Barcode Region Research Articles

DNA EXTRACTION TECHNIQUES FOR SOME WILD SPECIES FROM UZBEKISTAN

Although DNA extraction is already a routine procedure and protocols have been developed for a large number of plant species, various plant tissues and organs, solving the problem of extracting pure undegraded DNA from plant is the main step in molecular approaches for study plant organisms. The extraction of high-quality DNA from plant materials is an important factor for DNA barcoding. The effective DNA extraction protocol for samples with high content of admixture, which prevents the restriction reaction and the PCR, or degraded DNA should be established for each case. This work presents the comparison of DNA isolation techniques from silica gel dried and herbarium samples of some wild species collected in Uzbekistan. In this work, we optimized the technique for isolating the total DNA, on the examples of 16 representatives of Salvia, Dracocephalum, Phlomoides, Tulipa, Iris, Allium genera from Uzbekistan. The results of a comparative analysis of three methods of DNA extraction from leaf tissues of wild plants are represented in this article. DNA amount and purity degrees were measured using spectrophotometer. The efficiency of the DNA extraction method was assessed according to two criteria: the quantitative parameters -DNA concentration (ng/μl), and the qualitative parameters - DNA purity (nm). The DNA concentration variate from 758.1 ng/μl obtained with the CTAB method to 13.89 ng/μl obtained with commercial column extraction kits. λ = 260/280 ratio is range from 1.7 to 1.98 nm. Additionally, PCR amplification was realized to reflect the validation of the DNA isolation protocol, using specific primers of four chloroplast (rbcL, matK, psbA, trnL-F) and one nuclear (ITS) plant DNA barcode regions. The sequence length was 300–800 bp appropriately.

Molecular analysis reveals multiple native and alien Phoxinus species (Leusciscidae) in the Netherlands and Belgium

Fresh waters are among the most endangered ecosystems, one of the problems being the lack of data on biodiversity. In the center of the missing knowledge are cryptic species, two (or more) species classified as a single one due to their (seemingly) indistinguishable morphology. Lack of research and stabilizing selection are reflected in the cryptic diversity of the genus Phoxinus (Leusciscidae), the studies of which have intensified over the last two decades and reveal undetected taxonomic complexity. Moreover, some of the Phoxinus lineages act as invasive species, while others are endangered by their alien counterparts. Minnows have been intentionally (as food for predatory fish species) or unintentionally (with other fries) stocked causing hybridisation zones in Norway, Portugal, Spain, France, Italy, Germany and Austria. Given that genetic identity and lineage assignment of Phoxinus from Belgium and the Netherlands have not been researched, the goal of the study was to examine available samples from known localities in the area in order to infer- whether they are native or not. For this purpose, the barcoding region cytochrome oxidase I, another mitochondrial gene cytochrome b, a nuclear recombination activating gene 1 and a combination of these markers from a wider neighboring region were analyzed. The study found four different Phoxinus species/lineages occurring in Belgium and the Netherlands: P. phoxinus, P. csikii, P. septimaniae and genetic lineage 11 (possibly P.cf. morella). While the first seem to be native, the other three were probably introduced.

The complete nucleotide sequence of Viburnum odoratissimum chloroplast genome

The complete chloroplast genome (cpDNA) Viburnum odoratissimumis sequenced and assembled from the whole genome data. The cpDNA of V. odoratissimum is 158,744bp in lengthwith the overall GC content of 38.1%. It consists of a pair of inverted repeats (IRa and IRb, 26,494bp) which separate a large single copy (LCS, 87,348 bp) and small single copy (SSC,18,408 bp). The complete chloroplast genome contains 129 genes, including 84 protein-coding genes, 8 rRNA genes and 37 tRNA genes. A phylogenetic tree of DNA sequences of barcoding regions, including rbcL, matK, psbA-trnHfrom 8 species of the Genus Viburnum shows that V. odoratissimum is closely related to V. furcatum and V. burejaeticum.

Molecular phylogeny and DNA barcode regions efficacy for identification of the cultivar of Capsicum annuum L. in Thailand

ISHS IX International Scientific and Practical Conference on Biotechnology as an Instrument for Plant Biodiversity Conservation (physiological, biochemical, embryological, genetic and legal aspects) Molecular phylogeny and DNA barcode regions efficacy for identification of the cultivar of Capsicum annuum L. in Thailand

DNA-Based Identification of Eurasian Vicia Species Using Chloroplast and Nuclear DNA Barcodes

Many legume species of the Vicia L. genus (Fabaceae Lindl.) are key components of the Mediterranean diet and have an integral role in sustainable agriculture. Given the importance of the Vicia species for Eurasian culture, it is necessary to implement methodologies, such as DNA barcoding, that can enable the effective authentication and identification of species in the genus. In this study, we analysed the chloroplast trnL and rpoC1, as well as the nuclear ITS2 DNA barcoding regions, to identify 71 Vicia specimens of Eurasian descent. Both the trnL and ITS2 regions were highly effective in discriminating the analysed taxa, while the more conserved rpoC1 region could not identify all of the selected species due to high sequence conservation or non-annotated or absent rpoC1 species sequences in GenBank. A dendrographic representation of the generated trnL data showed sufficient clustering for most of the analysed taxa, although some topological discrepancies were observed. ITS2 and rpoC1 reconstructions were also used for resolving the topological discrepancies observed in the trnL tree. Our analysis suggests that a combination of DNA barcoding regions is essential for accurate species discrimination within the Vicia genus, while single-locus analyses do not provide the necessary resolution.

Checklist of decapod crustaceans from the coast of the So Paulo state (Brazil) supported by integrative molecular and morphological data: V. Dendrobranchiata and Pleocyemata [Achelata, Astacidea, Axiidea, Caridea (Alpheoidea and Processoidea excluded), Gebiidea, Stenopodidea

This checklist is the fifth and last compilation on the decapod crustaceans reported to So Paulo (Brazil) coastal area, resulting from long-term multidisciplinary projects, which combined morphological analyses and molecular techniques. The current research includes 75 decapod species, herein referred as shrimps/lobsters-like (shrimps, ghost-shrimps, lobsters, and related groups), reported to So Paulo coastal area. These species occur in marine, estuarine, and amphidromous habitats and are classified into 21 families as follow: Aristeidae (2 spp., 2 genera), Atyidae (4 spp., 2 genera), Axianassidae (1 sp., 1 genus), Callianassidae (1 sp., 1 genus), Callichiridae (6 spp., 4 genera), Crangonidae (1 sp., 1 genus), Glyphocrangonidae (1 sp., 1 genus), Luciferidae (2 spp., 2 genera), Nephropidae (4 spp., 2 genera), Palaemonidae (15 spp., 9 genera), Palinuridae (2 spp., 1 genus), Pandalidae (1 sp., 1 genus), Pasiphaeidae (1 sp., 1 genus), Penaeidae (10 spp., 6 genera), Sergestidae (3 spp., 3 genera), Sicyoniidae (4 spp., 1 genus), Scyllaridae (5 spp., 3 genera), Solenoceridae (4 spp., 3 genera), Stenopodidae (2 spp., 1 genus), and Upogebiidae (6 spp., 1 genus). We generated new sequences of cytochrome oxidase subunit I (barcode region) and 16S genes (51 and 54, respectively) of 54 species. Our examination concluded that 75 shrimps/lobsters-like species are reported to the So Paulo coast. We excluded Leander tenuicornis (Palaemonidae), Penaeus setiferus (Penaeidae), Philocheras gorei (Crangonidae), and Rhynchocinetes typus (Rhynchocinetidae) from this list.

Efficient Localization of Multitype Barcodes in High-Resolution Images

Barcode positioning technology is one of the important components of barcode technology. However, most of the current algorithms are only applicable to a single type of barcode positioning or limited to low-resolution images due to a large amount of calculation, and thereby it is still a challenge to locate multitype barcodes in high-resolution images. In response to the above problems, this paper proposes a reliable multitype barcode localization method for multibarcode localization in high-resolution images where one-dimensional (1D) barcodes, two-dimensional (2D) barcodes, or multitype barcodes are present simultaneously. The method consists of three main steps: first, extracting multiple types of barcode features through a joint edge detection algorithm; next, marking target barcode regions with a bidirectional contour labeling method; and finally, extracting barcode regions by an improved affine transformation. The experimental results show that, in terms of localization accuracy, the proposed method has a better accuracy of 97.83% than existing algorithms in low-resolution images and can locate multitype barcodes in high-resolution images with an accuracy rate of 98.04%. Besides, in terms of time cost, the proposed method effectively reduces the time cost by 50% and improves the barcode localization efficiency.

Employing Barcode High-Resolution Melting Technique for Authentication of Apricot Cultivars

Fast, accurate and affordable identification of food products is important to ensure authenticity and safety. There are various apricot (Prunus armeniaca L.) cultivars are being produced in Turkey. Each cultivar differs in quality and purpose of use. In this paper, we aimed to develop an easy and reliable method, Barcode High-Resolution Melting (Bar-HRM), to distinguish apricot cultivars. We designed and tested novel Bar-HRM primer sets HRM-ITS1 and HRM-ITS2, targeting the most popular barcoding region ITS1 and ITS2, specific to apricot cultivars. According to the results, HRM analysis distinguished 31 cultivars of 35 for ITS1, and 35 for ITS2. We recommend using ITS2 barcode region, amplified with using HRM-ITS2 primer set, for Bar-HRM analysis of different apricot cultivars.

Characteristics and comparative analysis of Mesona chinensis Benth chloroplast genome reveals DNA barcode regions for species identification.

Mesona chinensis Benth (MCB) is an important medicinal and edible plant in Southern China and Southeast Asian countries. Chloroplast (cp) genome is usually used for plant phylogeny, species identification, and chloroplast genetic engineering. To characterize the cp genome and determine the evolutionary position and perform the genetic diversity analysis of MCB, we sequence and characterize the MCB cp genome. The results show that the cp genome of MCB is a single circular molecule with a length of 152,635bp. It is a typical quadripartite structure, comprising a large single-copy region (LSC, 83,514bp) and a small single-copy region (SSC, 17,751bp) separated by two inverted repeat regions (IRs, 51,370bp). It encodes 129 unique genes, including 84 protein-coding genes (PCGs), 37 transfer RNAs (tRNAs), and 8 ribosomal RNAs (rRNAs). Altogether 127 simple sequence repeats (SSRs) are identified in the MCB cp genome with 86.61% of mononucleotide repeats. Phylogenetic analysis reveals that MCB is most closely related to Ocimum basilicum based on the whole cp genomes. Several highly divergent regions are found, such as trnH_psbA, rps16_trnQ, trnS_trnG, trnE_trnT, psaA_ycf3, rpl32_trnL, ccsA_ndhD, ndhG_ndhI, and rps15_ycf1, which can be proposed for use as DNA barcode regions. Genetic diversity analysis unveils a relatively narrow genetic basis of MCB germplasm resources. Therefore, the innovative breeding of MCB is very urgent and necessary in future research.

Potamophylax idliri sp. nov. (Trichoptera: Limnephilidae), a new species from the Jastrebac Mountains in Serbia, with molecular and ecological notes.

In this paper, we describe a new species, Potamophylax idliri sp. nov., from the Jastrebac Mountains in Serbia, which is morphologically most similar to Potamophylax coronavirus Ibrahimi, Bilalli Vitecek 2021 (in Ibrahimi et al. 2021) and Potamophylax juliani Kumanski 1999 (in Kumanski Malicky 1999). The males of the new species differ from all known species of the Potamophylax winneguthi Species Group by morphological characters of the male genitalia. The genetic distinctiveness calculated based on sequencing of the barcode region of the cytochrome c oxidase subunit I gene (COI) between Potamophylax idliri sp. nov. and other species of the Potamophylax winneguthi Species Group is on par with those of other morphologically recognized species in the group and especially the other four species of the P. winneguthi Species Cluster. Potamophylax winneguthi Species Cluster is erected for the following species: P. idliri sp. nov., P. winneguthi, P. haidukorum Malicky 1999 (in Kumanski Malicky 1999), P. juliani, and P. coronavirus, characterized primarily by the bilobed apical margins of inferior appendages with pointed corners, in lateral and caudal views. The new species was found at two localities in the Jastrebac Mountains, at 988 and 891 m above sea level. Considering the knowledge about distribution and ecology of the other species of this group, we posit that P. idliri sp. nov. is a microendemic of the Jastrebac Mountains in Serbia.

In Silico Evaluation of the Taxonomic Resolution and Coverage of the COI Region and Alternative Barcode Markers for Echinoderms

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses.

First Records of the Palearctic Species Trechus obtusus Erichson from the Appalachian Region of the Southeastern United States (Coleoptera: Carabidae: Trechinae: Trechini)

The Palearctic species Trechus obtusus Erichson is reported for the first time from eastern North America. Recent collections in Virginia and North Carolina (new state records) indicate the establishment of the species in the Appalachian region. DNA was extracted from six individuals, and the COI barcoding region was sequenced. All but one of the COI barcode haplotypes were identical to those of T. obtusus collected in western North America. No matches were found with available Palearctic sequences. Individuals of T. obtusus were collected from caves and forest leaf litter, both habitats used by native trechine species. Sampled Appalachian populations of T. obtusus appear to be small, with the exception of the population in Mebane Saltpeter Cave (Pulaski County, VA), which is large and included teneral individuals. Currently, there is no evidence that the species is displacing or otherwise negatively affecting native trechines. The species should be monitored carefully going forward, and targeted sampling of synanthropic habitats will be the best method for early detection.

The taxonomic identity of the monocle bream Scolopsis vosmeri species complex (Perciformes: Nemipteridae), with comments on molecular phylogenetic relationships within the genus Scolopsis.

The monocle bream Scolopsis vosmeri species complex is revised. Three species in the complex are recognized: Scolopsis vosmeri (Bloch, 1792), widespread in the Indo-West Pacific, from the northern Indian Ocean (Pakistan, western India, Sri Lanka, Bay of Bengal, and the Andaman Sea, but not recorded from the Red Sea or Arabian Gulf, east African coast or Madagascar) to western Indonesia and Borneo; S. japonica (Bloch, 1793), restricted to the western Pacific Ocean from western Indonesia and north-western Australia east to the Philippines and north to southern Japan; and S. curite Cuvier, 1815, widespread from the western to the eastern Indian Ocean, including the Red Sea and Arabian Gulf. All three species are similar morphologically, and have been confused taxonomically, but phylogenetic analysis of the COI barcoding region shows they are evolutionarily divergent. The three species are redescribed in detail and characters found to distinguish them. Scolopsis vosmeri is easily distinguished from S. japonica and S. curite in having a white band along the side of the body; having a black spot on most body scales (versus greenish yellow spot in S. japonica and S. curite); in lacking a distinct black spot (sometimes a small and faint spot present) on the upper pectoral-fin base (versus small black wedge-shaped spot present in S. japonica and S. curite); caudal peduncle whitish in live individuals (versus caudal peduncle usually yellowish in S. japonica and S. curite); and pelvic and anal fins crimson to orange-red (versus yellow in S. japonica and S. curite). Scolopsis japonica and S. curite are indistinguishable by color pattern but differ in the degree of spination on the preopercular margin. Neotypes are designated for Scolopsis japonica and S. curite. Nomenclatural problems, including validity of the genus Scolopsis, are discussed. We regard Scolopsis curite Cuvier, 1815 as a valid binomial name and thus the type species of Scolopsis Cuvier, 1814 by subsequent monotypy.

Integrative Taxonomy Reveals a New Melitaea (Lepidoptera: Nymphalidae) Species Widely Distributed in the Iberian Peninsula

Abstract The Melitaea phoebe group is constituted by six species distributed throughout the Palearctic. One of the most widespread species is Melitaea ornata Christoph, 1893, present from France (Provence) to Central Asia. Recently, populations of M. ornata were discovered in a mountainous region of south-eastern Iberia, although doubts about their taxonomy existed. To clarify the taxonomic status of these populations and to revise the distribution of this taxon in Iberia, we have sequenced mitochondrial (COI barcode region) and nuclear (wg, RPS5, MDH, and EF-1α) markers, and analyzed the male genitalia for 72 Iberian individuals and for all the species of the M. phoebe group. This information was complemented with phenological and ecological data. Our results unveiled that the Iberian M. ornata-like taxon is in fact distributed through most of the Iberian Peninsula, except for the south-west and north-east. In contrast to the univoltine M. ornata, the Iberian taxon can be bivoltine in the wild. The Iberian taxon was retrieved to be related to M. ornata, but the differences in the genetic markers and genitalia were comparable to those found between species in the group. Based on the evidence here presented and according to species delimitation results, we propose to consider the Iberian taxon as a novel species, tentatively named Melitaea pseudornata Muñoz Sariot & Sánchez Mesa, 2019, stat. nov.

Genetic variation in Iranian populations of citrus cottony scale (Pulvinaria aurantii Cockerell) based on RAPD, mitochondrial DNA and RFLP markers

In the present study, we used RAPD marker, COI gene sequence and RFLP analysis to investigate the genetic variation of 11 Pulvinaria aurantii Cockerell populations from different regions in northern Iran. The twenty-one RAPD primers generated 834 amplification products, of which 702 (84.17%) bands were polymorphic. The genotypic similarity of the populations ranged from 0.46 to 0.85 based on the Dice similarity coefficient. UPGMA dendrogram indicated that there is no clear relationship between genetic distances and geographical distances. An 850 bp fragment of COI gene was sequenced for 33 samples, resulting in five haplotypes. COI sequencing indicated that 63.63% of the samples (seven populations) had the same nucleotide sequence as outgroup (Japan). Enzymatic digestion of P. aurantii COI gene (656 bp fragment) resulted in 315 restriction fragments with 11 different sizes. The results indicated genetically differences of the populations but the level of variations was independent of geographical distances. Successful amplification of the COI barcode region of Pulvinaria aurantii Twenty-one RAPD primers generated 834 bands with 84% polymorphism Identify five different haplotypes based on the COI barcode region Detection of genetic diversity among some populations based on RAPD, COI and RFLP No clear correlation was found between geographical distances and genetic distances

Pliocene-Pleistocene dispersal bring along low inter species diversity between Vimba species based on multilocus analysis

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.

DNA barcoding of the horsefly fauna (Diptera, Tabanidae) of Croatia with notes on the morphology and taxonomy of selected species from Chrysopsinae and Tabaninae

In the Croatian fauna, horseflies (Tabanidae) are represented by 78 species belonging to two subfamilies, five tribes, and 10 genera. Identification of these species is based on morphological characteristics. In this study, 43 species of horseflies were analyzed. The highest number of species (19) belongs to the genus Tabanus, followed by the genera Hybomitra with seven species, Haematopota with six species, Chrysops with four species, Atylotus and Philipomyia with two species each, and the genera Silvius, Dasyrhamphis, and Heptatoma with one species each. The standard DNA barcoding region of the mitochondrial cytochrome c oxidase gene, subunit I (COI), was sequenced and compared to the Barcode of Life Database (BOLD). Our analyses confirmed our morphological identifications and added 16 new Barcode Index Numbers (BINs) for Tabanidae to BOLD. Potential problems in the systematics and taxonomy of this family are highlighted.

The complete chloroplast genome of Onobrychis gaubae (Fabaceae-Papilionoideae): comparative analysis with related IR-lacking clade species

BackgroundPlastome (Plastid genome) sequences provide valuable markers for surveying evolutionary relationships and population genetics of plant species. Papilionoideae (papilionoids) has different nucleotide and structural variations in plastomes, which makes it an ideal model for genome evolution studies. Therefore, by sequencing the complete chloroplast genome of Onobrychis gaubae in this study, the characteristics and evolutionary patterns of plastome variations in IR-loss clade were compared.ResultsIn the present study, the complete plastid genome of O. gaubae, endemic to Iran, was sequenced using Illumina paired-end sequencing and was compared with previously known genomes of the IRLC species of legumes. The O. gaubae plastid genome was 122,688 bp in length and included a large single-copy (LSC) region of 81,486 bp, a small single-copy (SSC) region of 13,805 bp and one copy of the inverted repeat (IRb) of 29,100 bp. The genome encoded 110 genes, including 76 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosome RNA (rRNA) genes and possessed 83 simple sequence repeats (SSRs) and 50 repeated structures with the highest proportion in the LSC. Comparative analysis of the chloroplast genomes across IRLC revealed three hotspot genes (ycf1, ycf2, clpP) which could be used as DNA barcode regions. Moreover, seven hypervariable regions [trnL(UAA)-trnT(UGU), trnT(GGU)-trnE(UUC), ycf1, ycf2, ycf4, accD and clpP] were identified within Onobrychis, which could be used to distinguish the Onobrychis species. Phylogenetic analyses revealed that O. gaubae is closely related to Hedysarum. The complete O. gaubae genome is a valuable resource for investigating evolution of Onobrychis species and can be used to identify related species.ConclusionsOur results reveal that the plastomes of the IRLC are dynamic molecules and show multiple gene losses and inversions. The identified hypervariable regions could be used as molecular markers for resolving phylogenetic relationships and species identification and also provide new insights into plastome evolution across IRLC.

Species authentication of canned mackerel: Challenges in molecular identification and potential drivers of mislabelling

The market for canned mackerel is growing due to their competitive price and to the excellent properties of the meat. However, the weak rules of current legislation, coupled with the loss of discrimination power due to being obliged to use short barcode regions, open up new challenges for traceability in processed products. In this study, for the first time, we applied a two-step mini-barcoding approach to identify canned mackerel sold in Italian markets and make assumptions on the potential drivers of mislabelling. Preliminary identification was performed using mini-barcode universal primers targeting a COI-fragment; then, in order to discriminate within the complex Scomber colias/S. japonicus/S. australasicus, we applied a second step, using new specific primers designed in the mitochondrial control region (D-loop). Comparison between labels and molecular identifications highlighted that the chance of mislabelling could be either 45% or 0%, depending on the interpretation of the generic terms used on the labels. Therefore, the study revealed that the ambiguous use, in the ingredient list, of ‘umbrella’ terms for naming fish species, such as ‘sgombro’ could be related to geographic origin of products and creates opportunities for fraudulent activities, but also misleads consumers. In light of this pattern, an urgent update of European legislation is needed to face current and future challenges for full chain traceability.

DNA barcoding of hard ticks (Ixodidae), notes on distribution of vector species and new faunal record for Croatia

Molecular methods are increasingly being utilized for accurate identification of ticks (Acari: Ixodidae), especially in cases of morphologically highly similar species. In this study, we performed molecular research of the tick fauna in Croatia using DNA barcoding method. Ticks were sampled in three biogeographical regions and thirteen species were recorded: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma marginatum, Ixodes frontalis, Ixodes hexagonus, Ixodes kaiseri, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus sanguineus s.l. and Rhipicephalus turanicus. Ixodes kaiseri is for the first time recorded in the fauna of Croatia. Of the thirteen hard tick species analyzed in this study, pathogens from different groups (bacteria, protozoa and viruses) have been detected in eight species in Croatia so far. For the important vector species R. sanguineus s.s., new distributional data for Croatia are given. The standard COI barcoding region was amplified, and the sequences were analyzed by species delimitation methods together with the sequences of conspecific and congeneric species from the public BOLD database. Our specimens of H. punctata represent a new, genetically distinct MOTU. A brief overview of the available public DNA barcoding data for Ixodidae is presented, highlighting the need for an integrative approach for the clarification of the taxonomic status of problematic Ixodid taxa. The results provide a basis for the establishment of a molecular data platform for the Ixodidae of the Croatian fauna.