Biosyst. Divers., 2022, 30(1) Biosystems Diversity ISSN 2519-8513 (Print) ISSN 2520-2529 (Online) Biosyst. Divers., 2022, 30(1), 3–11 doi: 10.15421/012201 The biology of Simulium erythrocephalum and S. chelevini (Diptera, Simuliidae): Morphological, ecological and molecular data K. B. Sukhomlin*, M. O. Zinchenko*, O. P. Zinchenko*, V. S. Tepliuk*, Y. V. Biletskyi*, V. V. Ivantsiv*, M. G. Biletska*, L. V. Buslenko*, V. V. Ivantsiv**, S. V. Budnik* *Lesya Ukrainka Volyn National University, Lutsk, Ukraine **Lutsk National Technical University, Lutsk, Ukraine Article info Received 15.01.2022 Received in revised form 08.02.2022 Accepted 09.02.2022 Lesya Ukrainka Volyn National University, Voli av., 13, Lutsk, 43025, Ukraine. Tel.: +38-033- 272-01-23. E-mail: Sukhomlin.Katerina @vnu.edu.ua Lutsk National Technical University, Lvivska st., 75, Lutsk, 43000, Ukraine. Tel.: + 38-033-274-61-03. E-mail: v.ivantsiv71@gmail.com Sukhomlin, K. B., Zinchenko, M. O., Zinchenko, O. P., Tepliuk, V. S., Biletskyi, Y. V., Ivantsiv, V. V., Biletska, M. G., Buslenko, L. V., Ivantsiv, V. V., & Budnik, S. V. (2022). The biology of Simulium erythrocephalum and S. chelevini (Diptera, Simuliidae): Morphologi- cal, ecological and molecular data. Biosystems Diversity, 30(1), 3–11. doi:10.15421/012201 The subgenus Boophthora is a typical Palearctic taxon, which includes only 6 species, among them. Simulium erythrocephalum has a transpalearctic distribution. In Europe, Adler notes only the species S. erythrocephalum, and Yankovsky – two species S. ( Boophthora ) erythrocephalum and S. ( Boophthora) chelevini . According to morphological characteristics, these species differ in their life stages. We have studied the development of S. erythrocephalum and S. chelevini from three rivers of Volyn region, Ukraine (Styr, Chornoguzka, Putylivka) from 2017 to 2019. We used the EPPO PM7 / 129 standard. Collected samples, 615-bp fragments of the COI gene were sequenced from five individuals of S. erythrocephalum and five individuals of S. chelevini and compared with four samples of S. erythrocephalum from the GenBank. We obtained the nucleotide sequence of S. chelevini . All of the S. erythrocephalum samples from Ukraine had 692 bases, the S. erythrocephalum samples from Armenia had 673 bases. S. erythrocephalum and S. chelevini did not have any intraspecific variations. These intraspecific variations were not larger than the interspecific variations. It has been proved that the populations of S. erythrocephalum and S. chelevini from medium and small rivers of Volyn do not differ in biological, behavioural and genetic characteristics. Comparison of S. erythrocephalum and S. chelevini life stages showed clear differences in 20 morphological features, which are probably manifestations of phenotypic variability. Comparison of species with data from the GenBank from Spain and Armenia on the mitochondrial cytochrome c oxidase subunit I (COI) gene confirmed the opinion that S. erythrocephalum and S. chelevini are one species. On the phylogenetic tree, the data are not grouped, there is no clear separation of the clades. Bootstrap values are 95–100%, which may indicate a significant similarity of all studied samples and the lack of isolation of individual morphotypes from Volyn, Spain and Armenia. To finally confirm the taxonomic posi- tion of these two species, additional research is needed covering more individuals from different parts of Europe and analysis of more genes. Keywords : black fly; subgenus Boophthora; Ukraine; mitochondrial DNA; cytochrome c oxidase subunit I (COI); taxonomy; genetic differences; phylogenetic relationships. Introduction Black flies are amphibiotic two-winged insects. Immature stages (eggs, larvae and pupae) are attached to the substrate in flowing water- courses, and adults live in the terrestrial habitats. These are ectoparasites of humans, farm animals, vectors of dangerous parasitic and infectious dis- eases (Sukhomlin & Zinchenko, 2007). Black flies occupy a leading place among blood-sucking dipterans in natural and anthropogenic landscapes of Ukraine, so the study of Simuliidae requires a detailed investigation at the genetic and morphological levels. This work is a continuation of a series of articles on the confirmation of the taxonomic status of certain problem species using modern molecular genetics methods (Zinchenko et al., 2021). Black flies of the subgenus Boophthora live in the Palaearctic. The subgenus includes only 6 species (Adler, 2021. World black flies (Diptera: Simuliidae): A с omprehensive revision of the taxonomic and geographical inventory): S. bujakovi Rubtsov, 1940 (Siberia), S. erythro- cephalum (De Geer, 1776) (Transpalearctic), S. guiyangense Chen, Liu, Yang, 2016 and S. quattuorfile Chen, Wu, Yang, 2010 (China), S. ma- kunbei (Ono, 1977), 2010 and S. yonagoense Okamoto, 1958 (Japan). In Europe, Adler (2021) notes only the species Simulium erythrocepha- lum, and Yankovsky (2002) – two species Simulium ( Boophthora) eryth- rocephalum and S. ( B. ) chelevini Ivashchenko, 1968. Since these species are morphologically quite clearly different, we decided to find out if there were any genetic differences. For the first time in Ukraine, the species S. ( B. ) erythrocephalum was registered by Rubtsov (1940), and S. ( B. ) chelevini by Panchenko (2004). These are multivoltine (three generations are produced annually), eurytopic black flies which have exploited every conceivable habitat from trickles to rivers. Habitats of larvae and pupae of the subgenus Boophthora species are large (Pripyat, Zakhidnyi Bug), medium (Styr, Stokhid, Turia, Goryn, Pivdennyi Sluch, Zherev, Teterev) and small (Konopelka, Gapa, Vyzhivka, Veselukha, Lva, Putylivka, Rita, Tsyr, Kormin, Cherevakha, Rudka, Chernyavka, Ikva, Stubla, Ustya, Zamchysko, Serehivka, Zhylzhanka, Vyrka, Yazvynka, Rudynka, Mura- vynka, Ubort, Uzh, Zvyzdal, Loznytsia) rivers, streams and meliorative canals (Fig. 1). These species are registered as active bloodsuckers of farm animals and humans. According to Yankovsky (2002), Kaplich et al. (2015) S. ( B. ) erythro- cephalum and S. ( B. ) chelevini in morphological characteristics are diffe- rent species that differ in all life stages. Adults differ in the structure of the genitalia, colour of the legs; larvae – a pattern on the frontoclypeal apoto- ma, the shape of the postgenal cleft, the hypostomal teeth; pupae – by gills branching and the number of spines on the abdomen VIII tergite. Molecular data are becoming an increasingly important tool in insect taxonomy (Simon et al., 1994; Sebastiani et al., 2001; Szalanski et al., 2006; Ruiz-Arrondo et al., 2018). Studies of COI gene sequences for S. erythrocephalum were implemented for black flies of Armenia (Werner & Kampen, 2012; Andrianov et al., 2015) and Spain (Ruiz-Arrondo et al., 2018). For S. chelevini such studies were not conducted, which deter- mined the relevance of our work. In the literature there is only information about the biology of the black flies subgenus Boophthora , in particular, the distribution is de- 3