Chironomus sokolovae Istomina, Kiknadze et Siirin, 1999 (Diptera, Chironomidae) is recorded from Mongolia for the first time. Eleven banding sequences determined in the Mongolian population were previously known from Altai and Yenisei populations: sokA1, sokB1, sokB2, sokC1, sokC2, sokD1, sokD2, sokE1, sokF1, sokF2 and sokG1. The additional B-chromosomes are absent. DNA-barcoding of COI gene was carried out for this species for the first time. The phylogenetic tree estimated by Bayesian inference showed a high similarity of the studied species with Ch. acutiventris Wülker, Ryser et Scholl, 1983 from the Chironomus obtusidens-group. The estimated genetic distance K2P between Ch. sokolovae and Ch. acutiventris is much lower (0.38%) than the commonly accepted threshold of 3% for species of genus Chironomus Meigen, 1803. Our results show that the accepted cytogenetic criteria of species level in the genus Chironomus are more in accordance with morphological ones of the same level, than with molecular-genetic criteria accepted for species COI genetic distance.

Chironomidae, Chironomus sokolovae, COI, Diptera, DNA-barcode, karyotype, Mongolia

At present time, nine species of Chironomus Meigen, 1803 identified by imago (Hayford 2005; Shcherbina and Zelentsov 2008) are recorded from Mongolia. Seven additional species identified by imago were described from the country as new for science (Sasa and Suzuki 1997) but never found after the original description. Macrozoobenthos of the Orkhon (Kharkhorin) Reservoir on the Orkhon River near Kharkhorin city and river sections upstream and downstream of the reservoir, were studied for the first time during the fieldwork of the Joint Russian-Mongolian Complex Biological Expedition in 2017. Further research has shown that the species Chironomus sokolovae Istomina et al. 1999 was erroneously recorded by larvae as Ch. obtusidens Goethebuer, 1937 (Prokin and Sazhnev 2019) from the reservoir and connected sections of the Orkhon River with a total number of specimens up to 5000 ind/ m2 and the total biomass up to 3,75 g/m2. Larvae of Ch. commutatus Keyl, 1960 co-occurred with Ch. sokolovae (Prokin and Sazhnev 2019).

The species Ch. sokolovae belongs to Chironomus obtusidens-group including six species: Ch. acutiventris Wülker, Ryser et Scholl, 1983; Chironomus bavaricus Wülker, Ryser et Scholl, 1983; Ch. obtusidens Goethebuer, 1937; Ch. arcustylus Siirin, 2002; Ch. heterodentatus Konstantinov, 1956; Ch. sokolovae Istomina, Kiknadze et Siirin, 1999 (Siirin et al. 2002; Kiknadze et al. 2016). Chromosomal polymorphism and cytogenetic differentiation in this group are still poorly studied (Siirin et al. 2002; Kiknadze et al. 2007).

The known range of the species includes the Altai Krai, Altai Republic, and the Tyva Republic in Russia (Istomina et al. 2000; Siirin et al. 2002), and Mongolia (this publication). The species was described from the Chemal River (Altai Republic) and recorded from different water bodies in the Altai region and the Tyva Republic, where it co-occurred with Ch. acutiventris and Ch. heterodentatus (Istomina et al. 2000; Siirin et al. 2002). Numerous populations of Ch. sokolovae larvae inhabit silty sand of the Enisey River ripal zone, near the confluence of the Bolshoy and the Maliy Enisey Rivers (environs of Kyzyl city) (Siirin et al. 2002).

The karyotype and DNA barcoding of COI gene of the Ch. sokolovae from the Orkhon River (Mongolia) are described in this publication with the aim of clarifying the species position within the Ch. obtusidens-group.

Two larvae were collected from the bay of the Orkhon River upstream of the reservoir: 47°10.734'N,102°47.384'E, in September 2017. Depth 0.5 m, bottom – silty sand. Temperature 20.0 °C, pH 7.2, EC 172 mkSm/sm, TDS = 98 mg/l. For all analyses larvae were fixed in ethanol (95%). Two fourth instar larvae were used for karyotype analysis by the ethanol-orcein technique (Dyomin 1989). A Micromed-6C (LOMO, St. Petersburg) light microscope equipped with standart (kit) oil objective ×100, and camera ToupCam5.1 (China) were used for microscopy analysis. Cytomaps from Kiknadze et al. (2016), Keyl (1962), Wülker et al. (1983) and Dévai et al. (1989) were used to identify chromosome banding.

One larva which was studied karyologically was taken for the total DNA extraction using «M-sorb-OOM» (Sintol, Moscow) kit with magnet particles according to manufacturer’s protocol. For amplification of COI (cytochrome oxidase subunit I) we used primers LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA -3’) (Evrogen, Moscow) (Folmer et al. 1994). Amplification reaction was carried out in 25 μLreaction mixture (1× buffer, 1.5 μM MgCl2, 0.5 mM of each primer, 0.2 μM dNTP of each nucleotide, 17.55 μL deionized water, 1 μL template DNA, 1 unit Taq-polymerase (Evrogen, Moscow). PCR performed at 94 °C (3 min), followed by 30 cycles at 94 °C (15 s), 50 °C (45 s), 72 °C (60 s) and a final extention at 72 °C (8 min). PCR products were visualized on 1% agarose gels and later purified by ethanol and ammonium acetate (3 M). Both strands were sequenced on an Applied Biosystems 3500 DNA sequencer (Thermo Scientific, USA) following the manufacturer’s instructions.

For alignment of COI nucleotide sequences we used MUSCLE in the MEGA6 software (Tamura et al. 2013). The MEGA6 was used to calculate pairwise genetic distances Kimura 2-parameter (K2P) with codon position preferences: 1^st, 2^nd, 3^rd and noncoding sites (Kimura 1980). The Bayesian analysis was performed using the program MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2003; Ronquist et al. 2012) with settings suggested by Karmokov (2019), for 1,000,000 iterations and 1000 iterations of burn-in, nst = 6 (GTP + I + G). The phylogenetic trees resulting in Bayesian inference analyses were visualized and edited using FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

In addition, the 27 COI sequences of the genus Chironomus from “GenBank” and “Barcode of Life Data Systems” (BOLD; http://www.boldsystems.org) were analyzed. Accession numbers of used sequences in GenBank and BOLD: Chironomus acutiventris Wülker, Ryser et Scholl 1983 (AF192200.1), Ch. annularius Meigen, 1818 (AF192189.1), Ch. aprilinus Meigen, 1830 (KC250746.1), Ch. balatonicus Devai, Wulker et Scholl, 1983 (JN016826.1), Ch. bernensis Wülker & Klötzli, 1973 (AF192188.1), Ch. borokensis Kerkis, Filippova, Schobanov, Gunderina et Kiknadze, 1988 (AB740261), Ch. cingulatus Meigen, 1830 (AF192191.1), Ch. commutatus Keyl, 1960 (AF192187.1), Ch. curabilis Belyanina, Sigareva et Loginova, 1990 (JN016810.1), Ch. dilutus Shobanov, Kiknadze et Butler, 1999 (KF278335.1), Ch. entis Shobanov, 1989 (KM571024.1), Ch. heterodentatus Konstantinov, 1956 (AF192199.1), Ch. heteropilicornis Wülker, 1996 (MK795772.1), Ch. luridus Strenzke, 1959 (AF192203.1), Ch. maturus Johannsen, 1908 (DQ648204.1), Ch. melanescens Keyl, 1961 (MG145351.1), Ch. nipponensis Tokunaga, 1940 (LC096172.1), Ch. novosibiricus Kiknadze, Siirin & Kerkis, 1993 (AF192197.1), Ch. nuditarsis Keyl, 1961 (KY225345.1), Ch. obtusidens Goetghebuer, 1921 (CHMNO207-15*); Ch. piger Strenzke, 1959 (AF192202.1), Ch. pilicornis Fabricius, 1787 (BSCHI736-17*), Ch. plumosus (Linnaeus, 1758) (KF278217.1), Ch. riparius Meigen, 1804 (KR756187.1), Ch. tentans Fabricius, 1805 (AF110157.1), Ch. tuvanicus Kiknadze, Siirin et Wülker, 1993 (AF192196.1), Ch. whitseli Sublette & Sublette, 1974 (KR683438.1). The COI sequence of Ptychoptera minuta Tonnoir, 1919 (KF297888) was used as outgroup in phylogenetic analysis.

The Species Ch. sokolovae and Ch. acutiventris are similar in their karyotypes but differ in a few fixed inversions in arms B, C, D and F (Kiknadze et al. 2016). The COI sequences of these species are also similar, which could be the effect of a close relationship between the species, indicative of their recent origin (Michailova et al. 2021), or could be the result of interspecific hybridization with fixation of mtDNA in one of the parental species in the population (Guryev and Blinov 2002; Polukonova and Dyomin 2010, 2013). Study by Siirin et al. (2002) mentioned the existence of interspecific hybrids of Ch. sokolovae and Ch. heterodentatus, which means that hybridization between the Ch. sokolovae and Ch. acutiventris still occur as a result of living together in the same habitats and co-swarming. At the same time, the mtDNA sequences mostly allows the delimitation between sibling species in such groups of species as Ch. obtusidens, Ch. lacunarius, Ch. plumosus etc. (Fig. 2), that was originally founded based on morphological and cytogenetics traits. For a more detailed analysis of the species position within the group is needed to perform sequencing of mitochondrial and nuclear genomes, coupled with the preliminary cytogenetic and morphological analysis as obligatory.

Material was sampled during the expedition of the hydrobiological team of the “Joint Russian-Mongolian Complex Biological Expedition of the Russian and Mongolian Academies of Sciences”. The work was realized according to the Russia state projects AAAA-A19-119102890013-3, AAAA-A18-118012690106-7, 121050500046-8 and 121051100109-1. The authors are grateful to A.S. Sazhnev and A.V. Krylov (IBIW RAS) for help in fieldwork and to E.A. Movergoz, A.A. Bobrov, B.A. Levin (IBIW RAS) and M.Kh. Karmokov (IEMT RAS) for their help and consultations during all stages of the investigation and manuscript preparation; D.D. Pavlov (IBIW RAS) for the linguistic corrections of the text. The authors thank the reviewers for their valuable suggestions.