Karyotype and COI sequences of Chironomus sokolovae Istomina, Kiknadze et Siirin, 1999 (Diptera,Chironomidae) from the bay of Orkhon River, Mongolia

Abstract 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.


Introduction
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 andSazhnev 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 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.

Materials and methods
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

Results and discussions
Karyotype of Ch. sokolovae from the Orkhon River, Mongolia The chromosome set of the species is 2n = 8. The cromosome arm combination is AB, CD, EF and G (the Chironomus "thummi" cytocomlex). The additional B-chromosomes are absent. The chromosomes AB and CD are metacentric, EF is submetacentric, and G is telocentric. The karyotype of Ch. sokolovae is similar to Ch. acutiventris, but differs by fixed inversions in arms B, C, D and F   (Istomina et al. 1999;Siirin et al. 2002) (Fig. 1).
All 11 banding sequences found in Mongolian larvae were previously known for both the Enisey and the Altai populations of studied species (Istomina et al. 1999;Siirin et al. 2002). In Mongolian and Enisey populations sokB1 banding sequence was found only in the heterozygous state, whereas in the Altai population it was in the homozygous state (Istomina et al. 1999;Siirin et al. 2002).

DNA-barcoding and phylogenetic analysis
Single nucleotide sequence of Ch. sokolovae for the F6.2 gene from the tissue-specific Balbiani rings locus (Alieva et al. 2004) is accessible in GenBank (AF521040), while there is no sequences for barcoding. We obtained the COI barcode for Ch. sokolovae with the length of 665 nucleotides (percentage A: 16.9; T: 25.3; G: 11.8; C: 12.5) and deposited it into the GenBank database with accession number -MW471100.
According to Polukonova et al. (2009) andProulx et al. (2013) Chironomus COI interspecific sequence divergences is about 3%. In our study, interspecific divergence between Ch. sokolovae and Ch. acutiventris is 0.38%, that is much lower than the 3% accepted interspecific threshold. In most cases such low values of distances between species occur due to incorrect species identification when only morphological traits were used. To exclude a possibility of such mistake, we used karyological analysis and confirmed the accuracy of our species identification (Fig. 1).
Phylogenetic analysis using COI sequences showed groups of related species (Fig. 2), which concur with how these species were combined into the groups earlier on the basis of karyological and morphological traits Karmokov 2019). Obtained data are highly accurate (~100%) and show that Ch. sokolovae belongs to the Chironomus obtusidens group and is closest to Ch. acutiventris.

Conclusions
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 . 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;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.