Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.

Diverse partitiviruses hosted by the ectomycorrhizal agaric Hebeloma mesophaeum and the natural transmission of a partitivirus between phylogenetically distant, sympatric fungi.

Mycorrhizal fungi host diverse mycoviruses that contribute to our understanding of their diversity and evolution. Here we report on the identification and complete genome characterization of three novel partitiviruses naturally infecting the ectomycorrhizal fungus Hebeloma mesophaeum. During NGS derived viral sequence analyses, we identified a partitivirus that is conspecific with the previously reported partitivirus (LcPV1) described from a saprotrophic fungus Leucocybe candicans. The two distinct fungal specimens inhabited the same vicinity of a campus garden. RdRp sequences encoded by the LcPV1 isolates from both host fungi was found to be identical. Bio-tracking studies revealed that viral loads of LcPV1 drop significantly in L. candicans but not in H. mesophaeum within four years period. The physical proximity of the mycelial networks of both fungal specimens implied the occurrence of a virus transmission event with unknown mechanism. Nature of this virus transmission was discussed in relation to transient interspecific mycelial contact hypothesis.

Identification and complete genome sequencing of a novel betapartitivirus naturally infecting the mycorrhizal desert truffle Terfezia claveryi.

Viruses that naturally infect fungal species and capable of establishing mycorrhizae are largely unknown. In this study, we identified and characterized a new partitivirus inhabiting the ascomycete, mycorrhizal desert truffle species Terfezia claveryi, and named it "Terfezia claveryi partitivirus 1" (TcPV1). The entire genome of TcPV1, sequenced by both high throughput sequencing of the total dsRNA extracts and by Sanger sequencing of the RLM-RACE PCR products comprised two dsRNA segments of 2404 bp and 2374 bp, respectively. Both dsRNA genome segments harbored a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. The BLASTp search of the RdRp and CP sequences revealed the highest sequence identities (41.92% and 24.13% identity, respectively) to those of Bipolaris maydis partitivirus 2 and Plasmopara viticola lesion associated partitivirus 5. Molecular phylogenetic analyses of the RdRp sequence showed that TcPV1 fall within a clade composed entirely of members of the genus Betapartitivirus, belonging to the family Partitiviridae. In light of this molecular evidence, TcPV1 is a new member of the genus Betapartitivirus. This is the first report of a new partitivirus hosted by the ascomycete, mycorrhizal fungus T. claveryi.

Revealing the Microbiome of Four Different Thermal Springs in Turkey with Environmental DNA Metabarcoding.

One of the most significant challenges for detecting microbial life in thermal springs by conventional techniques such as culturing is these places' physicochemical (temperature, heavy metal content, pH, etc.) conditions. Data from several studies suggest that high-throughput DNA sequencing technologies can be used to perform more accurate and detailed microbiome analyses. The primary aim of this paper was to determine the microbiome in the thermal source by metabarcoding environmental DNA isolated from four different sources and reveal the reflection of differences caused by temperature and chemical content on the microbiome. DNA was extracted from water filtered with enclosed filters and using the Illumina high-throughput sequencing platform, V3 and V4 regions of the 16S rRNA gene were sequenced. The results showed a correlation between physicochemical conditions and microorganism composition of four different thermal springs. Springs with extremely high temperature (89-90 °C) were dominated by hyperthermophiles such as Hydrogenobacter and Thermus, while a spring with a high temperature (52 °C) was dominated by thermophiles such as Thermoanaerobaculum and Desulfurispora, and a spring with a low temperature (26 °C) and high salinity was dominated by halophiles and sulfur-oxidizers such as Hydrogenovibrio and Sulfirimonas. With this research, we observed many manipulable steps according to the work of interest. This study sought to obtain data that will help decide the right gene region and choose the optimal bioinformatic pipeline.

Molecular characterization of a new mitovirus hosted by the ectomycorrhizal fungus Albatrellopsis flettii.

The complete genome of a novel mycovirus, Albatrellopsis flettii mitovirus 1 (AfMV1), hosted by the basidiomycetous ectomycorrhizal fungus Albatrellopsis flettii (Morse ex Pouzar) Audet, was sequenced and analyzed. The full-length cDNA sequence, obtained from a dsRNA replication intermediate of the AfMV1 genome, is 3037 bp in length with a predicted G+C content of 40.66%. Sequence analysis revealed that a single large open reading frame (ORF) is present on the positive strand when the mold mitochondrial genetic code is applied. The single ORF encodes a putative RNA-dependent RNA polymerase of 859 amino acids with a predicted molecular weight of 97.05 kDa that shares the closest similarity with the corresponding protein of Entomophthora muscae mitovirus 7, with 43.38% sequence identity. Phylogenetic analysis showed that AfMV1 could be classified as a new member of the genus Mitovirus within the family Mitoviridae. This is the first report of the complete genome sequence of a new mitovirus, AfMV1, isolated from the basidiomycetous ectomycorrhizal fungus A. flettii.

Full-length genome characterization of a new fusagravirus hosted by the spring orange peel fungus Caloscypha fulgens.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.

Molecular characterization of the complete genome of a novel partitivirus hosted by the saprobic mushroom Leucocybe candicans.

Virus communities of uncultivated fungi stay largely unknown. In the current study, we characterized a new partitivirus species detected in the basidiomycetous, saprobic mushroom Leucocybe candicans, named "Leucocybe candicans partitivirus 1" (LcPV1). The full-length genome of LcPV1, determined using deep sequencing and RLM-RACE approaches, consists of two dsRNA segments with each having the same size of 1984 bp. Both dsRNA genome segments comprise a single open reading frame (ORF), encoding an RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP show the highest identity (50.09% and 35.71% similarity, respectively) to those of partitiviruses reported from an oomycetous, plant pathogenic, stramenopile algae Plasmopara viticola and basidiomycetous, plant pathogenic fungus Ceratobasidium sp., respectively. Phylogenetic analyses performed based on the RdRp and CP sequences revealed that LcPV1 falls within a cluster that includes different alphapartitivirus species from the family Partitiviridae. In this study, we propose that LcPV1 is a new member of a species belonging to the genus Alphapartitivirus. To our knowledge, this is the first study reporting on a new fungal virus (mycovirus) identified in the basidiomycetous, saprobic mushroom Leucocybe candicans.

The unique genome organization of two novel fusariviruses hosted by the true morel mushroom Morchella esculenta.

Two putative mycoviruses belonging to the proposed family "Fusariviridae" were identified in Morchella esculenta by sequencing of double-stranded RNAs extracted from the morel mushroom. These viruses were tentatively named "Morchella esculenta fusarivirus 1″ (MeFV1) and "Morchella esculenta fusarivirus 2″ (MeFV2). Including the poly(A) tail the complete genomes of MeFV1 and MeFV2 are composed of 9096 and 9011 nucleotides (nt) respectively. Both genomes contain four non-overlapping open reading frames (ORFs) in which the largest and the smallest ORFs are ORF2 and ORF3 for both genomes respectively. The ORF1 of MeFV1 and MeFV2 are preceded by the 5' untranslated regions (UTRs) of 27 and 37 nt respectively and encode 341 and 339 aa long proteins that do not exhibit significant similarity to any of the protein sequences present in GenBank database. The 1502 and 1511 aa long proteins encoded by ORF2 of MeFV1 and MeFV2 share 84.42% sequence identity to each other and are 58.54% and 58.57% identical to the RNA-dependent RNA polymerase (RdRp) of Morchella importuna fusarivirus 1 (MiFV1) respectively. Interestingly, a Promethin/LDAF1 protein domain that is associated with the endoplasmic reticulum (ER) and lipid droplet (LD) membranes was identified at the N terminal regions of MeFV1 and MeFV2 RdRps, implying that the replication of these viruses is linked to the lipid membranes. The ORF3 and ORF4 of MeFV1 and MeFV2 encode proteins (268 and 333 aa long, and 645 and 647 aa long respectively) that only share significant sequence similarities with the proteins encoded by the ORF2 and ORF3 of MiFV1 respectively. The 3' UTRs of MeFV1 and MeFV2 are 162 and 159 nt long respectively and both of them have 51 nt long terminal poly(A) traits. To our knowledge, MeFV1 and MeFV2 are the first fusariviruses identified in M. esculenta and this is the first study reporting on the presence of Promethin/LDAF1 domain in viral RdRps.

Molecular characterization of a new endornavirus inhabiting the ectomycorrhizal fungus Hygrophorus penarioides.

Viruses hosted by uncultivated fungi have been poorly studied. We carried out studies to characterize a large dsRNA segment (~20 kbp) detected in the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides. The dsRNA was gel-purified and its randomly amplified cDNA fragments were used for high throughput sequencing (HTS). Reads were de novo assembled and BLASTx analysis revealed sequence similarity to viruses of the family Endornaviridae. The 5' and 3' terminal sequences of the dsRNA segment were determined by performing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full-length cDNA sequence of the putative endornavirus comprises 16,785 nt and contains a single, long open reading frame which encodes for a polyprotein of 5522 aa with conserved domains for cysteine-rich region, helicase, glycosyltransferase, and RNA-dependent RNA polymerase. The virus was named Hygrophorus penarioides endornavirus 1 (HpEnV1). A BLASTp search performed using the polyprotein sequence revealed that the most closely related, fully sequenced endornavirus to HpEnV1 is Ceratobasidium endornavirus B.

Molecular characterization of a novel partitivirus hosted by the false morel mushroom Gyromitra esculenta.

Virus populations of uncultivated fungi remain scarcely studied. In the present study, we characterized a new partitivirus isolated from the false morel mushroom Gyromitra esculenta, named "Gyromitra esculenta partitivirus 1" (GePV1). The complete genome of GePV1, whose sequence was determined by combining high-throughput sequencing and RLM-RACE approaches, comprises two dsRNA segments of 1971 bp and 1799 bp, respectively. Each dsRNA genome segment contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. The sequences of the RdRp and CP exhibited the highest similarity (69.77% and 47.00% identity, respectively) to those of Rosellinia necatrix partitivirus 2 (RnPV2). Phylogenetic analysis based on the CP and RdRp sequences demonstrated that GePV1 clusters within a clade that includes members of the genus Alphapartitivirus, family Partitiviridae. We propose that GePV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified in the false morel mushroom Gyromitra esculenta.

Novel and diverse mycoviruses co-inhabiting the hypogeous ectomycorrhizal fungus Picoa juniperi.

Viruses hosted by ectomycorrhizal fungi remain poorly studied. In this study, we detected eight new fungal viruses co-infecting a single isolate of the hypogeous ectomycorrhizal fungus Picoa juniperi using high-throughput sequencing. Phylogenetic analysis of one identified virus abbreviated as PjMTV1 revealed its closest relatives as members of the newly proposed family "Megatotiviridae". Phylogenetic analyses of two identified viruses abbreviated as PjV1 and PjV2 showed that these viruses are associated with members of the proposed family "Fusagraviridae". Phylogenetic analysis of the identified one another virus abbreviated as PjYV1 demonstrated that this virus is related to the members of the proposed family Yadokariviridae. The remaining four identified virus-like contigs were determined as segments of the bipartite dsRNA mycoviruses from the family Partitiviridae. The mycoviruses reported in this study are the first viruses described in Picoa juniperi, and PjMTV1 characterized herein is the secondly reported member of the newly proposed family "Megatotiviridae".

Novel and divergent bipartite mycoviruses associated with the ectomycorrhizal fungus Sarcosphaera coronaria.

Members of the family Partitiviridae are reported from a variety of fungal and plant taxa. After dsRNA-preparation, deep sequencing, and bioinformatics, we here reveal the existence of various divergent partitiviruses co-infecting the ectomycorrhizal fungus Sarcosphaera coronaria, symbiotically associated with the pine species Pinus brutia in Turkey. A total of 75 complete or nearly complete sequences related to the members of Alphapartitivirus and Betapartitivirus, were detected from the ascocarp sample of the fungal isolate. Two of the identified partitivirus genome segments encoding for partitiviral capsid protein represent evolutionarily distinct members of Alphapartitivirus, indicating that they may have diverged in the presence of long spatial isolation. In an attempt to match the two genome segments of the identified partitiviruses and distinguish individual species co-inhabiting a single host, nine possible genome segment pairs were identified.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Mitochondrial genetic variations of an introduced freshwater fish, goldfish Carassius auratus at the frontier between Europe and Asia (western Anatolia, Turkey): proximity to Europe rather than East Asia?

Carassius auratus is one of the most significant ornamental and food fishes of the world that is globally distributed and well known. Although it is known to have existed at least for six decades and expanding its distribution range in Turkish waters, there is a dearth of information on genetic structure and variations of goldfish in Turkey. In this study, four mitochondrial genes (Cytochrome b, cytochrome oxidase II, 12S ribosomal RNA, and 16S ribosomal RNA) were used to infer the genetic variations of goldfish populations sampled from western part of Anatolia, Turkey. Three populations were clustered under three haplotypes for each gene and all haplotypes were special. Cytochrome b was found to have more variable sites and higher genetic diversity than other genes. According to the haplotype networks, goldfish populations in Turkey showed high level of genetic structuring and originated from the common haplotype known in native East Asian populations of the species. Extensive sampling scheme covering whole Anatolia should provide better understanding on the dispersal pattern of the species.

Molecular evidence for the predation of Critically Endangered endemic Aphanius transgrediens from the stomach contents of world wide invasive Gambusia affinis.

Predation and competition among native and invasive species are difficult to study in aquatic environments. Identification of preys from semi-digested body parts sampled from stomach contents of the predator is very challenging. Recent studies were mainly based on use of DNA extracted from stomach content to identify the prey species. This study presents the molecular evidence that reveals the predation of critically endangered Aphanius transgrediens by world-wide invasive Gambusia affinis for a better understanding of the link between the invasion and the extinction of native species in freshwater ecosystems. DNA samples were extracted from semi-digested stomach contents of the invader and short fragments of mitochondrial NADH1 gene were amplified using species-specific primers designed in this study to make identification at species level. Existence of both the prey and the predator species were also confirmed using environmental DNA extracted from water samples.

Molecular identification of Hysterothylacium aduncum specimens isolated from commercially important fish species of Eastern Mediterranean Sea using mtDNA cox1 and ITS rDNA gene sequences.

The presence of a Raphidascarid parasitic nematode Hysterothylacium aduncum (Rudolphi, 1802) in two sparid fish (Sparus aurata and Diplodus vulgaris) and one soleid fish (Solea solea) was investigated in this study. A total of 868 individuals; 385 S. aurata, 437 D. vulgaris and 46 S. solea were collected from the Mersin Bay between February 2013 and January 2014 and examined. Variations in the prevalence, mean intensity, and mean abundance of the parasite were 14.55%, 2.05, and 0.30 for S. aurata, 4.12%, 2.44, and 0.10 for D. vulgaris, and 15.22%, 3.29, and 0.50 for S. sole respectively. Nucleotide sequences of 1398 base pair long fragment of 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA region and 641 base pair long fragment of mtDNA cytochrome c oxidase I (cox1) gene were used in molecular identification of isolated parasites at species level. All the parasite samples were identified as H. aduncum based on nucleotide sequence comparisons. Both ITS rDNA and mtDNA cox1 sequences revealed a genetic variation among H. aduncum specimens isolated from different fish species, while only mtDNA cox1 sequences were indicating a mean genetic distance of 0.010 among H. aduncum specimens of the same host species.

DNA barcoding commercially important fish species of Turkey.

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654-bp-long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2-parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour-joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.

Genetic structuring of Anguis fragilis (L., 1758) inhabiting in the north of 40° north latitude in Turkey.

The taxonomic situation of Anguis fragilis species is still unclear in Turkey. In order to clarify this situation, we used the DNA sequences of 16S rRNA and cytochrome b genes to analyze the phylogenetic relationship among A. fragilis populations. A total of 13 haplotypes in 16S rRNA dataset and 20 haplotypes in cytochrome b dataset were detected. Kimura 2-parameter genetic distance was found to be 0.012 for 16S rRNA and 0.026 for the cytochrome b dataset. Neighbor joining (NJ) trees were constructed to analyze phylogenetic relationship among specimens and were supported with median joining networks. Results indicate a clear genetic structuring in A. fragilis populations sampled from north of 40° north latitude of Turkey. Both mitochondrial gene sequences successfully detected the intraspecific variation among specimens of different populations. Genetic structuring, correlated with geographic distance, was found to be significant at the specimens sampled from edge populations of peripherally isolated climatic conditions.

DNA barcoding commercially important aquatic invertebrates of Turkey.

DNA barcoding was used in order to identify aquatic invertebrates sampled from fisheries bycatch and discards. A total of 440 unique cytochrome c oxidase sub unit I (COI) barcodes were generated for 22 species from three important phyla (Arthropoda, Cnidaria, and Mollusca). All the species were sequenced and submitted to GenBank and Barcode of Life Database (BOLD) databases using 654 bp-long fragment of mitochondrial COI gene. Two of them (Pontastacus leptodactylus and Rapana bezoar) were first records of the species for the BOLD database and six of them (Carcinus aestuarii, Loligo vulgaris, Melicertus kerathurus, Nephrops norvegicus, Scyllarides latus, and Scyllarus arctus) were first standard (>648 bp) COI barcode records for the GenBank database. COI barcodes were analyzed for nucleotide composition, nucleotide pair frequencies, and Kimura's two-parameter genetic distance. Mean genetic distance among species was found increasing at higher taxonomic levels. Neighbor-joining trees generated were congruent with morphometric-based taxonomic classification. Findings of this study clearly demonstrate that DNA barcodes could be used as an efficient molecular tool in identification of not only target species from fisheries but also bycatch and discard species, and so it could provide us leverage for a better understanding in monitoring and management of fisheries and biodiversity.

DNA barcoding common non-native freshwater fish species in Turkey: low genetic diversity but high population structuring.

Negative impacts of introduced non-native freshwater species on native species have been increasingly recognized in the world as well as in Turkey. However, there has been relatively little attention on genetic characterization of alien freshwater fishes in their non-native distribution range and virtually no study has been conducted in Turkey despite its crucial importance in invasion biology. The purpose of this study was to elucidate genetic diversity of common non-native freshwater fish species (Carassius auratus, Carassius gibelio, Gambusia holbrooki, Lepomis gibbosus, and Pseudorasbora parva) using mitochondrial Cytochrome c oxidase subunit I (COI) sequences; known as DNA barcodes. Through the whole dataset, seventeen haplotypes (haplotype diversity = 0.8908) were found containing 145 COI sequences. Mean Kimura two-parameter genetic distances were calculated as 0.209 for interspecific distance and 0.009 for intraspecific variation. COI barcode diversity among populations of the same species was found to be low, especially for C. gibelio, G. holbrooki, and L. gibbosus populations which were 0.5%, 0.6%, and 0.3%, respectively. Our results clearly demonstrate the effectiveness of the DNA barcoding approach both for identifications at species level and revealing intraspecific variation among populations, which could be used for effective management measures for invasive species and conservation strategies for indigenous and endemic species.