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Article

DNA Barcoding of Black Flies (Diptera: Simuliidae) in Slovakia and Its Utility for Species Identification

by
Tatiana Kúdelová
1,
Samuel Krčmárik
1,*,
Ivona Lužáková
1,
Bibiana Bujačková
2,
Karin Matická
1 and
Matúš Kúdela
1
1
Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, SK84215 Bratislava, Slovakia
2
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, SK81108 Bratislava, Slovakia
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(5), 661; https://doi.org/10.3390/d15050661
Submission received: 30 March 2023 / Revised: 28 April 2023 / Accepted: 8 May 2023 / Published: 13 May 2023
(This article belongs to the Special Issue Diversity, Distribution and Phylogeny of Vector Insects)
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Abstract

:
DNA barcoding based on the cytochrome oxidase I gene is increasingly used in black flies (Diptera: Simuliidae), but extensive data for larger areas are still rare. Slovakia, with well-explored black fly fauna, was chosen to verify the reliability of DNA barcoding for species identification. The DNA barcoding region of the COI gene of 235 individuals of 25 black fly species from Slovakia was sequenced. Among them, 30 sequence clusters with assigned Barcode Index Numbers (BINs) were identified, and 5 of them were recorded for the first time. The average intraspecific genetic divergence was 0–3.24%, whereas the average interspecific divergence was 12.3–17.8%. Based on the barcode sequence, 14 species could be identified unambiguously, and 3 of them (Prosimulium latimucro, Simulium costatum, S. degrangei) are split into two or more barcode clusters. In eleven species, some degree of barcode sharing occurred, often combined with barcode splitting. The results showed hidden diversity as well as cases of shared barcode sequences among the studied species. Further investigation using other molecular markers is necessary due to the overlap of intraspecific and interspecific variability.

1. Introduction

Black flies (Simuliidae) are mainly known as pests and vectors of parasitic diseases. More than 2000 out of 2398 known species feed on the blood of birds or mammals [1]. About 28 species of vertebrate parasites have been recorded in black flies [2]. The main scientific interest has been focused on the vectors of human onchocerciasis. According to WHO estimates from 2017, 220 million people in the world need preventive drugs against onchocerciasis, 14.6 million have skin symptoms of the disease, and 1.15 million have impaired vision [3]. The most common diseases of veterinary importance transmitted by Simuliidae are bovine onchocerciasis [4,5] and avian leukocytozoonosis [6]. In addition to transmitting parasitic diseases, blackfly outbreaks are also problematic. The saliva of black flies contains anesthetic and anticoagulant substances, which can cause allergic reactions; numerous attacks can cause fever (so-called black fly fever), toxic shock, and in some cases, even death [7]. Grazing farm animals on pastures in the vicinity of black fly breeding sites are often stressed by constant attacks, which also causes a decrease in production and associated economic losses [8,9]. In addition to their great importance as a blood-sucking group, the larvae and pupae of black flies also represent an important part of flowing water communities, which also contributes to the need for better knowledge of their diversity. Cytogenetic studies covered about one quarter of known black fly species and revealed that the described morphospecies are often complexes of two or more sibling species. These species might have different habitat preferences and often differ in their ability to transmit parasitic diseases [10,11].
Although cytotaxonomy provides valuable information supplementing the classical taxonomy, molecular methods are being widely used as well. The chromosomal analysis requires a lot of experience, and it can be performed only on the last instar larvae, which were specifically fixed in an ad hoc solution of ethanol and acetic acid (3:1). Therefore, reliable molecular methods for species identification are needed. The most used molecular marker is the barcoding region of the mitochondrial COI gene. Comprehensive studies on the barcoding of black flies were performed for example in North America [12,13], Central America [14], and Thailand [15,16,17,18]. Several studies showed the applicability of barcoding in resolving a particular problem with the identity or identification of European black fly species. Barcoding helped to resolve the problem of the identity of Simulium reptans and confirmed the presence of a second similar species, Simulium reptantoides [19,20]. Used together with cytotaxonomy, the DNA barcoding unambiguously identified all four species of the Simulium aureum group in Great Britain [21] and it confirmed that the Italian endemic species Prosimulium italicum is not a synonym of the widely distributed and morphologically very similar Prosimulium hirtipes [22]. In Europe, larger studies on the barcoding of black flies are still missing, except for Spain, with a barcoding project covering 22 species and 199 individuals [23]. In this study, Slovakia, with its relatively well-explored black fly fauna consisting of 46 species [24], is used as an example area for verifying the reliability of the use of barcoding for black fly species identification.

2. Material and Methods

The larvae and pupae of black flies were collected from branches, rocks, and vegetation in streams and rivers at 26 locations in Slovakia (Table 1, Figure 1). Collected individuals were fixed in 96% ethanol. Larvae and pupae were identified with Zeiss SteREO Discovery.V12 stereoscopic microscope and Zeiss AxioLab microscope using multiple identification keys for European black fly taxa [25,26,27,28,29,30]. All specimens were identified to the species level based on the morphological characters; the only exceptions are two pupae of the subgenus Eusimulium, which were identified to the species level as Simulium angustipes Edwards, 1915 and Simulium rubzovianum (Sherban, 1961) based on their barcode sequence because no reliable morphological characters are known for identification of the pupae. Nomenclature follows the recent inventory of black flies [1]. Material was deposited at the Department of Zoology of Comenius University in Bratislava, Slovakia.
A small piece of larval or pupal muscle tissue from each specimen was used for DNA extraction. The rest of each specimen was stored in 96% ethanol at −20 °C for further analysis. The DNA was extracted using commercial kit prepGEM Insect (Zygem), following the instructions of the manufacturer.
The barcoding region of the cytochrome c oxidase subunit I (COI) fragment was amplified using the primers HCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and LCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [31]. The PCR reaction was performed in a total volume of 25 µL: DNA or 2.5 μL Buffer Dream (Fermentas), 2.5 μL MgCl2, 2.0 μL dNTPs, 0.5 μL of each primer, and 0.4 μL DNA polymerase DreamTaq (Fermentas), or 10 μL Red Taq 2X Master Mix, (1.5 mM MgCl2 included); 0.4 µL of each primer; 12.2 µL of Nuclease-Free water, and 2 µL of extracted DNA. The PCR program for COI gene fragment consisted of initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 30 s; the last polymerization was at 72 °C for 5 min.
The quality of PCR product was checked on 1% agarose gel stained with GoldView. PCR products were purified and sequenced by Macrogen Europe, Amsterdam, The Netherlands.
Sequences were aligned and modified using Geneious 6.1.8 [32]. Genetic distances were calculated using the program MEGA11 [33] using Kimura 2-parameter (K2P) with bootstrap support values estimated 500 replicates. The most suitable evolutionary models were also calculated in the jModelTest2 [34]. Phylogenetic trees were created using the maximum parsimony (MP) method in PAUP 4.0 [35] with 100 random additions, nearest neighbor interchange (NNI) algorithm, and heuristic search approach. Bootstrap support values were estimated 1000 replicates. MrBayes v.3.1.2 [36] program was used for Bayesian phylogenetic inference (BI), with four simulations of Markov chains, 5 M generation, and sampling every 100 generations, with 25% of trees discarded as burn-in. Maximum likelihood (ML) analysis was performed in RAxML [37] through raxmlGUI 2.0 [38] interface.
Two sequences of Drosophila melanogaster (Genbank Access Numbers: HM102299.1; HM102298.1) were used as outgroups [39].
All sequences were uploaded to the Barcode of Life Database (BOLD) into dataset DS-SIMSK—Black Fly Barcoding Slovakia (Diptera: Simuliidae), and subsequently to the GenBank database (accession numbers: OQ922995—OQ923229).
Haplotype networks for selected groups were constructed in the PopART (Population Analysis with Reticulate Trees) software [40], using TCS network method [41].
Maps were created in QGIS 3.30.0 [42], using basemaps from https://www.naturalearthdata.com (accessed on 15 February 2023) and https://www.eea.europa.eu/data-and-maps (accessed on 15 February 2023).

3. Results

The DNA barcoding region of 25 black fly species and 235 specimens was successfully amplified. The COI barcodes for three species and one unnamed taxon are published for the first time here (S. argenteostriatum, S. maximum, S. sp. aff. monticola, S. colombaschense). In 216 specimens of 23 species, we obtained the barcoding region’s full length (658 bp). The only exceptions were the species S. reptans and S. reptantoides, sequenced one-sided in the pilot step of the project, which produced at least 625 bp long sequences. None of the sequences contained stop codons, deletions, or insertions.

3.1. Genetic Distances

In the genus Prosimulium, the average intraspecific genetic distances ranged between 0.06% (P. rufipes) and 1.81% (P. latimucro), and the maximal value of intraspecific genetic distance was found in P. latimucro (3.64%). The minimal average interspecific genetic distance was found in P. hirtipes (5.08%) and the maximal average interspecific genetic distance was found in P. tomosvaryi (10.80%).
In the genus Simulium, the average intraspecific genetic distances ranged between 0 (S. sp. aff. monticola) and 3.24% (S. ornatum). The maximal intraspecific genetic distance was found in S. ornatum (7.79%), followed by S. costatum (7.13%). The average values of interspecific genetic distance varied between 15.85% and 20.28%.
The overlap of intraspecific and interspecific genetic distance occurred in ten species of the genus Simulium and two species of the genus Prosimulium (Table 2). The genetic distances and possible relations of the haplotypes for selected taxa are visualized in haplotype networks (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7).

3.2. Phylogenetic Trees

All phylogenetic trees (ML, MP, and BI) yielded similar topologies. All species groups formed well-supported clades (Figure 2), and 14 species formed monophyletic clades with high statistical support. On the other hand, 11 morphologically distinct species did not form monophyletic clades. Non-monophyletic clades formed one species of the genus Prosimulium (P. hirtipes), all species of S. variegatum group (S. argyreatum, S. monticola, S. variegatum, S. maximum, and S. sp. aff. monticola), two species of S. ornatum group (S. ornatum and S. trifasciatum), and two species of subgenus Wilhelmia (S. lineatum and S. balcanicum).

3.3. Assignment to Barcode Index Numbers (BINs)

Sequences of all 25 species were classified into 30 BINs. Five BINs were identified as unique (new in the database, Table 1), and three of them were assigned to species for which the COI barcodes are published for the first time. Ten species were assigned a single matching BIN each (P. tomosvaryi, S. erythrocephalum, S. angustipes, S. rubzovianum, S. cryophilum, S. argenteostiatum, S. reptans, S. reptantoides, S. colombaschense, and S. equinum). Three species (P. latimucro, S. vernum, S. degrangei) split into two or more BINs, yet those BINs were species-specific and formed monophyletic clades (Figure 2), thus enabling reliable identification of these taxa. In the case of S. costatum, two species-specific BINs were identified but the species appeared to be paraphyletic regarding S. cryophilum (Figure 2).
Two species of the subgenus Wilhelmia (S. lineatum, S. balcanicum) shared the same BIN (merge situation).
Nine of the species were mixtures, i.e., split into two or more BINs and at the same time shared at least one BIN with another species. P. hirtipes split into two BINs, one of them shared with the single BIN of P. rufipes. The same situation was observed in S. maximum with two BINs and one of them shared with S. sp. aff. monticola. S. ornatum split into four BINs and two of them also included specimens of S. trifasciatum, split into these two BINs. Three species (S. argyreatum, S. monticola, and S. variegatum) shared the same BIN, and one additional BIN was found for S. argyreatum.

4. Discussion

Since its establishment in 2003, DNA barcoding has become an effective method for specimen identification [43,44]. Thanks to the accessible public databases, the pace of specimen identification to the species level has greatly accelerated. The success rate of specimen identification based on DNA barcodes was nearly 100% in many taxonomic groups [43,45,46,47]. However, in some groups, the efficiency of DNA barcoding in species identification was lower [48,49,50].
In black flies, several comprehensive studies showed a high success rate of species identification. A study of 75% of the North American simuliid genera correctly identified nearly 100% of the morphologically distinct species based on DNA barcodes [12]. In Thailand, DNA barcodes provided 96% correct identification of 41 black fly species. Barcodes also successfully differentiated cytoforms of some species complexes; however, in S. siamense complex, the levels of success were only 33% [51]. A more recent study from Thailand revealed a 90% level of success in species identification but in several species groups, the efficiency of COI sequences for species identification was very low [18]. In subgenus Gomphostilbia, the values of intraspecific and interspecific genetic divergence overlapped in 7 out of 13 species implying that DNA barcoding to identify these species will be ambiguous [52].
Based on COI barcodes, we were able to unambiguously identify only 56% (14 out of 25) of species from our sample. The other 11 species shared a barcode with at least one other species and the values of intraspecific and interspecific K2P genetic distance were overlapping. Sharing of mitochondrial DNA between valid morphological, cytotaxonomic, and biological species has often been recorded across the animal kingdom and represents a serious disadvantage of mitochondrial barcoding [53,54,55]. The successful identification of species based on DNA barcoding relies on the differences between intraspecific and interspecific genetic divergence and their overlap could lead to errors in species identification. Species non-monophyly (i.e., paraphyly, polyphyly) is the main reason for overlap of intraspecific and interspecific genetic divergence [56].

4.1. Prosimulium, Hirtipes Group

All four species of genus Prosimulium reliably present in Slovakia were successfully sampled. Specimens of Prosimulium tomosvaryi formed a single well-defined haplotype group, listed under the BIN BOLD:AEA2402. In addition to our samples, the group also includes conspecific specimens from Germany and Romania [57]. Its nearest neighbor BOLD:ADJ9213 represents the British samples of P. tomosvaryi.
The alpine species Prosimulium latimucro (s.lat.) with intraspecific genetic distance of up to 3.64% shows remarkable mitochondrial diversity, being attributed to three BINs—BOLD:AEI9525, AEF3934, and BOLD:AEF3642. At this point, we consider these groups to be intraspecific diversity, being neighbors of one another and distant to other central European members of the genus. The overall number of BINs for this species, therefore, rises to six public [57]. Two new BINs are currently formed mostly by our specimens from the Tatra Mountains in Slovakia. The exceptions are one single unidentified specimen from Montenegro [57], and one specimen from Spanish Pyrenees (Prosimulium sp.) [23,57], both in BIN BOLD:AEF3642. These records indicate a much wider distribution of the clade containing our new BINs. The closest relative of this triplet of BINs as a whole, is Prosimulium petrosum Rubtsov, 1955 from Armenia (BOLD:ACQ0837) which is also considered a close relative of P. latimucro based on cytotaxonomy and morphology [58].
Two BINs have been identified within samples of Prosimulium hirtipes and P. rufipes (s.lat.). First of them, BOLD:AAB9204, a widespread haplotype group exclusive to P. hirtipes and recorded across its range from the United Kingdom, through Scandinavia to Romania, was present only in two Slovak specimens of P. hirtipes. BIN BOLD:AER9302, hitherto including a sole sample of P. hirtipes from Spain [23], was present in both, P. hirtipes and P. rufipes (s.lat.). The BIN was shared almost equally (15 specimens of P. hirtipes and 20 of P. rufipes), with most specimens carrying a fully identical sequence, not differing in even a single mutation. These unexpected results were consistent in independent DNA isolations and PCR reactions and across localities. The morphological identification of specimens was double-checked by the authors and permanent microscopic slides were made to avoid any ambiguity.
To conclude, in Slovakia the DNA barcoding based on COI 5P region allows reliable identification of P. tomosvaryi and P. latimucro (s.lat), however, it is not possible to distinguish P. hirtipes and P. rufipes (s.lat.) sharing a common haplotype group.

4.2. Subgenus Boophtora

Simulium erythrocephalum is the only European member of the subgenus Boophtora and has an extremely wide distribution, reaching from the United Kingdom in the west up to the Russian Far East (Adler 2022). Both samples from Slovakia matched the only BIN recorded for this species BOLD:AAJ6649, containing 66 records from various parts of Europe, Armenia, and China [57]. Despite the extant range of the species, so far, there is no indication of higher genetic diversity for this species; however, more detailed sampling is needed.

4.3. Subgenus Eusimulium

We analyzed only two specimens of the subgenus Eusimulium, representing two of three species recorded in Slovakia so far. Simulium angustipes fits with the only BIN recorded for this species containing 29 samples and distributed from the United Kingdom over Sweden, Finland, Germany, and Armenia up to China. Similarly, S. rubzovianum fits with the only BIN recorded for this species containing 50 samples (however, listed under different names) and recorded in various parts of Europe, Morocco, and Turkey. A study of all four species of the subgenus Eusimulium in Great Britain showed that the morphologically and chromosomally well-defined populations differed markedly in their COI barcodes [21] and therefore barcoding seems to be suitable for the identification of these species, which are morphologically extremely similar and except for adult males, not possible to identify based on morphological characters only. Therefore, it would be useful to verify if barcoding allows the identification of the Eusimulium species also outside of Great Britain.

4.4. Subgenus Nevermannia, Vernum Group

Within subgenus Nevermannia, we sampled and sequenced only three species of the vernum species group, therefore Nevermannia had the smallest representation among all analyzed groups. Two of the analyzed species had a significant divergence of the COI barcode, each consisting of two BINs, with a genetic distance of 3.12% in S. vernum and 7.13% in S. costatum. The BIN of S. costatum BOLD:AEH7122 with six samples from Slovakia is unique, the only sample of S. costatum BOLD:AEH4753 fits with five samples from Turkey, additionally S. costatum BOLD: AAD1733 was reported from Sweden, United Kingdom, Germany, and Austria but not found in Slovakia. Twelve specimens of S. vernum belong to the large group BOLD:AAB8624 consisting of 65 samples distributed across large areas of Europe, and one sample represents a new unique BIN (BOLD:AET1431). In addition, five more BINs were reported where samples identified as S. vernum occur in Europe and in southwestern Asia. The eight specimens of S. cryophilum showed quite small interspecific genetic distance of 0.48% and all of them are assigned to the BOLD:ACU9243, distributed across Europe.
Higher levels of intraspecific genetic distance usually indicate the presence of species complexes or sibling species [12,14]. In black flies, the value of maximal intraspecific genetic distance around 5% (4.58–6.5%) is typically linked to the species complexes [12]. Therefore, the high level of genetic distance within S. costatum (7.13%) indicates the presence of two species.
In the checklist of black flies of Slovakia, 13 species of the subgenus Nevermannia are listed [24], and 12 of them belong to the vernum species group. It is likely that other species of the vernum group occur in Slovakia, as seven of them have been recorded in the surrounding countries and several others in the Alps within Germany, Italy, or France [1]. A more detailed study of the barcodes of the vernum group based on a larger dataset is strongly needed.

4.5. Subgenus Simulium, Argenteostriatum Group

Simulium argenteostriatum COI sequences are published for the first time here and they represent a new and unique BIN BOLD:AEH6008, which is quite distant from all the other known barcodes. The most similar BIN is BOLD:ACQ0608 represented by S. aureofulgens (Terteryan, 1949) from Armenia with a minimal genetic divergence of 8.66% [57,59]. There is no record of a barcode for the other two species from the group. The closest species within our samples was S. degrangei with a minimal genetic divergence of 9.77%.

4.6. Subgenus Simulium, Bukovskii Group

Simulium degrangei, the only representative of this group in Slovakia, showed a high intraspecific variability (up to 1.63%) and consisted of three BINs (Table 1). The COI sequences, including samples from Slovakia, have already been studied [60], and apart from the higher genetic divergence, no indications of the presence of cryptic species were found, therefore we consider the variability to be intraspecific.

4.7. Subgenus Simulium, Ornatum Group

Only two species of the ornatum group, S. ornatum and S. trifasciatum, are known in Slovakia [24]. We morphologically identified both in our samples. The three individuals of S. trifasciatum were assigned to the BINs BOLD:AEW0869 and BOLD:AEN0363; both also contained individuals which we determined as S. ornatum. The 34 individuals of S. ornatum showed very high intraspecific variability up to 7.79% and were split into four BINs. The majority of them (20) belong to BOLD:AEN0363, which is distributed from the United Kingdom up to Iran and Georgia; however, it is missing in northern Europe. Three individuals in our sample were assigned to BOLD:AAN3313, a smaller group recorded only in the United Kingdom and in Spain so far and being close to the previous BIN. The second largest group (10) was assigned to BOLD:AEW0869, a group common in the United Kingdom and northern Europe and reaching up to Romania in the south and east. Finally, one individual is assigned to BOLD:AAN3313, a group containing mostly individuals identified as S. intermedium Roubaud, 1906 and with most sequences recorded from Finland, followed by Spain and the United Kingdom. For further analyses, a complex taxonomic revision of the S. ornatum complex and the entire ornatum group is essential.

4.8. Subgenus Simulium, Variegatum Group

The five species of the variegatum species group formed a paraphyletic clade. All species split into four groups corresponded to different BINs, however, the BINs and the species did not match at all. Simulium maximum and S. sp. aff. monticola formed a separate well-supported clade of two BINs (Table 1, Figure 2 and Figure 6), which was quite distant from the other species with a genetic divergence of at least 6.20%. BOLD:AES0919 consisted of five samples of S. maximum; BOLD:ACV0745 included all samples of S. sp. aff. monticola and four samples of S. maximum, this BIN showed only minimal diversity, sharing a fully identical haplotype among all samples. Simulium argyreatum, S. monticola, and S. variegatum presented the second well-supported clade within the variegatum group (Figure 2) consisting of three BINs. BOLD:ADK2119 contained two specimens of S. argyreatum, all the other 42 samples represented the BIN BOLD:AAB8783 and included all three species. The internal variability within this BIN was relatively high. The samples of these three species included pupae only; thus, the morphological identification of specimens does not leave any space for misidentification. A similar result was shown for these three species in Spain [23], and the other unpublished samples in BOLD confirm this pattern. In summary, DNA barcoding based on COI 5P is completely unusable for species identification within the variegatum species group in Europe.

4.9. Subgenus Simulium, Reptans Group

All three species of the reptans group form separate monophyletic clades with high bootstrap support in the phylogenetic trees and one matching BIN was assigned to each species (Figure 2). All species of reptans group can be unambiguously identified by the COI barcodes. The COI sequences of S. reptans and S. reptantoides have been studied across Europe [19,20,61], and both species differ consistently to a similar extent. Simulium reptantoides is missing in the Scandinavian and Baltic countries; however, two lineages of S. reptans occur in Sweden and Great Britain, and only one of them was recorded in central Europe and Balkan countries [19,20,61]. Simulium colombaschense COI sequences are published for the first time here. Previous chromosomal studies revealed the presence of five cytoforms with different geographical distributions, at least some of which represent reproductively isolated species. According to the place of origin of the sequenced S. colombaschense samples located in the Danube river, we assume that they belong to the cytoform ‘A’, which probably represents the nominal species [62].

4.10. Subgenus Wilhelmia, Lineatum Group

All three specimens of S. equinum represented a well-supported sister branch to S. balcanicum and S. lineatum in the phylogenetic trees (Figure 2), and they were assigned to BOLD:AAM3554, a barcode recorded in 89 samples distributed across Europe and reaching Turkey but missing in northern Europe. Simulium equinum in northern Europe is assigned to BOLD:AAP9428 (five specimens in Finland), and this BIN is recorded also for Turkey (three specimens). The other two species of Wilhelmia, S. balcanicum and S. lineatum, formed one well-supported branch in the phylogenetic trees. Despite a few differences between the species in the phylogenetic trees and haplotype network, we could not distinguish between them based solely on the COI barcode sequence. Both species shared one BIN (BOLD:AAM4036), although small differentiation between the species could be found in the phylogenetic trees and haplotype network (Figure 7). This result agrees with the previous study of these two species in larger areas of Europe and Turkey [63]. According to the chromosomal study [64] and the small but consistent morphological difference between the pupae of both species, they can be considered closely related sister species, and the overlap in their barcodes could be the consequence of incomplete lineage sorting.
Mitochondrial haplotypes mixed and shared between valid morphological and cytotaxonomic species have been repeatedly recorded across the black fly family [23,65,66,67] hindering the ability of DNA barcoding based on COI-5P region to reliably identify species. Our study increases the number of such cases.
In some cases, e.g., S. degrangei, despite high genetic diversity and multiple BINs, there are no indications of the existence of several taxa [60]. In others, such as S. ornatum group and S. vernum group, multiple cryptic species are expected to exist, which may correspond to separate haplotype groups after resolving. The most intriguing situations are those where haplotype groups are partially or fully shared between well-defined species, presumably due to phenomena such as retention of ancestral polymorphism and introgression [68,69].
In the future, it will be necessary to increase the sampling of taxa poorly represented and missing in this study. Nuclear markers may be required for problematic groups to resolve taxonomy and allow molecular identification.

Author Contributions

Conceptualization, T.K.; material collection, T.K., M.K., S.K. and I.L.; material identification, M.K., T.K., K.M. and S.K.; molecular methods, T.K., B.B., I.L. and S.K.; data processing and analyses, T.K., M.K. S.K. and I.L.; figures preparation, T.K., S.K. and I.L.; writing and editing manuscript, M.K., T.K., S.K. and I.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Slovak Research and Development Agency under the Contract no. APVV-19-0076 and by the VEGA grant No. 1/0704/20 of the Ministry of Education, Science, Research and Sport of the Slovak Republic.

Data Availability Statement

Data are publicly available in BOLD (Dataset DS-SIMSK) and GenBank Databases.

Acknowledgments

We are grateful to Ladislav Jedlička for his comments on the preliminary results of this study and to Zuzana Čiamporová-Zaťovičová for providing the material of Prosimulium latimucro from three sites in the Tatra Mountains. We thank the three anonymous reviewers, who helped to improve the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Sampling sites for analyzed specimens. The localities are listed and numbered in Table 1.
Figure 1. Sampling sites for analyzed specimens. The localities are listed and numbered in Table 1.
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Figure 2. Maximum parsimony (MP) tree based on 235 mitochondrial cytochrome c subunit I haplotypes of 25 black fly species based on 625 bp long alignment. Bootstrap support values for maximum parsimony (MP) and maximum likelihood (ML), and posterior probability values for Bayesian inference (BI) are shown above branches or near branches.
Figure 2. Maximum parsimony (MP) tree based on 235 mitochondrial cytochrome c subunit I haplotypes of 25 black fly species based on 625 bp long alignment. Bootstrap support values for maximum parsimony (MP) and maximum likelihood (ML), and posterior probability values for Bayesian inference (BI) are shown above branches or near branches.
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Figure 3. TCS haplotype network of COI 5P haplotypes from 58 individuals of four species of genus Prosimulium. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
Figure 3. TCS haplotype network of COI 5P haplotypes from 58 individuals of four species of genus Prosimulium. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
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Figure 4. TCS haplotype network of COI 5P haplotypes from 29 individuals of three species of subgenus Nevermannia. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
Figure 4. TCS haplotype network of COI 5P haplotypes from 29 individuals of three species of subgenus Nevermannia. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
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Figure 5. TCS haplotype network of COI 5P haplotypes from 28 individuals of two species of ornatum species group. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
Figure 5. TCS haplotype network of COI 5P haplotypes from 28 individuals of two species of ornatum species group. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
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Figure 6. TCS haplotype network of COI 5P haplotypes from 66 individuals of two species of variegatum species group. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
Figure 6. TCS haplotype network of COI 5P haplotypes from 66 individuals of two species of variegatum species group. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
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Figure 7. TCS haplotype network of COI 5P haplotypes from 15 individuals of three species of subgenus Wilhelmia. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
Figure 7. TCS haplotype network of COI 5P haplotypes from 15 individuals of three species of subgenus Wilhelmia. Mutational steps are represented by ticks across network connections and also by the number if higher than five.
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Table
Table 1. Species and BIN classification of the studied dataset.
Table 1. Species and BIN classification of the studied dataset.
Species BIN N Locality
Waterbody,
Municipality 		Coordinates
N, E 		Date No. of Specimen (Per Date)
Prosimulium hirtipes (Fries, 1824)AER930271 Čierny potok, Dolný Harmanec48.8201,
19.0342		1 May 2021
11 May 2021
10 June 2021		4
2
1
12 Kamenný potok, Častá48.3940,
17.3037		24 April 20211
73 Potok Račková, Pribylina49.1055,
19.8069		11 May 2021
10 June 2021		2
5
AAB920421 Čierny potok, Dolný Harmanec48.8201,
19.0342		11 May 2021
10 June 2021		1
1
Prosimulium latimucro  (Enderlein, 1925)AEF393414 Malé Žabie Javorové pleso, Tatranská Javorina49.2023,
20.1500		14 September 20091
15 Prostredné Spišské pleso, Vysoké Tatry49.1910,
20.1972		12 August 20091
26 Malé Spišské pleso, Vysoké Tatry49.1901,
20.2004		12 August 20092
AEI9525 *26 Malé Spišské pleso, Vysoké Tatry49.1901,
20.2004		12 August, 20092
AEF364226 Malé Spišské pleso, Vysoké Tatry49.1901,
20.2004		12 August 20092
Prosimulium rufipes  (Meigen, 1830)AER9302111 Čierny potok, Dolný Harmanec 48.8201,
19.0342		1 May 2021
11 May 2021
10 June 2021		3
6
2
93 Potok Račková, Pribylina49.1055,
19.8069		11 May 2021
10 June 2021		2
7
Prosimulium tomosvaryi  (Enderlein, 1921)AEA240221 Čierny potok, Dolný Harmanec48.8201,
19.0342		1 May 20212
47 Drieňovka, Bratislava-Nové Mesto48.1877,
17.1234		5 May 20214
32 Kamenný potok, Častá48.3940,
17.3037		24 April 20213
48 Závada, Kšinná48.8302,
18.3631		1 April 20174
Simulium erythrocephalum  (De Geer, 1776)AAJ664929 Vojčianske rameno, Kyselica47.9751,
17.3702		16 September 20202
Simulium angustipes  Edwards, 1915AAF4267110 Bíňovce48.5092,
17.4772		6 March 20221
Simulium rubzovianum  (Sherban, 1961)AAP9556113 Hydina, Uhrovec (Látkovce)48.6929,
18.3499		1 April 20175
Simulium costatum  Friederichs, 1920AEH7122 *612 Sučiansky potok, Nitrianske Sučany48.7431,
18.4564		1 April 20176
AEH4753112 Sučiansky potok, Nitrianske Sučany48.7431,
18.4564		1 April 20171
Simulium cryophilum  (Rubtsov, 1959)ACU9243513 Hydina, Uhrovec (Látkovce)48.6929,
18.3499		1 April 20175
314 Vríca, Vrícko48.9764,
18.6875		12 September 20203
Simulium vernum  Macquart, 1826AAB862417 Drieňovka, Bratislava-Nové Mesto48.1877,
17.1234		21 March 20221
113 Hydina, Uhrovec (Látkovce)48.6929,
18.3499		1 April 20171
415 Struha, Bratislava-Vajnory48.2258,
17.1815		21 March 20224
612 Sučiansky potok, Nitrianske Sučany48.7431,
18.4564		19 May 20186
AET1431115 Struha, Bratislava-Vajnory48.2258,
17.1815		21 March 20221
Simulium argenteostriatum  Strobl, 1898AEH6008 *83 Potok Račková, Pribylina49.1055,
19.8069		17 July 20198
Simulium degrangei  Dorier & Grenier, 1960ACD5131316 Belá, Liptovský Hrádok49.0448,
19.7227		13 June 20133
ACQ6722116 Belá, Liptovský Hrádok49.0448,
19.7227		13 June 20131
Simuium ornatum  Meigen, 1818AEN0363117 Dunaj, Bratislava-Devín48.1681,
16.9873		18 March 20221
311 Nitrica, Diviacka Nová Ves48.7339,
18.4993		27 July 20173
218 Nitrica, Liešťany (Lomnica)48.8436,
18.4701		27 July 20172
519 Striebornica, Uhrovec48.7530,
18.3625		20 September 20175
220 Váh, Liptovský Hrádok49.0349,
19.7112		13 June 20132
314 Vríca, Vrícko48.9764,
18.6875		12 September 20203
AEW0869521 Rudava, Veľké Leváre48.4888,
17.0096		25 September 20205
119 Striebornica, Uhrovec48.7530,
18.3625		20 September 20171
AAV2392111 Nitrica, Diviacka Nová Ves48.7339,
18.4993		27 July 20171
120 Váh, Liptovský Hrádok49.0349,
19.7112		13 June 2013
1
AAN3313120 Váh, Liptovský Hrádok49.0349,
19.7112		13 June 2013
1
Simulium trifasciatum  Curtis, 1839AEN0363219 Striebornica, Uhrovec48.7530,
18.3625		20 September 20172
AEW0869119 Striebornica, Uhrovec48.7530,
18.3625		20 September 20171
Simulium colombaschense  (Scopoli, 1780)ADZ9523 *522 Dunaj, Medveďov47.7887,
17.6651		16 May 20135
Simulium reptans  (Linnaeus, 1758)AAA9951923 Morava, Vysoká pri Morave48.3219,
16.9081		30 April 20139
Simulium reptantoides  Carlsson, 1962AAA9950724 Váh, Ivachnová49.0960,
19.4118		19 July 20147
316 Belá, Liptovský Hrádok49.0448,
19.7227		19 July 20143
Simulium argyreatum  Meigen, 1838AAB878371 Čierny potok, Dolný Harmanec48.8201,
19.0342		1 May 20217
325 Podhradský potok, Zliechov48.9699,
18.3714		30 June 20183
33 Potok Račková, Pribylina49.1055,
19.8069		17 July 20193
ADK211921 Čierny potok, Dolný Harmanec48.8201,
19.0342		1 May 20212
Simulium maximum  (Knoz, 1961)ACV074543 Potok Račková, Pribylina49.1055,
19.8069		10 June 20214
AES0919 *43 Potok Račková, Pribylina49.1055,
19.8069		10 June 20214
Simulium monticola  Friederichs, 1920AAB8783141 Čierny potok, Dolný Harmanec 48.8201,
19.0342		24 September 2020
1 May 2021		6
8
Simulium sp. aff. monticolaACV0745141 Čierny potok, Dolný Harmanec 48.8201,
19.0342		24 September 2020
1 May 2021		4
10
Simulium variegatum   Meigen, 1818AAB8783625 Podhradský potok, Zliechov48.9699,
18.3714		30 June 20186
93 Potok Račková, Pribylina49.1055,
19.8069		17 July 20199
Simulium balcanicum  (Enderlein, 1924)AAM4036426 Priesakový kanál, Bratislava-Rusovce48.0729,
17.1395		23 March 20194
Simulium equinum  (Linnaeus, 1758)AAM3554311 Nitrica, Diviacka Nová Ves48.7339,
18.4993		27 July 20173
Simulium lineatum  (Meigen, 1804)AAM4036117 Dunaj, Bratislava-Devín48.1681,
16.9873		18 March 20221
711 Nitrica, Diviacka Nová Ves48.7339,
18.4993		27 July 20177
*—unique (new) BIN in BOLD database. N—number of specimens per BIN and locality. 1–26—numbers of localities used in Figure 1.
Table
Table 2. Genetic distances of analyzed species calculated by genera.
Table 2. Genetic distances of analyzed species calculated by genera.
SpeciesnIntraspecific Genetic DistancesInterspecific Genetic Distances
MinMaxAverageStdMinMaxAverageStd
P. hirtipes *170.00000.01950.00470.00720.00000.11870.05080.0491
P. latimucro80.00000.03640.01810.01220.06780.09970.07770.0099
P. rufipes *200.00000.00480.00060.00110.00000.11470.05430.0487
P. tomosvaryi130.00000.00640.00230.00160.08860.11870.10800.0071
S. erythrocephalum20.00320.00320.00320.00000.11840.18800.14290.0175
S. angustipes1N/AN/AN/AN/A0.07650.19420.15800.0149
S. rubzovianum1N/AN/AN/AN/A0.07650.19140.16450.0132
S. costatum *70.00000.07130.02250.03360.05860.20280.14530.0253
S. cryophilum80.00000.00480.00160.00150.05860.19420.15180.0279
S. vernum130.00000.03120.01170.00700.08770.18150.14430.0196
S. argenteostriatum80.00160.01460.00580.00360.09770.18140.14290.0125
S. degrangei40.00000.01630.00920.00650.09770.18340.14580.0144
S. ornatum *250.00000.07790.03240.02460.00000.19230.12750.0236
S. trifasciatum *30.00480.05670.01390.02680.00000.18580.11390.0399
S. colombaschense50.00160.00320.00260.00080.06700.15850.12390.0205
S. reptans90.00000.01300.00320.00470.06000.16650.11640.0219
S. reptantoides100.00000.01300.00670.00440.06000.17310.12340.0218
S. argyreatum *150.00000.04150.01430.01420.00000.19210.10490.0530
S. maximum *80.00000.01630.00880.00730.00000.20060.11060.0483
S. monticola *140.00000.00810.00340.00230.00000.19840.10420.0536
S. sp. aff. monticola *140.00000.00000.00000.00000.00000.19840.11490.0447
S. variegatum *150.00000.01130.00550.00240.00000.18980.10400.0531
S. balcanicum *40.00000.01630.01030.00590.00810.18080.15000.0295
S. equinum30.00000.00160.00110.00080.11670.20280.17770.0185
S. lineatum *80.00000.02960.01720.00880.00810.18130.15180.0229
*—species with overlapped maximal intraspecific and minimal interspecific genetic distances, overlapping values in bold. Std—standard deviation. n—number of specimens.
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Kúdelová, T.; Krčmárik, S.; Lužáková, I.; Bujačková, B.; Matická, K.; Kúdela, M. DNA Barcoding of Black Flies (Diptera: Simuliidae) in Slovakia and Its Utility for Species Identification. Diversity 2023, 15, 661. https://doi.org/10.3390/d15050661

AMA Style

Kúdelová T, Krčmárik S, Lužáková I, Bujačková B, Matická K, Kúdela M. DNA Barcoding of Black Flies (Diptera: Simuliidae) in Slovakia and Its Utility for Species Identification. Diversity. 2023; 15(5):661. https://doi.org/10.3390/d15050661

Chicago/Turabian Style

Kúdelová, Tatiana, Samuel Krčmárik, Ivona Lužáková, Bibiana Bujačková, Karin Matická, and Matúš Kúdela. 2023. "DNA Barcoding of Black Flies (Diptera: Simuliidae) in Slovakia and Its Utility for Species Identification" Diversity 15, no. 5: 661. https://doi.org/10.3390/d15050661

APA Style

Kúdelová, T., Krčmárik, S., Lužáková, I., Bujačková, B., Matická, K., & Kúdela, M. (2023). DNA Barcoding of Black Flies (Diptera: Simuliidae) in Slovakia and Its Utility for Species Identification. Diversity, 15(5), 661. https://doi.org/10.3390/d15050661

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