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 phylogeographic inferences, the Vimba genus is monophyletic, and its species dispersed in the Pleistocene era.

cyt b, genetic diversity, Phylogeography, Vimba bream, Vimba mirabilis

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 Vimba mirabilis. V. vimba was initially described as Cyprinus vimba L. from several Swedish lakes in Scandinavia, the North Sea, coastal waters of Baltic Sea basins, and, subsequently, after the description, it was also found in the Caspian, Black Sea, Marmara Sea basins, and the Rhine River. In Anatolia, V. vimba is distributed from the Marmara basin up to Büyük Menderes, Eğirdir Lake, Köprüçay and Eşen rivers in the south, and Kızılırmak in the east. V. melanops was described initially from the Meriç (Evros) River and its distribution now extends within the borders of Turkey, Greece, Bulgaria, and Macedonia in the North Aegean basin from Meriç to the Pinios River. The Anatolian endemic V. mirabilis was detected only in the type locality Büyük 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 considered 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 in the Caspian Sea and Caspian bream, has been identified as Vimba persa (Hänfling et al. 2009; Naseka and Bogutskaya 2009; Chaichi et al. 2011a, 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), Hänfling et al. (2009), Perea et al. (2010), Triantafyllidis et al. (2011), Geiger et al. (2014), Schönhuth et al. (2018). In particular, Hänfling et al. (2009) discovered 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 Alburnoides, Barbus, Capoeta, Gobio, Squalius, Telestes and typically uncovered distinct patterns according to mitochondrial 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 markers. (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 Bektaş 2019).

In European rivers, freshwater fishes have largely colonized the Black Sea upstream, up rivers such as the Dnieper 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 Western 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 Pliocene 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.

The present study investigates the phylogeny and phylogeography of three Vimba species to provide the first comprehensive molecular study on the genus. Although some work has been conducted on Vimba species, there are no comprehensive studies on their phylogeography, which is provided in the present study.

Here, the phylogeny constructed by ML and BI approaches is 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 (V. vimba (Western), V. vimba (Caspian), V. melanops, and V. mirabilis) with high-reliability BI Posterior probability value (BI = 0.98) although 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 geographical 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.

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 characters. Species with high haplotype diversity and low nucleotide diversity are usually thought to originate through small founder populations during the expansion period, in accordance with previous analyses of Anatolian freshwater 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 Miocene (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 consistent with a dispersal from the Danube basin to Anatolia via river capture during this period (Levy et al. 2009). Similarly, Hänfling et al. (2009) estimated the time of separation of Vimba genus in Pleistocene as well. The divergence time between the two main clades shows that the divergence among the Vimba species started in 2.27 (0.57–4.02) mya, early Pleistocene (Gelasian) (Fig. 5).

According to Hänfling 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, Hänfling et al. (2009), Naseka and Bogutskaya (2009), Jouladeh-Roudbar et al. (2015), and Esmaeili et al. (2018) stated that the Caspian Vimba should be considered as a distinct species corresponding to V. persa, which is 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 phylogenetic and StarBEAST analyses support Caspian Vimba corresponding to a different linage from the Western lineage of V. vimba. However, advanced research and comprehensive sampling of Caspian and Western populations are required to clarify this. V. vimba in other regions has relatively low intraspecific diversity, except for the Caspian 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).

Phylogeography of V. vimba was investigated by Hänfling et al. (2009), and it reflected the presence of two refugia 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 separation 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 (Vanhove et al. 2012). In addition, multiples fish groups in 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; Kotlík et al. 2008; Ninua et al. 2018; Bartáková et al. 2019; Levin et al. 2019). Due to the migration 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 Marmara Sea and the uplift of the Aegean mountains contributed greatly to the separation of V. vimba and V. mirabilis. Furthermore, while the water of the Büyük Menderes River was flowing in the north-south direction because of the western Anatolian Mountains barrier, fractures and foldings 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 (Akçer-Ön 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 suture, which leads to distinct species of Vimba between the Büyük 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 Alburnoides 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 Paratethys into the Aegean Sea. Divergence time estimated between V. melanops and V. mirabilis as 1.69 (1.07–2.38) mya is consistent with this event. Durand et al. (2000), Hewitt (2000), and Hewitt (2004) further highlighted that the Danube was an essential component of the Chub (Squalius cephalus) evolutionary history, especially during the Pleistocene glaciations as it acted as a refugium for this species. Our molecular data confirm this influence on the divergence of V. melanops and V. mirabilis.

I am pleased to thank Davut Turan and Cüneyt Kaya for providing samples. Also, I extend my special thanks to the Editor, Nicolas Hubert and two anonymous reviewers for their valuable comments and suggestions that greatly improved the manuscript.