Tropidophoxinellus_spartiacus_Evrotas_Greece_Ex11_F3_59007 96 Tropidophoxinellus_spartiacus_Evrotas_Greece_Ex11_F4_59008 Tropidophoxinellus_spartiacus_Evrotas_Greece_Ex11_F2_59006 Tropidophoxinellus_hellenicus_Pinios_Greece_Ex12_H2_59001 Tropidophoxinellus_hellenicus_Pinios_Greece_Ex12_H3_59002 99 Tropidophoxinellus_hellenicus_Pinios_Greece_Ex11_F12_59004 Tropidophoxinellus_hellenicus_Pinios_Greece_Ex11_G1_59005 99 Tropidophoxinellus_hellenicus_Pinios_Greece_Ex11_F11_59003 Tropidophoxinellus_hellenicus__Trichonis_Greece_Ex35_A3_D2136 Tropidophoxinellus_hellenicus__Trichonis_Greece_Ex35_A4_D2137 Tropidophoxinellus_hellenicus_Trichonis_Greece_Ex11_D10_55885 Tropidophoxinellus_hellenicus_Trichonis_Greece_Ex11_D8_55883 Tropidophoxinellus_hellenicus_Trichonis_Greece_Ex11_D9_55884 93 Delminichthys_jadovensis_Jadova_Croatia_Ex47_C4_435 87 Delminichthys_jadovensis_Jadova_Croatia_Ex47_C6_435 Delminichthys_jadovensis_Jadova_Croatia_Ex01_D12_435 Delminichthys_krbavensis_Krbava_Croatia_Ex01_D10_433 Delminichthys_ghetaldii_Vrijeka_BiH_Ex35_F6_A1793 99 Delminichthys_ghetaldii_Vrijeka_BiH_Ex35_F5_A1792 Delminichthys_ghetaldii_Ljubomir_BiH_Ex01_D8_431 Delminichthys_ghetaldii_Fatnicko_BiH_Ex35_F4_A1745 94 Delminichthys_ghetaldii_Fatnicko_BiH_Ex35_F3_A1744 Delminichthys_adspersus_Neretva_Croatia_Ex01_E2_437 Delminichthys_adspersus_Trebizat_BiH_Ex35_F2_A2026 Delminichthys_krbavensis_Krbava_Croatia_Ex60div_G5_433 98 Pelasgus_minutus_Ohrid_Macedonia_RS_C4_A2904 99 Pelasgus_minutus_Ohrid_Macedonia_RS_C3_A2878 Pelasgus_minutus_Ohrid_Macedonia_RS_C2_A2877 Pelasgus_minutus_Morinjska_Montenegro_Ex05_C1_443 Pelasgus_prespensis_Prespa_Greece_Ex11_B1_55927 93 Pelasgus_prespensis_Prespa_Albania_WH04_F4_D300 99 78 Pelasgus_prespensis_Prespa_Albania_WH04_F5_D305 91 Pelasgus_laconicus_Evrotas_Greece_Ex11_F6_55917 Pelasgus_laconicus_Evrotas_Greece_Ex11_F7_55918 94 Pelasgus_laconicus_Evrotas_Greece_Ex11_F5_55916 Pelasgus_laconicus_Alfios_Greece_Ex11_E8_55941 99 Pelasgus_laconicus_Alfios_Greece_Ex11_E10_55943 Pelasgus_laconicus_Alfios_Greece_Ex11_E7_55940 Pelasgus_laconicus_Alfios_Greece_Ex11_E9_55942 Pelasgus_marathonicus_Sperchios_Greece_Ex10_G7_55922 Pelasgus_marathonicus_Sperchios_Greece_Ex10_G8_55924 99 Pelasgus_marathonicus_Sperchios_Greece_Ex10_G6_55920 Pelasgus_marathonicus_Sperchios_Greece_Ex10_F11_55926 Pelasgus_marathonicus_Sperchios_Greece_Ex10_F9_55923 Pelasgus_marathonicus_Sperchios_Greece_Ex10_F8_55921 Pelasgus_marathonicus_Sperchios_Greece_Ex10_F7_55919 Pelasgus_stymphalicus_Trichonis_Greece_Ex10_C11_55937 Pelasgus_stymphalicus_Pinios_Greece_Ex12_G5_55938 99 Pelasgus_stymphalicus_Pinios_Greece_Ex12_C9_55934 Pelasgus_stymphalicus_Pinios_Greece_Ex12_C11_55936 Pelasgus_stymphalicus_Pinios_Greece_Ex12_G6_55939 Pelasgus_sp._Acheron_Greece_Ex48_F11_55891 99 Pelasgus_sp._Acheron_Greece_Ex48_G1_55893 Pelasgus_sp._Acheron_Greece_Ex48_F9_55889 99 Pelasgus_sp._Acheron_Greece_Ex48_F8_55888 Pelasgus_sp._Acheron_Greece_Ex48_F10_55890 Pelasgus_sp._Acheron_Greece_Ex48_F12_55892 Pelasgus_sp._Kalamas_Greece_Ex10_H9_55931 Pelasgus_sp._Kalamas_Greece_Ex10_H11_55933 99 Pelasgus_sp._Kalamas_Greece_Ex10_H7_55929 Pelasgus_sp._Kalamas_Greece_Ex10_H6_55928 Pelasgus_sp._Kalamas_Greece_Ex10_H10_55932 Pelasgus_sp._Kalamas_Greece_Ex10_H8_55930 98 Pelasgus_thesproticus_Arachthos_Greece_Ex10_E12_55948 Pelasgus_thesproticus_Arachthos_Greece_Ex10_E8_55944 Pelasgus_thesproticus_Arachthos_Greece_Ex10_E9_55945 99 Pelasgus_thesproticus_Arachthos_Greece_Ex10_E10_55946 Pelasgus_thesproticus_Arachthos_Greece_Ex10_E11_55947 Pelasgus_thesproticus_Arachthos_Greece_Ex48_F7_55886 99 Leuciscus_vorax_Homs_Syria_Ex57_H8_1224 Leuciscus_vorax_Homs_Syria_Ex57_H7_1224 99 Leuciscus_aspius_Strymon_Greece_boldgr_na_182 Leuciscus_aspius_Strymon_Greece_boldgr_na_184 84 Leuciscus_aspius_Strymon_Greece_boldgr_na_185 Leuciscus_burdigalensis_Agly_France_boldfr_FFFtag4110 99 Leuciscus_burdigalensis_Agly__France_Ex75_D10_CMK17613 Leuciscus_burdigalensis_Agly_France_boldfr_FFFtag4059 Leuciscus_burdigalensis_Aude_France_boldfr_na_FFFtag4322 99 88 Leuciscus_idus_Rhone_France_Ex26_E7_55631 Leuciscus_idus_Rhone_France_Ex26_E8_55632 Leuciscus_leuciscus_Ouche_France_boldfr_FFFtag4400 98 Leuciscus_leuciscus_Rhone_France_boldfr_na_FFFtag4418 Leuciscus_leuciscus_Lez_France_boldfr_FFFtag4318 Pachychilon_pictum_Ohrid_Macedonia_WH08_D9_A2861 99 Pachychilon_pictum_Devolli_Albania_WH08_D7_A555 Pachychilon_pictum_Bistrica_Albania_WH08_D5_D400 Pachychilon_pictum_Kiri_Albania_WH08_D8_D115 Pachychilon_pictum_Mat_Albania_WH08_D6_Bos749 Pachychilon_macedonicum_Vardar_Greece_boldgr_AUTH06-37 Pachychilon_macedonicum_Vardar_Greece_boldgr_AUTH06-39 Pachychilon_macedonicum_Vardar_Greece_boldgr_AUTH06-36 99 Pachychilon_macedonicum_Pinios_Greece_Ex05_A9_658 Pachychilon_macedonicum_Pinios_Greece_Ex05_A10_658 Pachychilon_macedonicum_Pinios_Greece_Ex05_A11_658 Pachychilon_macedonicum_Pinios_Greece_Ex08_D11_50768 Pachychilon_macedonicum_Pinios_Greece_Ex08_D12_50769 99 Pachychilon_macedonicum_Pinios_Greece_Ex08_E1_50770 Pachychilon_macedonicum_Pinios_Greece_Ex08_E2_50771 Pachychilon_macedonicum_Pinios_Greece_Ex08_F1_50782 Pachychilon_macedonicum_Pinios_Greece_Ex08_F2_50783 Pachychilon_macedonicum_Pinios_Greece_Ex08_F3_50784 Pachychilon_macedonicum_Vardar_Macedonia_Ex35_G12_A2829 Pachychilon_macedonicum_Pinios_Greece_Ex08_F3_50784 Pachychilon_macedonicum_Vardar_Macedonia_Ex35_G12_A2829 Alburnoides_thessalicus_Pinios_Greece_Ex08_E8_50777 Alburnoides_thessalicus_Pinios_Greece_Ex10_C10_55757 Alburnoides_thessalicus_Pinios_Greece_Ex10_C9_55756 Alburnoides_thessalicus_Pinios_Greece_Ex10_C8_55755 Alburnoides_thessalicus_Pinios_Greece_Ex08_E7_50776 Alburnoides_thessalicus_Pinios_Greece_Ex08_E5_50774 99 Alburnoides_thessalicus_Pinios_Greece_Ex08_E4_50773 Alburnoides_thessalicus_Pinios_Greece_Ex08_E3_50772 Alburnoides_thessalicus_Pinios_Greece_Ex08_E9_50778 Alburnoides_thessalicus_Pinios_Greece_Ex08_E6_50775 Alburnoides_thessalicus_Vardar_Greece_Ex48_E2_55860 Alburnoides_thessalicus_Vardar_Greece_Ex48_E4_55862 99 Alburnoides_thessalicus_Vardar_Greece_Ex12_B2_55754 76 Alburnoides_thessalicus_Aliakmon_Greece_Ex12_A6_55748 Alburnoides_thessalicus_Aliakmon_Greece_Ex12_A7_55749 Alburnoides_thessalicus_Aliakmon_Greece_Ex12_A8_55750 Alburnoides_thessalicus_Vardar_Greece_Ex48_E3_55861 Alburnoides_thessalicus_Vardar_Greece_Ex48_E5_55863 Alburnoides_strymonicus_Strymon_Greece_Ex12_A10_55752 Alburnoides_strymonicus_Strymon_Greece_Ex48_E8_55855 99 99 Alburnoides_strymonicus_Strymon_Greece_Ex12_A9_55751 Alburnoides_strymonicus_Strymon_Greece_Ex48_E7_55854 Alburnoides_strymonicus_Strymon_Greece_Ex48_E6_55853 Alburnoides_sp._Sperchios_Greece_Ex12_G9_55747 88 Alburnoides_bipunctatus_Rhone_France_Ex26_A5_55581 99 Alburnoides_bipunctatus_Rhone_France_Ex56_G4_23 Alburnoides_bipunctatus_Rhone_France_Ex56_G3_23 75 Alburnoides_ohridanus_Ohrid_Albania_Ex35_C12_D3748 Alburnoides_ohridanus_Ohrid_Albania_RS_F2_D3743 99 Alburnoides_ohridanus_Erzen_Albania_Ex35_D2_A338 85 Alburnoides_ohridanus_Ohrid_Albania_RS_F1_D3742 Alburnoides_ohridanus_Erzen_Albania_Ex35_D1_A337 93 Alburnoides_ohridanus_Erzen_Albania_Ex35_D3_A339 Alburnoides_devolli_Devolli_Albania_Ex35_B5_A2973 Alburnoides_devolli_Devolli_Albania_Ex35_B6_A2974 94 Alburnoides_prespensis_Prespa_Greece_boldgr_274 Alburnoides_fangfangae_Osumi_Albania_Ex35_C9_D635 Alburnoides_fangfangae_Osumi_Albania_Ex35_C10_D636 Alburnoides_fangfangae_Osumi_Albania_Ex35_C6_D337 98 Alburnoides_fangfangae_Osumi_Albania_Ex35_C4_D335 Alburnoides_fangfangae_Osumi_Albania_Ex35_C7_D338 Alburnoides_prespensis_Prespa_Greece_boldgr_273 Alburnoides_devolli_Devolli_Albania_Ex35_B2_A2970 Alburnoides_prespensis_Prespa_Greece_Ex07_F12_50698 Alburnoides_devolli_Devolli_Albania_Ex35_A12_A2968 Alburnoides_devolli_Devolli_Albania_Ex35_B1_A2969 Alburnoides_devolli_Devolli_Albania_Ex35_B3_A2971 Alburnoides_devolli_Devolli_Albania_Ex35_B4_A2972 Alburnoides_fangfangae_Osumi_Albania_Ex35_C8_D634 Alburnoides_prespensis_Prespa_Greece_Ex07_G1_50699 Alburnoides_prespensis_Prespa_Greece_Ex07_G2_50700 Alburnoides_prespensis_Prespa_Greece_boldgr_275 Abramis_brama_Rhone_France_Ex26_A4_55580 Abramis_brama_Volvi_Greece_boldgr_AUTH07-120 Abramis_brama_Volvi_Greece_boldgr_AUTH07-118 Abramis_brama_Volvi_Greece_Ex35_B9_D2575 99 Abramis_brama_Volvi_Greece_Ex35_B8_D2574 Abramis_brama_Sapanca_Turkey_Ex13_D9_90 Abramis_brama_Rhone_France_Ex26_A1_55577 Abramis_brama_Volvi_Greece_boldgr_AUTH07-119 Abramis_brama_Tiber_Italy_Ex40_D2_50353 Abramis_brama_Tiber_Italy_Ex40_D1_50352 Acanthobrama_lissneri_Jordan_Syria_WH02_B5_1219 79 Acanthobrama_telavivensis_Yarkon_Israel_Ex33_D6_1121 Acanthobrama_lissneri_Jordan_Syria_WH01_F8_1230 99 Acanthobrama_lissneri_Jordan_Syria_Ex18_F8_1230 Mirogrex_terrasanctae_Muzarib_Syria_Ex18_G6_1237 Mirogrex_terrasanctae_Muzarib_Syria_Ex18_G5_1237 Acanthobrama_orontis_Ceyhan_Turkey_WH09_A10_1481 Acanthobrama_orontis_Ceyhan_Turkey_Ex53_A7_1481 Acanthobrama_orontis_Seyhan_Turkey_Ex53_A4_395 98 Acanthobrama_orontis_Seyhan_Turkey_Ex53_A3_395 Acanthobrama_orontis_Seyhan_Turkey_Ex53_A2_395 Acanthobrama_orontis_Ceyhan_Turkey_Ex53_A11_1481 99 Acanthobrama_orontis_Ceyhan_Turkey_Ex53_A10_1481 Acanthobrama_orontis_Ceyhan_Turkey_Ex53_A9_1481 Acanthobrama_orontis_Ceyhan_Turkey_Ex53_A8_1481 Acanthobrama_orontis_Seyhan_Turkey_Ex53_A5_395 98 Acanthobrama_orontis_Seyhan_Turkey_Ex53_A6_395 99 Mirogrex_terrasanctae_Tiberias_Israel_WH08_B9_1122 Mirogrex_terrasanctae_Tiberias_Israel_WH08_B10_1122 Mirogrex_terrasanctae_Tiberias_Israel_Ex51_G11_1122 Blicca_bjoerkna_Rhone_France_WH03_C12_FRHP-024 Vimba_vimba_Köprü_Turkey_WH07_F10_1563 Vimba_vimba_Köprü_Turkey_WH07_F11_1563 Vimba_vimba_Biga_Turkey_Ex13_G10_270 Vimba_mirabilis_BüyükMenderes_Turkey_Ex37_H10_1071 Vimba_melanops_Vardar_Greece_Ex37_E9_50755 Vimba_melanops_Vardar_Greece_WH08_E1_50754 99 Vimba_melanops_Strymon_Greece_boldgr_187 Vimba_melanops_Strymon_Greece_boldgr_188 Vimba_melanops_Strymon_Greece_boldgr_183 Vimba_melanops_Volvi_Greece_boldgr_AUTH07-127 Vimba_melanops_Volvi_Greece_boldgr_AUTH07-128 Vimba_melanops_Volvi_Greece_boldgr_AUTH07-129 Vimba_melanops_Evros_Bulgaria_WH08_C12_C1331 Vimba_vimba_Gönen_Turkey_Ex48_C7_314 Vimba_vimba_Gönen_Turkey_Ex48_C4_314 Vimba_melanops_Evros_Bulgaria_WH08_D2_C1333 Vimba_melanops_Evros_Bulgaria_WH08_C11_C1320 Vimba_melanops_Evros_Bulgaria_WH08_D1_C1332 Alburnus_qalilus_Sanawbar_Syria_Ex15_F12_1151 99 Alburnus_qalilus_Kabir_Syria_Ex19_A2_1327 Alburnus_qalilus_Hawaiz_Syria_Ex19_A4_1327 99 Alburnus_qalilus_Hawaiz_Syria_Ex19_A3_1327 Alburnus_qalilus_Hawaiz_Syria_Ex15_G1_1154 Alburnus_cf.qalilus_Orontes_Turkey_Ex14_G6_389 99 Alburnus_cf.qalilus_Orontes_Turkey_Ex14_G7_389 Alburnus_adanensis_Seyhan_Turkey_Ex56_F8_2329 83 Alburnus_adanensis_Seyhan_Turkey_Ex56_F6_2329 99 Alburnus_adanensis_Seyhan_Turkey_Ex56_F7_2329 Alburnus_kotschyi_Arzus_Turkey_RS_G6_286 Alburnus_kotschyi_Ceyhan_Turkey_Ex16_B12_1479 Alburnus_kotschyi_Ceyhan_Turkey_Ex16_C1_1479 99 Alburnus_orontis_Orontes_Turkey_Ex27_B9_312 Alburnus_orontis_Orontes_Turkey_RS_G10_312 99 Alburnus_baliki_Ilica_Turkey_Ex27_B11_311 Alburnus_baliki_Aksu_Turkey_Ex19_F1_1554 99 Alburnus_baliki_Ilica_Turkey_RS_G9_311 99 Leucaspius_delineatus__France_Ex26_E6_55630 Leucaspius_delineatus_Izmit_Turkey_Ex51_A11_2012-1045 84 99 Anaecypris_hispanica_Guadiana_Spain_Ex17_A6_AT8436 Anaecypris_hispanica_Guadiana_Spain_Ex17_A7_AT8435 Anaecypris_hispanica_Guadiana_Spain_Ex17_A5_AT8437 99 Anaecypris_punica_Mejerda_Tunisia_WH11_C8_1836 Anaecypris_punica_Mejerda_Tunisia_WH11_C6_1836 Anaecypris_punica_Mejerda_Tunisia_WH11_C7_1836 Alburnus_volviticus_Volvi_Greece_Ex05_A2_245 Alburnus_istanbulensis_Gönen_Turkey_RS_G8_307 Alburnus_vistonicus_Vistonis_Greece_Ex05_A1_235 Alburnus_istanbulensis_Menderes_Turkey_RS_G4_272 Alburnus_istanbulensis_Biga_Turkey_RS_G7_298 Alburnus_carinatus_Simav_Turkey_Ex19_B8_1507 Alburnus_carinatus_Simav_Turkey_Ex19_B7_1507 Alburnus_volviticus_Volvi_Greece_boldgr_AUTH07-124 Alburnus_istanbulensis_Menderes_Turkey_Ex27_C5_272 Alburnus_istanbulensis_Menderes_Turkey_Ex27_C6_272 Alburnus_volviticus_Volvi_Greece_boldgr_AUTH07-125 99 99 Alburnus_volviticus_Volvi_Greece_boldgr_AUTH07-126 Alburnus_istanbulensis_Istanbul_Turkey_Ex51_C9_2012-1047 Alburnus_carinatus_Simav_Turkey_RS_G2_126 Alburnus_battalgilae_Gediz_Turkey_RS_F11_77 Alburnus_attalus_Bakir_Turkey_Ex27_G6_83 Alburnus_attalus_Bakir_Turkey_Ex27_G8_83 Alburnus_attalus_Bakir_Turkey_Ex27_G7_83 96 Alburnus_battalgilae_Gediz_Turkey_Ex27_G12_77 Alburnus_battalgilae_Gediz_Turkey_Ex27_G11_77 Alburnus_attalus_Bakir_Turkey_Ex31_C8_1675 Alburnus_attalus_Bakir_Turkey_RS_G1_83 99 Alburnus_demiri_Büyük_Menderes_Turkey_RS_G5_274 91 Alburnus_demiri_Dalaman_Turkey_WH03_E10_332 Alburnus_demiri_Zeytinkoy_Turkey_albg_A2_109 Alburnus_arborella_Tiber_Italy_Ex04_G11_59061 Alburnus_cf._maximus_Lugano_Switzerland_CE_nn_2737 Alburnus_arborella_Po_Italy_Ex12_F6_59032 Alburnus_arborella_Po_Italy_Ex12_F5_59031 78 Alburnus_arborella_Po_Italy_Ex12_F4_59030 Alburnus_arborella_Po_Italy_Ex12_F3_59029 Alburnus_arborella_Po_Italy_Ex12_F2_59028 Alburnus_arborella_Po_Italy_Ex12_F1_59027 Alburnus_arborella_Po_Italy_Ex12_E12_59026 Alburnus_arborella_Po_Italy_Ex03_A3_45968 Alburnus_arborella_Po_Italy_Ex03_A2_45967 Alburnus_arborella_Po_Italy_Ex03_A1_45966 Alburnus_arborella_Arno_Italy_Ex04_A5_50225 Alburnus_arborella_Arno_Italy_Ex04_A3_50225 Alburnus_arborella_Volturno_Italy_Ex04_G3_59064 Alburnus_neretvae_Neretva_Croatia_RS_G12_693 Alburnus_neretvae_Neretva_Croatia_Ex01_G1_693 Alburnus_albidus_Alento_Italy_Ex12_E6_59020 Alburnus_albidus_Alento_Italy_Ex12_E7_59021 Alburnus_albidus_Alento_Italy_Ex12_E8_59022 79 97 Alburnus_albidus_Alento_Italy_Ex43_H9_59086 99 Alburnus_albidus_Alento_Italy_Ex43_H10_59087 Alburnus_albidus_Alento_Italy_Ex43_H11_59088 Alburnus_albidus_Alento_Italy_Ex43_H8_59085 Alburnus_arborella_Zrmanja_Croatia_Ex20_D9_G312 Alburnus_arborella_Garda_Italy_Ex21_B1_1200 99 Alburnus_arborella_Garda_Italy_Ex21_B2_1200 Alburnus_arborella_Cavata_Italy_Ex04_F11_59050 Alburnus_arborella_Cavata_Italy_Ex04_F12_59051 Alburnus_cf._maximus_Lugano_Switzerland_CE_nn_2738 Alburnus_arborella_Cavata_Italy_Ex04_F12_59051 Alburnus_cf._maximus_Lugano_Switzerland_CE_nn_2738 Alburnus_arborella_Arno_Italy_Ex04_A4_50225 Alburnus_belvica_Prespa_Greece_boldgr_293 Alburnus_belvica_Prespa_Greece_boldgr_294 Alburnus_belvica_Prespa_Greece_boldgr_295 Alburnus_belvica_Prespa_Greece_Ex07_F7_50693 99 Alburnus_belvica_Prespa_Greece_Ex07_F8_50694 Alburnus_belvica_Prespa_Greece_Ex07_F11_50697 Alburnus_hybrid_Prespa_Greece_Ex07_F10_50696 Alburnus_scoranza_Skadar_Montenegro_Ex47_C2_460 Alburnus_scoranza_Skadar_Montenegro_Ex47_C3_460 99 Alburnus_scoranza_Ohrid_Albania_RS_E12_D3738 Alburnus_scoranza_Ohrid_Albania_RS_E11_D3737 Alburnus_scoranza_Ohrid_Albania_RS_E10_D3736 Alburnus_scoranza_Aoos_Greece_Ex07_C6_50656 Alburnus_escherichii_Beysehir_Turkey_Ex49_E7_1067 Alburnus_escherichii_Beysehir_Turkey_Ex49_E10_1067 Alburnus_escherichii_Sakarya_Turkey_Ex49_H8_128 Alburnus_escherichii_Beysehir_Turkey_Ex49_E8_1067 Alburnus_escherichii_Beysehir_Turkey_Ex49_E11_1067 Alburnus_escherichii_Beysehir_Turkey_Ex49_E9_1067 Alburnus_nasreddini_Ilgin_Turkey_Ex39_E12_1097 Alburnus_nasreddini_Aksehir_Turkey_Ex17_G3_125 79 Alburnus_nasreddini_Aksehir_Turkey_Ex17_G2_125 Alburnus_nasreddini_Aksehir_Turkey_Ex17_G4_125 95 Alburnus_macedonicus_Vardar_Greece_boldgr_AUTH06-28 Alburnus_macedonicus_Vardar_Greece_boldgr_AUTH06-31 Alburnus_macedonicus_Vardar_Greece_boldgr_AUTH06-27 Alburnus_thessalicus_Vardar_Greece_Ex08_C4_50749 Alburnus_thessalicus_Vardar_Greece_Ex08_C8_50753 85 Alburnus_thessalicus_Pinios_Greece_Ex43_C8_2115 Alburnus_thessalicus_Pinios_Greece_Ex43_C9_2115 Alburnus_thessalicus_Vardar_Greece_Ex08_C5_50750 Alburnus_thessalicus_Vardar_Greece_Ex08_C6_50751 84 Alburnus_sp._Volvi_Greece_boldgr_AUTH07-152 Alburnus_sp._Volvi_Greece_boldgr_AUTH07-153 Alburnus_sp._Volvi_Greece_boldgr_AUTH07-151 98 Alburnus_sp._Strymon_Greece_boldgr_221 Alburnus_sp._Strymon_Greece_boldgr_219 Alburnus_sp.__Strymon_Greece_Ex43_C3_2111 Alburnus_sp._Strymon_Greece_boldgr_220 Alburnus_sp.__Strymon_Greece_Ex43_C2_2111 Alburnus_sp.__Volvi_Greece_Ex43_B1_2100 94 Alburnus_alburnus_Apolyont_Turkey_Ex31_E7_130 Alburnus_alburnus_Simav_Turkey_Ex31_C9_1677 Alburnus_alburnus_Apolyont_Turkey_Ex31_E6_130 Alburnus_alburnus_Sado_Portugal_WH05_C6_FSJF Alburnus_alburnus_Rhone_France_Ex26_A8_55584 Alburnus_alburnus_Rhone_France_Ex56_G10_28 Alburnus_alburnus_Sado_Portugal_WH05_C4_FSJF Alburnus_alburnus_Sado_Portugal_WH05_C8_FSJF Alburnus_alburnus_Agly__France_Ex75_D8_CMK17612 Alburnus_alburnus_Agly__France_Ex75_D9_CMK17612 Alburnus_alburnus_Sado_Portugal_WH05_C5_FSJF Alburnus_alburnus_Evros_Bulgaria_DNADIV_A10_C1322 Alburnus_alburnus_Evros_Bulgaria_DNADIV_A11_C1323 Alburnus_alburnus_Evros_Bulgaria_DNADIV_A12_C1337 Alburnus_alburnus_Evros_Bulgaria_DNADIV_B1_C1338 Alburnus_alburnus_Evros_Bulgaria_DNADIV_A9_C1321 Rutilus_rubilio_Tiber_Italy_Ex04_D12_50417 Rutilus_rubilio_Volturno_Italy_Ex04_G5_59066 Rutilus_rubilio_Tiber_Italy_Ex04_D4_50396 Rutilus_rubilio_Crati_Italy_Ex12_D9_59136 Rutilus_rubilio_Crati_Italy_Ex12_D8_59135 Rutilus_rubilio_Crati_Italy_Ex12_D7_59134 Rutilus_rubilio_Cavata_Italy_Ex04_E3_50425 Rutilus_rubilio_Arno_Italy_Ex04_A2_50198 Rutilus_rubilio_Arno_Italy_Ex04_A1_50199 Rutilus_rubilio_Arno_Italy_Ex04_B6_50283 Rutilus_rubilio_Arno_Italy_Ex04_B7_50284 Rutilus_rubilio_Tiber_Italy_Ex04_D5_50397 Rutilus_rubilio_Tiber_Italy_Ex04_E1_50418 99 Rutilus_rubilio_Volturno_Italy_Ex04_G4_59065 95 Rutilus_rubilio_Volturno_Italy_Ex04_G7_59068 Rutilus_rubilio_Arno_Italy_Ex04_B1_50239 Rutilus_rubilio_Volturno_Italy_Ex04_G6_59067 Rutilus_rubilio_Lazio_Italy_Ex04_G2_59063 77 Rutilus_rubilio_Cavata_Italy_Ex04_E2_50424 98 Rutilus_rubilio_Lazio_Italy_Ex04_G1_59059 Rutilus_rutilus_Sapanca_Turkey_Ex13_D5_85 Rutilus_rutilu