Zoosyst. Evol. 98 (1) 2022, 65-75 | DOI 10.3897/zse.98.73937 Gp Musee TOR BERLIN Pliocene-Pleistocene dispersal bring along low inter species diversity between Vimba species based on multilocus analysis Gokhan Kalayci' 1 Recep Tayyip Erdogan University, Faculty of Fisheries, Department of Basic Sciences, 53100 Rize, Turkey http://zoobank.org/A65F'15B9-00BD-4F 26-BA 1D-4B 1EEC4AB30F Corresponding author: Gékhan Kalayci (gokhan.kalayci@erdogan.edu.tr) Academic editor: Nicolas Hubert # Received 23 October 2021 # Accepted 15 February 2022 Published 25 February 2022 Abstract This study investigates phylogenetic and phylogeographic relationships of Vimba species using mitochondrial cytochrome b (cyt b) (1023 bp) and cytochrome c oxidase subunit I (COI) barcoding region (652 bp) genes. Ninety-one samples from 36 populations for the cyt b gene and 67 samples from 20 populations for the COI were analyzed. We identified 29 haplotypes and calculated overall haplotype diversity as Hd: 0.907 + 0.015 for cyt b. We also identified 13 COI haplotypes and calculated overall haplotype diversity as 0.826 + 0.026 for this marker. The phylogenetic analysis of Vimba species reveals the presence of four clades, based on concatenated cyt b and COI sequences. The first and second clade consist of Vimba vimba Western lineage, and Vimba vimba Caspian lineage, while the third and fourth clade consist of Vimba mirabilis and Vimba melanops. Based on haplotype network analyses and phylo- geographic inferences, the Vimba genus is monophyletic, and its species dispersed in the Pleistocene era. Key Words cyt b, genetic diversity, Phylogeography, Vimba bream, Vimba mirabilis Introduction As a member of the Leuciscidae family, the genus Vimba is distributed throughout almost all Eurasia and consists of three species: Vimba vimba, Vimba melanops, and Vim- ba mirabilis. V. vinba was initially described as Cyprinus vimba L. from several Swedish lakes in Scandinavia, the North Sea, coastal waters of Baltic Sea basins, and, subse- quently, after the description, it was also found in the Cas- pian, Black Sea, Marmara Sea basins, and the Rhine River. In Anatolia, V. vimba is distributed from the Marmara basin up to Biiyuk Menderes, Egirdir Lake, Koprticay and Esen rivers in the south, and Kizilirmak in the east. Vv melanops was described initially from the Meri¢ (Evros) River and its distribution now extends within the borders of Turkey, Greece, Bulgaria, and Macedonia in the North Aegean ba- sin from Meric to the Pinios River. The Anatolian endemic V. mirabilis was detected only in the type locality Biytik Menderes and two individuals in Bafa Lake in Southwest Anatolia (Bogutskaya 1997). According to Crivelli (1996), V. mirabilis is under threat of extinction due to the water intake from the basin for drinking and irrigation. Since then, a few taxonomic developments of the genus Vimba have occurred, like the subalpine Vimba lineage previously identified as Vimba elongata, but now consid- ered a synonym of Vimba vimba. Acanthobrama mirabilis was synonymized with Vimba vimba tenella by Durand et al. (2002), as A. mirabilis belongs to the Vimba clade. But, Perea et al. (2010) found Acanthobrama mirabilis is the synonym of V. mirabilis based on genetic evidence. Also, in some literature, V vimba, distributed 1n the Caspian Sea and Caspian bream, has been identified as Vimba persa (Hanfling et al. 2009; Naseka and Bogutskaya 2009; Cha- ichi et al. 201 1a, 2011b; Mohamadian et al. 2012). Globally, Vimba genus has been well studied in terms of growth parameters and its parasites, but genetic studies are more limited (Zardoya and Doadrio (1999), Durand et al. (2002), Hanfling et al. (2009), Perea et al. (2010), Trian- tafyllidis et al. (2011), Geiger et al. (2014), Schonhuth et al. (2018). In particular, Hanfling et al. (2009) discovered Copyright Kalayci G. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 66 the phylogeographic origin and colonization factor of V. vimba, but other studies have quite limited samples. Phylogeographic and phylogenetic studies have been made on some European freshwater fishes such as A/- burnoides, Barbus, Capoeta, Gobio, Squalius, Telestes and typically uncovered distinct patterns according to mito- chondrial cytochrome b (cyt b) and cytochrome c oxidase I (COI) genes. In order to study systematics and phylogeny of Leuciscidae and its congeners, cyt b and COI sequence analysis have been demonstrated to be useful DNA mark- ers. (Zardoya and Doadrio 1999; Durand et al. 2000, 2002; Tsigenopoulos et al. 2002; Levin et al. 2012; Bektas et al. 2017; Turan et al. 2018; Aksu and Bektas 2019). In European rivers, freshwater fishes have largely colo- nized the Black Sea upstream, up rivers such as the Dnie- per and the Danube. Freshwater fishes contain numerous lineages with genetic divergences representing separation over the past 2 Myr in this region. Several of these have clear geographic distributions and provide evidence of older Black Sea-Caspian Sea divergence. Interglacial and postglacial expansions also indicate colonization of West- ern Europe from numerous major refugia, particularly the Black Sea, Dnieper- Volga, Danube, Rhine— Rhone, Elbe, and other rivers. The influence of older water bodies such as the Ponto-Caspian Sea and recent great periglacial lakes and floods is also apparent (Hewitt 2004). While it frequently highlighted that the Pleistocene increased speciation rates, molecular data have recently revealed that species diverged in the Pleistocene and Pli- ocene in Europe (Hewitt 2000). In the current study, mtDNA sequences were used (cyt b, COI barcoding) to examine the biogeography of Vimba populations, applying a phylogeographic approach. The objective is to reveal the phylogenetic relationships and genetic diversity of Vimba species whose populations are currently decreasing. Materials and methods Sample collection, DNA extraction, and sequencing All currently recognized taxa of Vimba were includ- ed in our dataset. We sequenced 68 Vimba specimens collected from their distribution ranges in Turkey and further included sequences of 23 specimens from NCBI GenBank. Vimba species of Turkey were collected from 14 sampling sites of drainages of the Black and Aegean and Marmara seas, comprising type localities or type basins (Fig. 1, Table 1). The animals were experiment- ed with as per the Republic of Turkey animal welfare laws, guidelines, and policies, and was approved by the Republic of Turkey Recep Tayyip Erdogan University Local Ethics Committee for Animal Experiments (De- cision No: 2014/72). For faunal surveys, fishes were collected, surgical procedures were performed only for excision of fin clips after anesthesia with MS222, zse.pensoft.net Kalayci, G.: Phylogeography of Vimba species and fin clips were further preserved in 70% ethanol for genetic studies. In our experiments, none of the fishes were distressed by the experimental conditions. Spec- imens and tissue vouchers from Turkey were kept in DNA Collection and Zoology Museum, Faculty of Fisheries, Recep Tayyip ErdoSan University, Rize. Total DNA was extracted from fin clips via Qiacube automated DNA/RNA purification system by using Qiagen DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). Both the quality and quantity of DNA were checked on a NanoDrop 2000/c spectrophotom- eter (Thermo Scientific, Rockford, IL, USA) and 0.8% agarose gel electrophoresis. Cyt b (1023 bp) gene was amplified by the newly designed primer set B-cytbF (5'-GAAGAACCACCGTTGTWVTTCAAC-3') — and the B-cytbR (5'- CGGATTACAAGACCGATGC -3'), and COI barcoding gene (652 bp) was amplified by the FishFl (5'-TCAACCAACCACAAAGACATTG- GCAC-3') and FishR1 (5'-TAGACTTCTGGGTGGC- CAAAGAATCA-3') (Ward et al. 2005). PCR reactions were performed in a 50 uL reaction volume containing 5 uL 10x PCR buffer, 100 ng template DNA, 0.5 mM dNTPs mix, 3 mM MgCl, 0.5 mM of each primer, and 1 uL Taq DNA polymerase (New England Biolabs). The polymerization was carried out under the following conditions: initial denaturation at 95 °C for 30 s, dena- turation at 95 °C for 30s, annealing at 55 °C for 50 s for cyt b and 58 °C for 45 s for COI, extension at 68 °C for 1 min through 35 cycles, and a final extension at 68 °C for 5 min using Biorad T100 (Bio-Rad, Hercules, CA, USA) thermal cycler. The PCR products were run and visualized under UV Quantum-Capt ST4 system (Vil- ber Lourmat, France) also, purified and sequenced at Macrogen Europa Inc. (Amsterdam, Netherlands) (Tur- an et al. 2018). Genetic structure and phylogenetic analysis Clustal W (Thompson et al. 1994) algorithm was im- plemented in Bioedit v7.2.5 (Hall 1999) software to align cyt b and COI barcoding regions. Haplotype number (H), polymorphic and variable sites, haplo- type diversity (Hd), and nucleotide diversity (2) were computed for each species by DnaSP version 6.12.03 (Rozas et al. 2017) program. Nucleotide frequencies and transition/transversion rate were calculated by MEGA X (Kumar et al. 2018). Molecular Variance (AMOVA) was analyzed using the Arlequin v3.5.1.2 (Excoffier and Lischer 2010) software to calculate genetic variation among and within the groups. The haplotypes were submitted to the NCBI GenBank with accession numbers OK493404—OK493416 for cyt b OK444819-OK 444823 for COI barcoding region. Cyt b and COI sequences were concatenated (1675 bp) for further phylogenetic inferences. The TrN+G mod- el:-In = 2905.1890 was the best-fit nucleotide substi- tution model for the concatenated dataset according Zoosyst. Evol. 98 (1) 2022, 65-75 67 Table 1. List of sequences analyzed in this study with information on drainage, GenBank no, haplotype no, and country of origin. Locality no 33 34 Species Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba vimba Vimba melanops Vimba melanops Locality Kirmir stream, Ankara, Turkey Binkilig stream, Black sea, Istanbul, Turkey Koca stream, Balikesir, Turkey Koca ¢ay! stream, Canakkale, Turkey Aydinlar stream, Zonguldak, Turkey Aksu stream, DUzce, Turkey Buyuk Melen stream, Duzce, Turkey Yenice stream, Zonguldak, Turkey Cayagzi stream, Duzce, Turkey Iznik lake, Kocaeli, Turkey Suat Ugurlu Dam lake, Samsun, Turkey Curonian Lagoon, Baltic Sea basin, Lithuania Danube |, Black Sea basin Germany Elbe, North Sea basin, Germany Olandsan, Baltic Sea basin, Sweden Mondsee, Danube, Black Sea basin, Germany Eder, Weser, North Sea basin, Germany Tuzlov, Don, Sea of Azov basin, Russia Seversky, Donetz Don, Sea of Azov basin, Russia Samur, Caspian Sea basin Russia Sea of Azov, Sea of Azov basin Ukraine Kuban, Sea of Azov basin, Russia Tsymlyansk Reservoir, Don, Sea of Azov basin, Russia Dagomys, northeastern Black Sea basin, Russia Libechovka river, Elbe basin, Czech Republic Bashly-chai, Caspian Sea Basin, Russia Lake Sapanca, Sakarya, Turkey Gonen drainage, Canakkale, Turkey Egirdir Lake, Isparta, Turkey Biga drainage, Bursa, Turkey Koeprue drainage, Isparta, Turkey Danube R., Slovakia Inece stream, Kirklareli, Turkey Evros, Aegean Sea basin, Greece Coordinate 40°14'10.5'N, 32°15'41.2°E 41°22'48.3'N, 28°17'46.0°E 39°46'55.2'"N, 27°35'46.2"E 39°48'52.9'N, 27°13'46.1"E 41°13'47.1'N, 31°27-1.1.2°E 40°45'49.0"N, 30°57'43.0"E 40°50'08.0"N, 31°06'35.0"E 41°20'27.6'N, 32°04'40.8"E 41°05'27.2'N, 31°13'18.5"E 40°26'18.1'N, 29°38'03.5"E 41°01'52.4'N, 36°38'33.4"E 55°42'18.0'N, 20°00'00.0"E 48°53'24.0'N, 11°48'54.0°E 51°28'30.0'N, 11°58'01.2"E 60°20'24.0'N, 173 LOZE 47°49'40.8'N, 13°23'02.4°E 51°09'18.0'N, 8°54'07.2"E 49°58'58.8'N, 42°01'04.8"E 47°37'37.2'N, 40°53'16.8"E 41°52'26.8'N, 48°33'34.9"E 46°03'50.4'N, 36°36'54.0"E 45°11'56.4'N, 37°42'54.0"E 47°45'56.2'N, 42°49'18.8°E 43°40'01.2'N, 39°39'07.2"E 50°28'45.0'N, 14°29'07.5"E, (predict) 42°20'37.0'N, 48°05'30.8"E 40°43'14'N, 30°17'41"E 39°56'45.6'N, 27°20'13.2"E 38°02'00.9"N, 30°52'24.3"E (predict) 40°12'18.0'N, 29°05'13.2"E 37°31'40.8'N, 31°16'08.4"E 48°04'04.2'N, 17°09'53.2"E (predict) 41°41'34.0'N, 27°04'59.0"E 40°50'42.0'N, 26°O1'22.8°E N 4 1 cytb Genbank no OK493404 OK493404, OK493407, OK493408 OK493404, OK493406, OK493409 OK493405, OK493406 OK493410 OK493411 OK493411 OK493410 OK493410, OK493411 OK493406 0K493412, 0K493413 GQ279763 GQ279762 GQ279761 GQ279756 GQ279755, AY026405 GQ279755 GQ279751 GQ279751 GQ279765 GQ279754, GOQ279752 GQ279753 GQ279751 GQ279750 HM560237 GQ279765 AY026404 OK493415, OK493416 GQ279757 Haplotype no H1 H1,H4,H5 H1,H3,H6 H2,H3 H7 H8 H8 H7 H16 H27 H28 H26 20 col Genbank no Haplotype no H7 Reference OK444821 This study OK444820 H6 This study OK444820, OK444821 H6, H7 This study OK444820, OK444821 H6, H7 This study This study This study This study This study This study This study This study Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Durand et al. 2002 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Hanfling et al. 2009 Perea et al. 2010 HM560383 H6 Hanfling et al. 2009 Keskin & Atar 2013 H11,H12 Geiger et al. 2014 KC501853- KC501872 KJ554799, KJ554924 MW940905, MW940906 H13 H10, H6 Eren,H. (unp.) KJ554609 H6 Geiger et al. 2014 KJ554606, KJ554754 H6 Geiger et al. 2014 Durand et al. 2002 OK444819 H1 This study Hanfling et al. 2009 zse.pensoft.net 68 Kalayci, G.: Phylogeography of Vimba species Locality Species Locality Coordinate cytb col Reference no N Genbank no Haplotype N Genbank no Haplotype no no 35 Vimba Biserska River, Greece 40°55'08.5"N, 1 MG806725 H25 1 MG806910 H1 Schonhuth et al. melanops 26°11'48.0"E 2018 (predict) 36 Vimba River Strymon, Greece 41°43'53.5"N, 2 AFOQ90778, H24 H21 1 HM560382 H4 Zardoya & melanops 23°09'30.9"E HM560236 Doadrio, 1999 (predict) Perea et al 2010 37 Vimba Pinios, Aegean Sea basin, 39°39'57.6'N, 2 GQ279758, H23 H22 Hanfling et al. melanops Greece 22°14'02.4°E GQ279759 2009 38 Vimba Aliakmon R. Kaloneri, 40°17'26.5"N, 1 HM560235 H21 Perea et al. 2010 melanops Greece 21°28'17.9"E (predict) 39 Vimba Volvi lake, Greece 40°39'36.0"N, 1 AYO26403 H29 3 HQ600801- H1 Triantafyllidis melanops 23°32'24.0°E HQ600803 et al. 2011 Durand et al. 2002 40 Vimba Kerkini lake, Greece 41°06'36.0'N, 3 HQ600804- H1, H3, Triantafyllidis et melanops 23°03'00.0"E HQ600806 H4 al. 2011 4l Vimba Biserska R., Evros drainage, 41°51'18.0'N, 3 KJ554935, H1 Geiger et al. 2014 melanops Bulgaria 25°55'22:3'E KJ554568, KJ554722 42 Vimba Charmanlijskaja drainage, 41°53'20.4'N, 1 KJ554876 H2 Geiger et al. 2014 melanops Bulgaria 25°41'13.2°E 43 Vimba Vardar drainage, Greece 40°59'16.8'N, 2 KJ554926, H5 Geiger et al. 2014 melanops 22°33'28.8'E KJ554576 44 Vimba Akcay stream, Buyuk 37°45'34.0"N, 9 OK493414, H20 H19 4 0kK444822, H8,H9 ‘This study, Durand mirabilis Menderes, Aydin, Turkey 28°20'07.0"E AY026410 OK444823 et al. 2002 45 Vimba Cine stream, Buyuk 37°45'43.8'N, 4 OK493414 H20 1 KJ554739 H8 This study, Geiger mirabilis Menderes, Aydin, Turkey 27°50'13.1"E et al. 2014 Figure 1. Map showing the analyzed population stations. to Akaike information criterion (AIC) and Bayesian information criterion (BIC), as implemented in jMod- eltest v. 0.0.1 (Posada, 2008). Phylogenetic relation- ships among haplotypes and species were estimated by the maximum likelihood (ML) method using PhyML (Guindon et al. 2010) with 1000 bootstrap. Similarly, the Bayesian inference (BI) analysis was run in the MrBayes 3.1.2 software (Ronquist and Huelsenbeck 2003), using the Metropolis-coupled Markov chain zse.pensoft.net Monte Carlo (MCMC) algorithm from randomly gen- erated starting trees for five million generations with sampling taking place in every 1000 generations. The initial 25% of saved trees sampled in each MCMC run were discarded as burn-in. In all phylogenetic analyses, Blicca bjoerkna (AP009304) was selected as outgroup taxa. Haplotypes’ network inference was constructed through a median-joining (MJ) algorithm (Bandelt et al. 1999) implemented in Network 5.0.0.1 software Zoosyst. Evol. 98 (1) 2022, 65-75 (www.fluxus-engineering.com). MEGA X (Kumar et al. 2018) software was used to calculate pairwise genetic distance among the species using the Kimura 2-parameter substitution model (Kimura 1980). We estimated divergence times using StarBEAST (Ogilvie et al. 2017), which was implemented in BEAST 2.6.0 (Bouckaert et al. 2014). Compared with standard BEAST, StarBEAST better accounts for species trees vs. gene trees and intraspecific vs. interspecific events. Spe- cies were delimited according to the individual grouping recovered by previous phylogenetic analyses. Thus, the analysis was conducted with 5 groups of individuals con- sisting of V. vimba (Caspian), V. vimba (Western), V. mira- bilis, V. melanops, and outgroup Blicca bjoerkna. The mo- lecular clock calibration was based on the divergence rate of cyt b gene in Leuciscinae of 0.4% per lineage per mil- lion years, as determined by Perea et al. (2010), and this rate was used by Buj et al. (2019) and Vifiuela Rodriguez et al. (2020). Based on a Yule speciation prior and a strict clock model, branch rate estimates were calculated. Parti- tioned cyt b and COI dataset were used for the molecular clock analysis and the partitions were linked except for substitution models. The substitution models were used the TrN+G:-In = 1878.5334 (Tamura and Nei 1993) and the HK Y:-In = 1063.2973 (Hasegawa et al. 1985) for cyt b and COI barcoding region, respectively, with Gamma site heterogeneity for both markers. The number of MCMC steps (chain length) was ten million, with parameters logged every 1000 generations. The residual parameters were default parameters of the software. Tree results were summarized in TreeAnnotator v.2.6.0 software with 10% burn-in to get a maximum clade credibility tree. Node bars, height median with height 95% HPD, and node la- bels were mapped on the tree with FigTree v1.4.4 (Ram- baut 2018). Geological scale was plotted using the geo- scalePhylo function in R package strap (Bell and Lloyd 2015). Effective sample size (ESS) and convergence of parameters was estimated using Tracer v.1.6 (Rambaut and Drummond 2013). The effective sample sizes for all parameters of interest were greater than 200. Results Genetic diversity and species divergence The nucleotide sequences of the cyt b gene region (1023 bp) were studied in 91 specimens from three species of Vimba (Additional Table 1). The average nucleotide fre- quencies were estimated as 26.9% A, 29.7% T, 28.0% C, and 15.4% G. The transition/transversion rate k] = 115.99 (purines), k2 = 34.563 (pyrimidines) and the overall tran- sition/transversion bias were estimated as R = 31.458. The haplotype number was identified as 29: 18 from V. vimba (N = 66), nine from V. melanops (N = 12), and two from mirabilis (N = 13) (Additional Table 1). We identified 65 variable sites, of which 36 were parsimony-informative. Species-specific nucleotide (SSN) positions in the mito- chondrial DNA cyt b gene sequences provide evidence for 69 genetic differentiation of three Vimba species, as in V. vim- ba, V. mirabilis, and V. melanops are determined: 7, 7, and 1, respectively. The haplotype and nucleotide diversity are Hd = 0.855 + 0.025 and x = 0.00175 + 0.00029 for Vimba vimba, Hd = 0.939 + 0.00333 and m = 0.00783 + 0.00119 for V. melanops, and Hd = 0.154 + 0.01590 and m= 0.00015 + 0.00012 for V. mirabilis. The overall haplo- type and nucleotide diversity are Hd = 0.907 + 0.015 and m= 0.00994 + 0.00104. According to the AMOVA, 88.34% of all genetic variation occurs among three species. In the haplotype network analysis, the 29 distinct haplotypes are separated by one up to nine mutations. The most common haplotype was H1, shared by many populations belonging to V. vimba. Many haplotypes were distributed in either one or two populations (Fig. 2). Average pairwise genetic distances between species were estimated as 0.0212 (V. vimba and V. melanops), 0.0198 (V. vimba and V. mirabi- lis), and 0.0171 (Vv. melanops and V. mirabilis). Average pairwise genetic distance was estimated as 0.0101 be- tween V. vimba Caspian (H16) and West lineages. Average intraspecific variations within V. vimba, V. mirabilis, and V. melanops were estimated as 0.002, 0.000, and 0.008, respectively. Nucleotide sequences of the COI barcod- ing region (652 bp) were obtained in 67 specimens from three species of Vimba (Additional Table 1). The nucleo- tide frequencies were calculated as 25.28% A, 29.00% T, 27.71% C, and 18.01% G. The transition/transversion rate k1 = 3.588 (purines), k2 = 1.848 (pyrimidines), and the overall transition/transversion bias was calculated as R = 1.27. The haplotype number was identified as 13: six from V. vimba (N = 46), five from . melanops (N = 16), and two from V. mirabilis (N = 5) (Table 1). A total of 21 vari- able sites were recognized, and 10 of them were parsimo- ny-informative. The haplotype and nucleotide diversity with the standard deviation are Hd = 0.680 + 0.00156 and m= 0.00308 + 0.00021 for V. vimba, Hd = 0.608 + 0.01695 and 2 =0.00204 + 0.00065 for V. melanops, and Hd =0.400 + 0.237 and a = 0.00123 + 0.00073 for V. mirabilis. The overall haplotype and nucleotide diversity are Hd: 0.826 + 0.026 and a = 0.00457 + 0.00041. According to the AMOVA, 58.52% of all observed genetic variations in three species are occurring within species. Haplotype network analysis has 13 unique haplotypes with at least one mutational step, and no haplotype 1s shared between species that are separated from each other by at least one mutational step. The most common haplotypes were H13, represented by 20 specimens sampled by Iznik Lake. All haplotypes were distributed in either one or two popula- tions (Fig. 3). Average pairwise genetic distances between species were estimated as 0.0054 (V. vimba and V. mela- nops), 0.0093 (V. vimba and V. mirabilis), and 0.0088 (V. melanops and V. mirabilis). Phylogenetic relationships and divergence times Based on ML and Bayesian phylogenetic inferences, four mitochondrial clades of Vimba species are observed. The first and second clades include distinct V. vimba zse.pensoft.net 70 H27 H28 H25 H29 H26 H24 H23 H21 H22 O Vimba vimba @ Vimba melanops © Vimba mirabilis Kalayci, G.: Phylogeography of Vimba species HS H2 H4 H9 H10 H16 H19 Figure 2. Median-joining network of the cyt b haplotypes. Circle size corresponds to sample size; one bar indicates an additional mutational step. Each small line represents one nucleotide difference. populations, while the other clades comprise V. mirabilis and . melanops. V. vimba is not monophyletic because the Western (Pontic) and Caspian basin haplotypes of V. vimba are located in two different clades. As shown in Fig. 4, the maximum likelihood and Bayesian inference analyses of concatenated data of mitochondrial cyt b and COI barcoding region trees were supported by high boot- strap values (>75) for the distinction of species and Vimba vimba lineages. According to the divergence time between the two main clades, the divergence among the Vimba spe- cies starts in 2.27 (0.57-4.02) mya, early Pleistocene (Ge- lasian), and differentiates into four clades in early Pleisto- cene (Calabrian) based on StarBEAST analysis (Fig. 5). Discussion The present study investigates the phylogeny and phy- logeography of three Vimba species to provide the first comprehensive molecular study on the genus. Although some work has been conducted on Vimba species, there zse.pensoft.net are no comprehensive studies on their phylogeography, which 1s provided in the present study. Here, the phylogeny constructed by ML and BI ap- proaches 1s based on concatenated mtDNA cyt b and COI sequences of three Vimba species. The phylogenetic tree topology revealed that species were clustered into four well-supported clades (Vv. vimba (Western), Vi. vimba (Caspian), V. melanops, and V. mirabilis) with high-re- liability BI Posterior probability value (BI = 0.98) al- though low ML bootstrap value (ML < 50). In V. vimba, two lineages are observed, the Caspian and the Western, monophyly of both group supported by high BI (0.98) value. V. melanops and V. mirabilis belong to different clades with high BP (97) and BI posterior probability (1.00) (Fig. 4). According to tree topology, a clear geo- graphical pattern emerged, which is also supported by the median joining network. However, variability at cyt b and COI markers varied, the AMOVA analysis found almost all (88.34%) of the genetic variations observed for the cyt b gene occurred between species, while this value drops to 58.52% for the COI barcoding region. Zoosyst. Evol. 98 (1) 2022, 65-75 H2 HS H4 H3 ) O Vimba vimba @ Vimba metanops @ Vimba mirabilis 71 H10 H12 Hil H8 H9 Figure 3. Median-joining network of the COI haplotypes. Circle size corresponds to sample size; one bar indicates an additional mutational step. Each small line represents one nucleotide difference. Geiger et al. (2014) similarly reported that V vimba, V. melanops, V. mirabilis are closely related species, with relatively low interspecific genetic distances, but they can be distinguished from each other by morphological char- acters. Species with high haplotype diversity and low nu- cleotide diversity are usually thought to originate through small founder populations during the expansion period, in accordance with previous analyses of Anatolian freshwa- ter fish species (Hrbek et al. 2004; Bektas et al. 2017). A small independent evolution of similar origin (originating from the Pliocene/Pleistocene) may account for the low intraspecific diversity and corresponds with tectonic or glacial activity in the area. Anatolia and Central Europe were connected from the late Oligocene to the late Mio- cene (23—5.33 mya) (Popov et al. 2004). The estimated separation time of Vimba from sister genus Blicca was calculated as 6.16 (1.42—13.11) mya, a timeframe con- sistent with a dispersal from the Danube basin to Anatolia via river capture during this period (Levy et al. 2009). Similarly, Hanfling et al. (2009) estimated the time of separation of Vimba genus in Pleistocene as well. The di- vergence time between the two main clades shows that the divergence among the Vimba species started 1n 2.27 (0.57-4.02) mya, early Pleistocene (Gelasian) (Fig. 5). According to Hanfling et al. (2009), V. vimba lineages were defined as Pontic and also Caspian clade, including Caspian Sea haplotype which is highly divergent from the other haplogroup. This suggests V. vimba originated from two refugial regions located in the Danubian drainage and the northern Pontic regions. Moreover, Hanfling et al. (2009), Naseka and Bogutskaya (2009), Jouladeh-Roud- bar et al. (2015), and Esmaeili et al. (2018) stated that the Caspian Vimba should be considered as a distinct spe- cies corresponding to V. persa, which 1s anadromous and endemic to the Caspian Sea. Similarly, in this study, it is supported that Caspian Vimba should be classified as V. persa, not V. vimba considering intra and interspecies divergence of Vimba species. Furthermore, both phyloge- netic and StarBEAST analyses support Caspian Vimba corresponding to a different linage from the Western lin- eage of V. vimba. However, advanced research and com- prehensive sampling of Caspian and Western populations are required to clarify this. Vv vimba in other regions has relatively low intraspecific diversity, except for the Cas- pian haplogroup. Natural distribution records for V. vimba in Europe, the former USSR, are most likely due to stock translocations and introductions from other areas due to their economic value (Freyhof 1999). zse.pensoft.net 72 Kalayci, G.: Phylogeography of Vimba species K. vimba Black Sea basin, Turkey 71/0.54 K. vimba Black Sea basin, Turkey K. vimba Baltic Sea basin, Lithuania V. vimba Black Sea basin, Turkey V. vimba North Sea basin, Germany K. vimba Azov Sea basin, Russia KV. vimba Baltic Sea basin ,Sweden Ve vimba Marmara Sea basin, Turkey V. vimba Black Sea basin, Germany V. vimba Marmara Sea basin, Turkey K vimba Black, Marmara and Azov Sea basin, Turkey, Ukraine K vimba Black and Azov Sea basin, Turkey, Ukraine K. vimba Marmara Sea basin, Turkey -/0.85 ; FV. vimba Marmara Sea basin, Turkey F. vimba Black Sea basin, Turkey F. vimba North Sea basin, Germany k. vimba Marmara Sea basin, Turkey F. vimba Black Sea basin, Turkey V. vimba Black Sea basin, Turkey -/0.98 K vimba Elbe basin, Czech Republic V vimba Black Sea basin, Turkey 60/0.98 | mirabilis Biyuk Menderes River basin, Turkey 100/1.00 K. mirabilis Buyuik Menderes River basin, Turkey K. mirabilis Biyiik Menderes River basin, Turkey V. mirabilis Buyik Menderes River basin, Turkey V. melanops Aegean Sea basin, Turkey KF. melanops Aegean Sea basin, Greece 0.98 93/0.99 97/1.00 KF. melanops Aegean Sea basin, Turkey 95/1.00 F. melanops Aegean Sea basin, Greece V. melanops Aegean Sea basin, Greece V. melanops Aegean Sea basin, Greece 87/0.99 V. melanops Aegean Sea basin, Greece V. melanops Aegean Sea basin, Greece 94/1.00 K. melanops Aegean Sea basin, Greece V. vinba Caspian Sea Basin Russia Blicca bjoerkna AP009304 0.010 -—————__—_—_—__| Figure 4. Maximum likelihood tree based on the two concatenated mitochondrial genes (cyt b and COI) (1675 bp) sequences of Vimba species. Maximum likelihood and Bayesian inference analyses resulted in congruent trees. Bootstrap and posterior probabil- ity values are shown above nodes on a tree if 50% or higher. Phylogeography of V. vimba was investigated by Han- fling et al. (2009), and it reflected the presence of two ref- ugia and recolonization of V. vimba as Caspian sea, and Western or Pontid clade, dating back 1—2 mya during the early Pleistocene. According to the present study, the sep- aration of Vimba, dated in Pleistocene, occurred at 1.06 (0.2—2.55) mya. Correspondingly, in view of the timing, Pleistocene events must have played a central role in structuring the Balkan’s marbled goby populations (Van- hove et al. 2012). In addition, multiples fish groups in zse.pensoft.net the Black and Caspian Sea basins display similar patterns of Pleistocene divergence such as the Black Sea roach, Rutilus frisii, salmon, barbell, and dreissenid mussels (Stepien et al. 2003; Kotlik et al. 2008; Ninua et al. 2018; Bartakova et al. 2019; Levin et al. 2019). Due to the mi- gration of Vimba using brackish waters, it spread all over Eurasia also in Northern Germany and Netherlands, using the Danube River and old canal system (Freyhof 1999). In the Early Pleistocene, the depression of the Marma- ra Sea and the uplift of the Aegean mountains contributed Zoosyst. Evol. 98 (1) 2022, 65-75 6,16 (1,42-13,11) 13 10 73 Vimba vimba (Western) 1,06 (0,1-2,55) Vimba vimba (Caspian) 2,27 (0,57-4,02) Vimba mirabilis 1,55 (0,3-3,12) Vimba melanops Blicca bijoerkna Piacenzian Gelasian Calabrian Pleistocene 5 0) Figure 5. Divergence timescale for the Vimba species inferred under Bayesian strict clock method from two concatenated mitochon- drial genes (cyt b and COT) (1675 bp) sequences. Numbers in front of the node represent divergence times in million years (Ma) and their HPD 95% credibility intervals. greatly to the separation of V. vimba and V. mirabilis. Fur- thermore, while the water of the Btiywk Menderes River was flowing in the north-south direction because of the western Anatolian Mountains barrier, fractures and fold- ings which occurred in the Early Pleistocene caused the river to turn west and take its present form. V. mirabilis is present in Bafa Lake because of alluvium brought in by the Menderes River that blocked the old sea gulf and separated it from the sea, creating Bafa Lake (Akcer-On et al. 2020). Por, (1989) specified that a line drawn from west to east in the middle of Turkey could be considered as a major su- ture, which leads to distinct species of Vimba between the Buytk Menderes and northern Aegean. The differences between the Balkan and Anatolian species can primarily be associated with the formation of the Aegean in the late Pliocene (Kosswig 1955; Bilgin 2011). Also, Bektas et al. (2019) discovered that dispersal of A/burnoides symrnae and Alburnoides economui was dated in 5.42—2.31 Ma (Early Pliocene), when the former Aegeopotamus River was a very large river that discharged the waters of Pa- ratethys into the Aegean Sea. Divergence time estimated between . melanops and V. mirabilis as 1.69 (1.07—2.38) mya 1s consistent with this event. Durand et al. 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