Zoosyst. Evol. 99 (1) 2023, 185-194 | DOI 10.3897/zse.99.94333 yee BERLIN DNA barcoding of the genus A/burnoides Jeitteles, 1861 (Actinopterygii, Cyprinidae) from Anatolia, Turkey Halim Canoglu', Ismail Aksu*, Davut Turan’, Yusuf Bektas? 1 Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, 53100, Rize, Turkiye 2 Department of Basic Sciences, Faculty of Fisheries, Recep Tayyip Erdogan University, 53100, Rize, Turkiye 3 Deparment of Molecular Biology and Genetic, Faculty of Sciences, Karadeniz Technical University, 61080, Trabzon, Turkiye https://zoobank. org/6F BE3E0A-9CE9-4189-AC0C-736A58762B9B Corresponding author: Yusuf Bektas (ybektas@ktu.edu.tr) Academic editor: Nicolas Hubert # Received | September 2022 Accepted 20 February 2023 @ Published 6 March 2023 Abstract The present study investigated the ability of DNA barcoding to reliably identify the endemic freshwater species in Turkey, known as biodiversity hotspots. The barcode region (652 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) was used to barcode 153 individuals from 13 morphologically identified species of the genus A/burnoides. Based on the Kimura two-parameter (K2P) evolution model, the average interspecific distance (0.0595) was 31-fold higher than the average intraspecific distance (0.0019). There was a clear-cut barcode gap (0.0158—0.0187) between maximum intraspecific distance (A. ‘zanevi and A. veliog/ui) and min- imum nearest-neighbour distance (A. frevhofi and A. kurui) for Anatolian A/burnoides species and a common genetic threshold of 0.0158 sequence divergence was defined for species delimitation. The multiple species delimitation methods (ABGD, ASAP, GMYC and bPTP) revealed a total of 11 molecular operational taxonomic units (MOTUs) for 13 morphospecies. Neighbour-joining (NJ), Maximum Likelihood (ML) and Bayesian Inference (BI) tree analysis indicated that all haplotypes were clustered into two major clades, which corresponded to eleven A/burnoides species clusters, with strong bootstrap support. Furthermore, all the specimens clustered in concurrence with the morpho-taxonomic status of the species, except for two species (A. coskuncelebii and A. emineae) that were morphologically differentiated, but showed overlap in variation for COI-based DNA barcode data with other species. Overall, present results identified that COI-based DNA barcoding is effective for species identification and cataloguing of genus Alburnoides in Turkey. Key Words COI, mtDNA, species delimitation, Spirlin Introduction Turkey is located at the intersection of three (Caucasus, the Irano-Anatolian and the Mediterranean) of the world’s 35 biodiversity hotspots (Mittermeier et al. 2004; Fricke et al. 2007). Turkish freshwater wetlands contain numerous lakes, rivers and reservoirs, which host over 180 Cyprinid species (Freyhof et al. 2014; Kuru et al. 2014; Cicek et al. 2020). However, recent reports have shown a decline in the number of fish caught from most Turkish inland wa- ters due to habitat degradation, pollution, climate change, inadequate management of fisheries, highly-regulated large river drainages, such as dams and hydropower plants and the introduction of non-native fishes (Fricke et al. 2007; Cicek et al. 2015; Tarkan et al. 2015; Freyhof et al. 2020). Despite being faced with serious and increas- ing human activities-induced threats to fish biodiversity, especially in recent years, Turkey is not yet at the level it should be in terms of biodiversity and habitat conserva- tion activities (Sekercioglu et al. 2011). Phylogeny and phylogeography of A/burnoides species in Turkey were revealed by analysing the mitochondrial cytochrome b (Cyt b) gene sequences (Bektas et al. 2019a). However, COI-based DNA barcoding has not been performed for Copyright Canoglu, H. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 186 the detection of intraspecific diversity and species de- limitations in the genus A/burnoides, most of which are endemic to Anatolia. Hebert et al. (2003) proposed DNA barcoding technology using mitochondrial cytochrome c oxidase subunit I (COI) gene sequence, for which the intraspecific diversity 1s significantly lower than the inter- specific diversity, as a barcode for species identification and classification. For over a decade, the COI gene has been widely used for classifying and identifying Cyprinid fishes (Hubert et al. 2008, 2015; Keskin and Atar 2013; Chakraborty and Ghosh 2014; Geiger et al. 2014; Karim et al. 2016; Lakra et al. 2016; Baycelebi et al. 2018; Aksu and Bektas 2019; Bektas et al. 2019b; Rahman et al. 2019; Bahadir et al. 2020; Bektas et al. 2022). So far, the COI-based DNA barcode has been used to identify a limited number of cyprinid fish genera or species in Tur- key (Keskin et al. 2012; Keskin and Atar 2013; Geiger et al. 2014; Bektas et al. 2019b) in Turkey. However, Alburnoides populations in Iran (Jouladeh-Roudbar et al. 2016; Eagderi et al. 2019) and the Caucasus (Levin et al. 2018), which are adjacent regions to Turkey, were bar- coded, based on the COI sequences, but DNA barcodes of the A/burnoides species in Anatolia, an important centre of endemism, have not yet been generated. This paper aims to determine intraspecific and inter- specific genetic distances and to identify DNA barcodes of Alburnoides species from Anatolia. Materials and methods Sample collection and morphological identification One hundred and fifty-three freshwater fish samples be- longing to 13 Alburnoides species were collected from 47 different sites (Some from type localities) in Turkish inland waters between 2016 and 2020 (Fig. 1, Suppl. ma- terial 1). Morphological identification was performed in situ by following the counts and measurements of Turan et al. (2016, 2017). After each specimen was morpholog- ically identified, tail fin tissue was taken, preserved in 95% ethanol and stored at —20 °C. DNA extraction, PCR amplification and DNA sequencing Genomic DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in an automated DNA extraction robot (QIAcube). DNA yield and purity were quantified using a NanoDrop 2000C spectrophotometer (Thermo Scientific). Extracted DNA were stored at -20 °C. Universal PCR primers FishF1 and FishR1 (Ward et al. 2005) were used for COI amplifi- cation. Polymerase chain reaction (PCR) was performed on BIORAD T100TM Gradient thermocycler (Biorad, Hercules, CA, USA) following the protocol of Ward et al (2005). Thermal cycling conditions were as follows: zse.pensoft.net Canoglu, H. et al.: DNA barcoding of Alburnoides 3 min initial denaturation at 94 °C, followed by 35 cycles of 1 min at 94 °C, annealing for 40 s at 54 °C, elongation for 1 min at 72 °C and final extention for 7 min at 72 °C. PCR products were electrophoresed on a 1.4% agarose gel containing ethidium bromide and visualised under UV using a gel documentation system. PCR products were purified using the QIAquick kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and purified PCR products were directly sequenced in both directions using an ABI PRISM 3730x1 automated DNA sequencer (Applied Biosystem, Foster City, USA) following the manufacturer’s instruction. Genetic distance, haplotyping and phylogenetic analyses Sequences were edited using DNASTAR software (Ed- itSeq V.7.1.0 (Lasergene, DNASTAR) and aligned using the MegAlign V.7.1.0 (Lasergene, DNASTAR), based on the Clustal W (Thompson et al. 1994). The mitochondrial COI sequences were aligned and adjusted to have an equal length of 652 bp. Pairwise genetic distance amongst and within species, polymorphic sites and nucleotide com- position were computed using MEGA X (Kumar et al. 2018) with the Kimura 2-parameter (KK2P) model (Kimu- ra 1980). The haplotype data were generated using the DnaSP v.5 (Librado and Rozas 2009) software. All hap- lotype sequences were submitted to GenBank under the accession MZ539436—MZ539471 (Suppl. material 1). Based on the COI barcode region, phylogenetic relation- ships amongst the Spirlin haplotypes were reconstructed using three methods, Neighbour-joining (NJ), Bayesian Inference (BI), and Maximum Likelihood (ML). The pro- gramme jModel Test 0.1.1 (Posada 2008) was used to obtain the appropriate model of evolution using the Akaike Infor- mation Criterion (AIC; Akaike (1973)) and the Bayesian Information Criterion (BIC; Schwarz (1978)). The NJ tree was generated using MEGA X (Kumar et al. 2018) with the Kimura-2 parameter (K2P). The node support was evaluat- ed with 1000 bootstrap pseudo-replicates. Bayesian Infer- ence analysis was conducted by using BEAST 1.8.2 (Drum- mond et al. 2012), setting a coalescent tree prior and an uncorrelated lognormal relaxed clock. Each Bayesian tree was run for 10 million generations with a sampling frequen- cy of 1,000 and were combined with a burn-in set to 25% using LogCombiner 1.8.2 (Drummond et al. 2012). A ma- jority rule consensus tree was constructed using TreeAnno- tator 1.8.2 (Rambaut and Drummond 2015). In BEAST, the divergence date of A/burnoides and Rutilus (approximately 27 Mya; Levy et al. (2009); Perea et al. (2010)) was used as a calibration point for estimating the divergence times of Alburnoides species. The ML method was performed with RAXML v.8.1.21 (Stamatakis 2014). The support of each node was estimated using a rapid bootstrap analysis with 1,000 replicates. Rutilus rutilus (GenBank Accession No. MW473258) was used as an outgroup. The obtained trees were visualised using FigTree v.1.4.3 (Rambaut 2012) and modified using Adobe Photoshop CS6 software. Zoosyst. Evol. 99 (1) 2023, 185-194 Figure 1. Map showing the range of distribution for each species and sampling points within each species. Species delimitation methods To estimate Molecular Operational Taxonomic Units (MOTUs), two distance-based methods, Automatic Bar- code Gap Discovery (ABGD; Puillandre et al. (2012)) and Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. (2020)) and two tree-based approaches, the General Mixed Yule-coalescent (GMYC; Pons et al. (2006)) and Bayesian poisson tree process (bPTP; Zhang et al. (2013)), were applied to the COI dataset. For the species delimitation, we used Automatic Bar- code Gap Discovery (ABGD) (Puillandre et al. 2012) and Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2020) (both distance-based methods) on the alignment of CO1 sequences. The ABGD species de- limitation was performed via the ABGD web server with default settings (P_. = 0.001, P_ =0.1, Steps = 10, X rel- ative gap width = 1.5, Nb bins = 20) and testing of two substitution models (JC69 and K2P) for calculation of the Kimura 2-parameter distances (https://bioinfo.mnhn.fr/abi/ public/abgd/abgdweb.html, accessed on 3 March 2022). ASAP species delimitation was performed via the ASAP web server with testing of all three substitution models for calculation of the distances (https://bioinfo.mnhn.fr/ abi/public/asap/asapweb.html, accessed on 19 December 2022) using the distance matrix, generated through MEGA X (Kumar et al. 2018). The single-locus and tree-based delimitation approach- es, the General Mixed Yule-coalescent (GMYC; Pons et al. (2006)) and Bayesian poisson tree process (bPTP; Zhang et al. (2013)) models, distinguish specimens in species level using coalescence theory. The ultrametric tree required for the GMYC method was obtained using BEAST 1.8.2 (Drummond et al. 2012) on COI dataset. The MCMC chain was run for 10 million generations with the best substitution model calculated in jModelTest 0.1.1 (Posada 2008) and the Yule speciation model. The MCMC log-on prior and posterior values was examined in Tracer 1.6 (Rambaut et al. 2014) and a burn-in of 1 mil- lion generations was used to avoid suboptimal trees in the final consensus tree. The single-threshold GMYC meth- od was run using ultrametric haplotype BEAST trees on the GMYC web server (species.h-its.org/gmyc, accessed on 06 May 2022). For PTP-based MOTU estimation, the needed rooted phylogenetic input-tree was constructed with RAXML v.8.1.21 (Stamatakis 2014) with the best substitution model. The PTP model was implemented following the default parameters and 500,000 MCMC generations on the bPTP web server (http://species.h-its. org/ptp/). We based species delimitation on a majority consensus of the results inferred from all four methods: two species were considered a single species if at least three out of four methods harmoniously merged two species as one. Distance-based species assignment Distance-based methods of species assignments in con- junction with computer simulations can determine the statistical significance of species identification success rates (Huang et al. 2013). To evaluate the potential of the COI dataset for correct species identification and to select the best threshold value, these were tested using the “Best Close Match’ (BCM), implemented in Species Identifier 1.8 programme of TaxonDNA software (Meier et al. 2006). Results Sequence variation of morphospecies A total of 36 distinct haplotypes for COI barcode region were identified (Suppl. material 1). The average nucleo- tide frequencies were 29.71 (T), 25.98 (C), 19.20 (G) and zse.pensoft.net 188 25.11 (A)%. The mean ratio is approximately 6:1. In this study, the average intraspecific K2P distance was 0.0019, compared with the 0.0595 intraspecific value. The mean interspecific distance was found to be 31-fold higher than the mean intraspecific distance. Phylogenetic relationships within A/burnoides Phylogenetic relationships amongst A/burnoides species were investigated using TIM3+I+G, which was selected as the best evolution model in BI and ML approaches for our dataset. Neighbour-joining, Maximum Likelihood and Bayesian Inference analyses generated similar tree topol- ogies and two main clades were detected consisting of eleven deeply divergent haplogroups within A/burnoides (posterior probabilities > 0.95 and 68% < bootstrap values for ML and NJ, Fig. 3). The dataset was analysed using the strict molecular clock in BEAST. The timing of split- ting-events of A/burnoides lineages are shown in Fig. 3. Species delimitation The morphological species hypothesis was not supported by our analyses using ABGD, ASAP, bPTP and GMYC, which delimited 13 species. Nevertheless, 11 putative spe- cies were recognised by all the methods that we applied to the COI dataset. We detected a distinct gap between in- tra- and interspecific genetic distances for 11 hypothetical species of A/burnoides, ranging from 0.0158 to 0.0187 for K2P distances (Fig. 2). Results from the four species delimitation analyses, ABGD, ASAP, bPTP and GMYC are summarised in Fig. 3. Relationships amongst the ma- jor clades, identified using the GMYC and bPTP analyses (Fig. 3), were identical to each other and to the concate- nated phylogenies. Nodal support was high (> 0.95) for most nodes of the BEAST consensus tree (Fig. 3). Results from GMYC and bPTP tests corroborated the presence of eleven independent lineages in Al/burnoides (Fig. 3). Ac- cording to the four methods of MOTU delimitation, there was an overlap between A. tzanevi-A. coskuncelebii and A 895 Barcoding gap Number of runs moo & oo F oreo CODoe tee £.¢ oo ceo PRP oO Of £2 foc Oo 8 oe 20 FPrRFrPFNMON NW WW woe Se SOO U AHA KDA aA DO Distance value Canoglu, H. et al.: DNA barcoding of Alburnoides A. velioglui-A. emineae belonging to the genus A/burnoi- des. Therefore, the A/burnoides dataset was reduced to eleven groups, based on the results of four delimitation methods (Fig. 3). Species identification with BCM Due to the observed discrepancy between taxa delimited, based on the four MOTU delimitation methods described above and morphospecies, all A/burnoides species in Turkey were assigned into eleven of thirteen categories. Moreover, the DNA barcoding gap, which was the max- imum intraspecific distance of each species against its minimum distance to the nearest neighbour, was calcu- lated for all species. The maximum sequence divergence amongst individuals of some species (A. tzanevi: 0.01583 and A. velioglui: 0.01582, Table 1) exceeded some of the nearest-neighbour distance values (A. veliog/ui & A. em- ineae: 0.0142 and A. tzanevi & A. coskuncelebii: 0.0120). Therefore, the identification success of the BCM method was re-evaluated by treating A. veliog/lui-A. emineae and A. tzanevi-A. coskuncelebii as a species in the dataset us- ing a final distance threshold of 0.0158 (Table 1) and the BCM approach returned 100% correct identifications. Table 1. Mean and maximum intraspecific and nearest-neigh- bour (NN) distance (percentage) for all species. Species Mean Max. Nearest Distance intraspecific intraspecific neighbour to NN distance distance A. smyrnae N/A N/A A. tzanevi 0.04801 A. tzanevi 0.00758 0.01583 A. freyhofii 0.02516 A. freyhofi 0.00145 0.00308 A. kurui 0.01875 A. turani N/A N/A A. eichwaldii 0.03885 A. manyasensis 0.00028 0.00153 A. turani 0.04611 A. velioglui 0.00862 0.01582 A. eichwaldii 0.02848 A. kosswigi 0.00329 0.00776 A. turani 0.05350 A. diclensis N/A N/A A. eichwaldii 0.03076 A. fasciatus N/A N/A A. eichwaldii 0.04945 A. kurui N/A N/A A. freyhofi 0.01875 A. eichwaldii 0.00168 0.00464 A. velioglui 0.02845 N/A denotes that species is a singleton. 0.06 0.06 0.05 0.04 0.03 Distance value 0.02 0.02 0.01 €LOT 9bT? o Ww a uo 61TZe c6CP Rank 8Er9 TISL ’8S8 LS96 OELOT Figure 2. Histogram (A) and ranked pairwise (K2P) distances (B) amongst COI-based barcode sequences by the automated barcode gap discovery (ABGD) approach. zse.pensoft.net Zoosyst. Evol. 99 (1) 2023, 185-194 189 | hAec3 hAec4 BI/ML/NJ 0.95/80/86 =a hAecl A. eichwaldii hAec? hAec5 hAemn A. emineae i 1.00/78/93 4 95/68/87 | hawiee | | hAvlg4 D6/87/90 hAvlg3 A. velioglui hAvlg5 1.00/80/9 hAvig1 hAdcl_ A. diclensis Ee a [| | hAtur A. turani ii [i i. (i [ hAkw3 hAkw4 1.00/76/90 hAkw5 5. A, kosswigt hAkw6 hAkw1 ¥.00/78/87 hAkw2 hAfsc A, fasciatus fi a l] {] {| hAmy2 , A. manyasensis hAmy1 hAtz7 hAtzs hAtz6é ae A. tzanevi hAtz3 hAtz2 hAtzl hAcsk A. coskuncelebii hAfry1 1.00/85/100 1|,.00/95/100 hAfy4 A. freyhofi 00/84/91 hAfry2 hAfy3 hAkur A. kurui hAsym A. smyrnae Rutilus rutilus Eocene Oligocene Pliocene Pleistocene S Holocene > 40 35 30 25 20 15 10 5 So Figure 3. Species delimitation results visualised as bars on an ultrametric Bayesian phylogenetic tree of A/burnoides COI gene. The tree phylogroups were obtained according to ABGD, ASAP, GMYC, bPTP and morphology. As outgroup, Rutilus rutilus was used. zse.pensoft.net 190 Discussion Phylogenetic reconstructions Morphological identification of Spirlin can be challeng- ing due to their similar morphology and limited mor- phological information. As maternally-inherited mtDNA evolves about four times faster than that of the nucle- ar genome, mtDNA can be used to track differences in closely-related taxa and even within species (Stepien and Kocher 1997; Desalle et al. 2017). In this study, the phy- logenetic relationships inferred using mitochondrial COI (Fig. 3) are consistent with previous studies using several mitochondrial markers (Jouladeh-Roudbar et al. 2016; Stierandova et al. 2016; Levin et al. 2018), but differ in some of the deepest nodes, with the currently accepted phylogenies of A/burnoides (Cyt b, Bektas et al. 2019a). As seen in the phylogenies of Bektas et al. (2019a), the phylogenetic reconstructions strongly support the topol- ogy of two clades (western and eastern), containing 13 Alburnoides lineages, each corresponding to the species, with moderate-high posterior probability and bootstrap values (> 0.95 pp, > 86 BT; Fig. 3). On the contrary, the analysis of the present COI dataset supports the existence of 11 instead of 13 major lineages in A/burnoides. The phylogenetic trees constructed (Fig. 3) showed that the most conspecific haplotypes cluster together, which sub- stantially confirmed previous taxonomic assignments, based on morphology (Turan et al. 2013, 2014, 2016, 2017; Kaya 2020). Nevertheless, our phylogenetic tree topologies are completely not congruent with the mor- phological data: A. emineae is located inside A. velioglui and A. coskuncelebii is located inside A. tzanevi. As most closely-related species (A. cockuncelebii / A. tzanevi and A. emineae / A. velioglui) do not share haplotypes and both species pairs are living allopatrically, the low genetic distances between them may be a result of recent speciation, thus representing the most recently diverged species in the study area. Alburnoides tzanevi is found in some rivers that flow into the Black Sea from the Bulgarian-Turkish border to Sinop, the northernmost point of Turkey, while A. coskuncelebii has only been re- ported from the Biytikmelen River, which is also the type locality. As can be seen in Fig. 1, the geographic distri- butions of A. tzanevi and A. coskuncelebii overlap. Cor- rected divergence estimates between the species (Fig. 3) indicate that this phylogeographical structure was initiat- ed by historical events of the Early to Middle Pleistocene. Current distributions of two species are restricted to the Thrace and south-western Anatolia coastal areas of the Black Sea (Fig. 1), where they, thus, most likely survived several later glacial cycles in separate allopatric refugia. The Tigris-Euphrates River system originated in the Late Miocene, developed from small and probably short-lived isolated channels in the Pliocene and became the main drainage system of the region at present (Stow et al. 2020). Alburnoides velioglui is found in the upper tributaries of the Euphrates, the Karasu and Murat and A. emineae 1s in zse.pensoft.net Canoglu, H. et al.: DNA barcoding of Alburnoides the Beyazsu Stream, which is the upper tributary of the Khabur River in the Euphrates drainage system, within the borders of Turkey. According to our divergence time estimates (Fig. 3), these species were probably isolated in the distant tributaries of the Euphrates after the Pliocene (Trifonov et al. 2018), when the drainage system of the region evolved into its present form. Species delimitations In our study, the combination of four species delimitation methods (ABGD, ASAP, bPTP and GMYC) (Fig. 3) im- plemented, suggested that our results generally confirm the high taxonomic utility of the COI-barcoding region in Alburnoides and most closely correspond to the current morphology-based classification of A/burnoides. Con- trary to the findings of Turan et al. (2014, 2019), which reported a total of 13 A/burnoides species, a total of elev- en putative partitions were identified amongst Spirlin samples, based on COJI-based analysis in this study. There was a strong consensus between the species delimitation results and the phylogenetic clustering, revealing the fol- lowing lineages: A. smyrnae, A. tzanevi, A. eichwaldii, A. freyhofi, A. kurui, A. turani, A. manyasensis, A. velio- glui, A. kosswigi, A. diclensis and A. fasciatus (Fig. 3). Algorithms like bPTP/GMYC help us to objectiv- ise species Or as an argument to drop poorly-described morphotaxa. The reason why we use different approach- es like bPTP and GYMC is that they may give different results. When we test the number of morphotaxa using both approaches for our dataset, we found that the results of bPTP and GYMC analyses are harmonised, but under- estimate the number of species. We need to discuss these morphospecies when the pPTP and GMYC do not match the tip labels (phenotypic) or give a lower number. bPTP and GMYC give 11 species standing against the 13 an- notated in ML and BI trees. Looking at the topology and branch-lengths distribution in NJ and BI trees, it shows 11 clades regarding intraclade coherence and interclade distinction (Fig. 3). This result tells us that the number of morphologically distinguished species is high. Most of the major clades include only one species, with only two including genetically quite similar species. These species (A. emineae and A. coskuncelebii) are poorly separat- ed from other ones, so reducing the number of species makes sense. A. emineae falls into the A. veliog/ui group, which has a higher intra-species diversity. Similarly, A. coskuncelebii is nearly identical to some tzanevi, but the A. tzanevi’s intra-species diversity is higher. Therefore, there is little reason molecular-wise to keep A. emineae and A. coskuncelebii as a different species. Following the bPTP/GMYC result, species epithets, A. ve/iog/ui and A. tzanevi, can be dropped as they have priority. Conse- quently, the species delimitation algoritm showed that A. tzanevi and A. coskuncelebii merged into a single puta- tive species, while A. velioglui and A. emineae were also merged into a single putative species. Zoosyst. Evol. 99 (1) 2023, 185-194 Our results revealed two cases of discordance be- tween morphology-based classification and COI-barcod- ing-based species delimitation. These discordances result- ed from the underestimation of possible species in which taxa, currently recognised as valid taxa, collapse in single barcode units. The first case when COI-barcoding failed to recognise morphologically distinct taxa as independent MOTUs includes the A. tzanevi and A. coskuncelebii from the Black Sea drainage in Thrace and western Anatolia. In our analyses, all methods recognised A. tzanevi and A. coskuncelebii, as a single unit (Fig. 3). Alburnoides coskuncelebii was recently recognised from the Biytik Melen, a catchment situated between the Sakarya and Kizilirmak Rivers, while A. tzanevi from small streams in Thrace and adjacent Anatolia (Turan et al. 2019). These results may be explained by allopatric speciation which led to initial differentiation in mtDNA and morpholo- gy, which was reported earlier for Spirlins (Bektas et al. 2019a). The second case 1s the allopatric A. veliog/ui and A. emineae, inhabiting Murat, Munzur, Sultansuyu and Beyazsu Rivers (Suppl. material 1) in the upper tributaries of the Euphrates River Basin. These two species are easily distinguished by several diagnostic morphological charac- ters (Turan et al. 2014), yet all species delimitation meth- ods applied herein failed to recognise them as independent groups (Fig. 3). Further studies, including examination of nuclear DNA markers, are required to clarify evolutionary relationships and taxonomic status of these species. Barcoding gap and automated identification Species identification, using DNA barcoding, 1s based on the principle that the genetic distance between two species is much greater than that within a species (Ge et al. 2021). A 10-fold sequence difference between the average interspecific and the average intraspecific differences has been suggested as the standard COI threshold for animal species identification (Hebert et al. 2004). In our present study, the mean interspecific distance was found to be 31-fold higher than the mean intraspecific distance, which was higher than the 23-fold difference observed in Caucasian Alburnoides species (Levin et al. 2018) and the 15-fold differences observed in Iranian A/burnoides species (Jouladeh-Roudbar et al. 2016). This difference is due to the fact that the average interspecies genetic distance calculated in this study (0.0595) is higher than those (0.0395 for 13 Caucasian species and 0.0386 for 11 Iranian species) in previous studies (Jouladeh-Roudbar et al. 2016; Levin et al. 2018). In other words, our results point to clear genetic differences between the Anatolian species of Alburnoides. The high species diversity and clear interspecies genetic differentiation detected for Alburnoides can be attributed to the presence of three biodiversity hotspots (Mediterranean, Caucasus and Irano-Anatolian) that coincide in Anatolia (Mittermeier et al. 2011) and the fact that Turkey served as the refugium for southern European taxa during the last glacial age (Reyjol 191 et al. 2007; Bilgin 2011). Anatolia, indeed, exhibits high species diversity and endemic species richness compared to its neighbouring regions (Levy et al. 2009; Giannetto and Innal 2021). The families Nemacheilidae, Gobiidae, Cobitidae and Salmonidae provide evidence that Anatolia is such a centre of diversity (Kuru et al. 2014). Based on mitochondrial Cyt b gene sequences, Stierandova et al. (2016) reported that A/burnoides lineag- es in Albania show low mitochondrial distances between 0.83% and 1.43%, which are more likely to be evaluated at an intraspecific level. On the other hand, Levin et al. (2018) reported a COI-based threshold value of 1.3% for Alburnoides species in the Caucasian and Middle East regions. Our results indicate that there is a clear barcod- ing gap (0.0158—0.0187) separating the intraspecific and interspecific distances. Consequently, a common genetic distance threshold of 0.0158 was observed for rapid and reliable identification of A/burnoides species. We think that our threshold value, which is higher than that report- ed by Levin et al. (2018), is due to the complex topogra- phy and geomorphology of Anatolia (EEA 2002). Conclusion Our study demonstrates the usefulness of DNA barcod- ing for the identification of A/burnoides species in Tur- key. This study contributes to the construction of DNA reference barcode data for Turkish fish fauna. We also confirm that DNA barcoding could assist in resolving issues of ambiguity in morphological identification of species. Finally, it has been concluded that COI-based DNA barcoding is a reliable and useful approach for the genetic identification of morphospecies belonging to the genus Alburnoides. Acknowledgements We thank Dr. Cuneyt Kaya and and Dr. Esra Baycelebi (Faculty of Fisheries, Recep Tayyip Erdogan University) for their assistance during the field sampling. This work was supported by the Scientific and Technical Research Council of Turkey (KBAG-113Z102) and by Recep Tayyip Erdogan University Research Fund Grants (FYL- 2021-1240). References Akaike H (1973) Information theory and an extension of the maxi- mum likelihood principle. 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The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.99 94333 .suppl1