An analysis on the divergence of Chironomid spp. based on the study of 18S rRNA and polytene chromosome organization in the species revealing the role of environment on speciation

Nine species of Chironomus evolved throughout the world were measured for their divergence with regard to their DNA sequences concerning 18S rRNA since it is conserved for a specific species. With the advancement of the field of molecular evolution, cytogenetics requires further correlation between molecular architecture and morphological features of a species to compare amongst others to decipher their role in speciation. Therefore, divergence of DNA sequences of the Chironomus were compared with differences in the polytene chromosome features of most of the species under this investigation to evaluate underlying correlation among them, if any, to finally establish a novel method of molecular classification broadly applicable in cytogenetics studies. When Chironomus javanus Kieffer was considered as a reference organism, an in silico pair-wise alignment of sequences for the 18S rRNA gene regions of the other eight different species of the same genus exhibited nucleotide sequence homology ranging from 67 to 98%. This divergence of the species under consideration might be due to environmental impact causing alteration of nitrogenous bases probably due to mismatch pairing in DNA replication. This may be suggested as a cause of evolution of species in nature. A concomitant study on the polytene chromosome band patterns of majority of these species belonging to this series also indicated a divergence ranging from 10% to 30%. Sequence analysis based on 18S rRNA of nine species of Chironomus under this investigation shows a similarity in the polytene chromosome organization in most of the Chironomid species of the series. Hence, molecular divergence in the species is consistent with cytological difference among Chironomid species. Therefore, molecular data based on 18S rRNA and cytological characters based on the polytene chromosome features of the Chironomid species may be useful for their taxonomical recognition. Moreover, variations concerning two aspects of this study may be correlated to their environmental distinctions.


Background
The Chironomids belong to the insect order Diptera and they represent the family Chironomidae (Ashburner, 1970;Maitra, 2001). The members of the family are small fly like mosquitoes. Chironomid flies are known as 'blind mosquitoes', but they are neither blind and nor mosquitoes. The dipteran insects belonging to the genus Chironomus are considered as excellent biological indicators (El-Shenawy, Ahmed, Ismail, & Abo-Ghalia, 2010;Lotfi, Ahmed, El-Shatoury, & Hanora, 2016;Midya, Bhaduri, Sarkar, & Ghosal, 2013;Sarkar, Bhaduri, Ghosh, & Midya, 2011;Vermeulen, 1995;Warwick, 1985) inhabiting almost all ecological zones. They cause little harm to human life or other animals of interest. Known harmful impact of these flies refers to a variety of nuisance problems, health hazards, and human diseases.
Chironomids respond to a wide range of sensitivities to environmental parameters such as dissolved oxygen, acidity or alkalinity, salinity, water current, food, temperature, humidity, depth, pollution by organic wastes as well as by inorganic contaminants like heavy metals.
Based on their morphological analysis many species of the genus could be recognized by the taxonomists. However, in the current state of progression of biological studies, the morphotaxonomical analysis needs a correlation with molecular analysis of the species. When the data obtained through morphometric analysis, cytological analysis, and molecular analysis are clubbed together to designate a species, those may be of great help for using one organism for many biological investigations. In this regard, the species of Chironomus being ubiquitous in inhabiting various ecological habitats in the environment may be studied in terms of their molecular organization.
Therefore, in the present study, a comparison of the molecular data obtained from several species of Chironomus was considered to measure the degree of divergence of 18S rRNA. Concomitant with this, a comparison of cytological features of several Chironomid species has been carried out to find out their homology and divergence. Both the molecular and cytological analyses could show a correlation deciphering related features based on which the role of environmental impact on divergence among the Chironomus species may be suggested.

Methods
Sequence-based molecular analysis of 18S rRNA genes of Chironomid spp.
Nine species of Chironomus were considered for homology analysis for their 18S rRNA based on scientific reports of their worldwide ubiquitous distribution differing in ecological conditions. The species taken under consideration in the present study were Chironomus javanus Kieffer; Chironomus xanthus Rempel, 1939; Chironomus transvaalensis Kieffer; Chironomus (Lobochironomus) dorsalis Meigen, 1818; Chironomus riparius Meigen, 1804; Chironomus matures Johannsen, 1908; Chironomus maddeni sp.; Chironomus duplex sp. and Chironomus crassiforceps Kieffer, 1916. The 18S rRNA sequences of the studied species were reported by a number of investigators (Martin, Blinov, Alieva, & Hirabayashi, 2007). Experimentally obtained latest nucleotide sequence data for 18S rRNA genes of all of these nine species were retrieved from the NCBI website (https://www.ncbi.nlm.nih.gov/) for their pair-wise alignment using standard methods as briefly described below (Dey, Ganguli, Basu, Roy, & Datta 2010). Sequences were searched using the terms "18S rRNA" and "Chironomus" under the gene category of the NCBI website resulting in 18S rRNA gene sequences for a diverse Chironomid species from which the aforesaid nine species including Chironomus javanus were selected for the present analysis. Accession numbers and nucleotide sequences of these retrieved data are listed in the Supplementary Data 1.
The above-mentioned nucleotide sequences for this rRNA gene as obtained for different species under investigation were considered for pair-wise alignment taking the sequence of C. javanus as the reference one. Clustal Omega online server (https://www.ebi.ac.uk/Tools/msa/ clustalo/) was used to conduct the pairwise alignment of the retrieved nucleotide sequences of the 18S rRNA genes of the nine Chironomus species under study. Retrieved sequences in FASTA format were utilized to calculate percent sequence similarity and divergence, if any using standard protocols (Dey et al., 2010;Dey, Ganguli, Roy, & Basu, 2011).

Collection and rearing of Chironomus javanus
To investigate whether the obtained nucleotide sequence divergence of the said Chironomid spp. shows similar pattern in terms of cytological features, their polytene chromosomes were analyzed and compared taking Chironomus javanus as a reference species. The egg masses laid by females of Chironomus javanus were collected from outskirts of Kolkata, West Bengal, India. These egg samples were randomly collected throughout the year and from different water bodies to ensure sufficient sample size and remove time dependent bias, if any. They were made to hatch in the laboratory and the larvae developed from the eggs were reared in the culture trays with sufficient sterilized pond (or waterbody) water over the soil so that water submerge base of the culture trays at least by 1/2 inch. The larvae were  allowed to grow for about three weeks to achieve the penultimate fourth instar stage. The penultimate fourth instar larvae were taken from the culture trays to observe their polytene chromosomes in the salivary gland cells using below mentioned protocol.
Preparation of polytene chromosomes of Chironomus javanus and their cytological comparison with other Chironomid spp.
A comparison of the polytene chromosome arms of six species belonging to the above-mentioned series was also made to find out the homology or similarity between the species. For polytene chromosome morphology comparison, the species taken under consideration were Chironomus javanus Kieffer; Chironomus dorsalis Meigen, 1818; Chironomus riparius Meigen, 1804; Chironomus maturus Johannsen, 1908; Chironomus maddeni sp. and Chironomus duplex sp.
For this analysis, a comparison was made pair-wise also taking C. javanus as the reference organism as mentioned above. Polytene chromosomes of C. javanus as obtained from natural habitats of West Bengal, India, and reared as above, was characterized as per the formula given by Keyl (1957Keyl ( , 1962, Devai, Miskolczi, and Wülker, (1989) and Kerkis, Kiknadze, Filippova, and Gunderina, (1989).
Polytene chromosome from C. javanus were prepared from the salivary gland cells and stained with 2% acetoorcein using standard protocols (Hollenberg, 1976;Zacharopoulou, 1987). However, the chromosomes were studied under high power objective for determining its cytological features. The polytene chromosome features of the other Chironomus spp. used in this study have been taken from the works of some other investigators (Kiknadze, Michaĭlova, Istomina, Golygina, Int, & Krastanov, 2006;Kiknadze, Broshkov, Istomina, Gunderina, &   Vallenduuk, 2008; Martin, 1971;Martin et al., 2007;Yamamoto, 1977). The polytene chromosomes exhibiting major bands, Balbiani rings, constricted sites (waists), centromeric region, and NOR along each of the chromosome arms were taken into consideration for the comparative analysis. Both the data obtained from molecular analysis and cytological analyses were assessed for the degree of divergence of these species of Chironomus under consideration using Microsoft Excel software (Niglas, 2007).

Generation of dendrogram for the nine Chironomid spp. under study
A dendrogram or phylogram was generated on the basis of divergence and least distances between 18S rRNA nucleotide sequences of the above mentioned nine Chironomid species from the mini-matrices calculated based on sequence dissimilarity matrices. For this analysis, 18S rRNA nucleotide sequences of all nine Chironomid spp. were first aligned together and % identity was calculated. % dissimilarity was then calculated by subtracting the similarity values from 100%. Using similar methods, 18S rRNA nucleotide sequences of individual Chironomid species under study were then analyzed and % dissimilarity was calculated to draw the dendrogram thereby identifying the nearest neighbors with evolutionary relevance. GraphPad Prism and Microsoft Excel software were used for these calculations and representations (Motulsky, 2007;Niglas, 2007).    Fig. 1 and Supplementary Figure 1). Therefore, the two species diverged by 3%. Similar comparison between C. javanus and C. transvaalensis showed 81% homology and therefore they are 19% diverged from each other, C. javanus and C. dorsalis showed 68% homology and so they diverged by 32%, C. javanus and C. riparius showed 98% homology and so they possess 2% divergence, C. javanus and C. maturus showed 98% homology and so they have 2% divergence, C. javanus and C. maddeni showed about 67% homology, C. javanus and C. duplex showed homology value nearing 67%, C. javanus and C. crassiforceps showed 98% homology and so they have 2% divergence (Fig. 1). Hence, with regard to the homology of the sequences among these species indicated a nearness relation in the order as Chironomus javanus, Chironomus crassiforceps, Chironomus maturus, Chironomus riparius, Chironomus xanthus, Chironomus transvaalensis, Chironomus  dorsalis, Chironomus maddeni, and Chironomus duplex (Figs. 1 and 11). The polytene chromosome features from different arms of the chromosomes as obtained from six different species considered in this investigation have been shown in Table 1 below. Along with this table, the figures of the polytene chromosome arms of different species are showing there polytene chromosome features under consideration (Figs. 2, 3, 4, 5, 6, 7, and 8).
Pair-wise comparison of the polytene chromosome features as found in different species was performed with those obtained from C. javanus obtained in our laboratory. Comparison of the polytene chromosome features was mainly based on band number (major), Balbiani Ring (BR), Nucleolar Organizer (Lotfi et al., 2016), number of waists and centromeric heterochromatin region. The data obtained in relation to the above-noted polytene chromosome features of different species under investigation are shown in Table 2.
Therefore, the divergence of each of these species from C. javanus with regard to their polytene chromosome parameters may be indicated as under (Table 3, Fig. 9).
These macromolecular elements in the living organisms create hope among the taxonomists for characterizing species at the molecular level. The DNA segment promoting the synthesis of 18S rRNA of the ribosome has been found to be a conserved region in the DNA. Many investigators have tried to characterize the 18S rRNA gene of different species of Chironomus to reveal the variation in the nucleotide sequence usable for taxonomic categorization of the species (Gunderina et al., 2015;Martin et al., 2007;Michailova, Petrova, Bovero, Cavicchioli, Ramella, & Sella, 2000).
The present study dealt with the molecular features concerning the 18S rRNA gene sequences of nine chironomid species and the cytological features concerning    the polytene chromosome organization of majority of these species. A comparative analysis of the two aspects, i.e., molecular sequence and cytological characteristics as polytene chromosome bands, constrictions, Balbiani Rings were made in order to quantify their importance in speciation of Chironomids.
In this comparison and for easy analysis, C. javanus has been taken as the reference one and a comparison at random among the species have been done. For the sake of simplicity, a pair wise alignment of the sequence of C. javanus and that of any of the other considered species namely, Chironomus xanthus, Chironomus transvaalensis, Chironomus dorsalis, Chironomus riparius, Chironomus maturus, Chironomus maddeni, Chironomus duplex, and Chironomus crassiforceps was made to measure the range of their divergence in the organization of 18S rRNA gene (Supplementary Figure  1). The comparative molecular analysis of the species in terms of their polytene chromosome features as the number of bands (major), BR, NOR, waists, and centromere along each of the chromosome showed that the percentage of divergence from C. javanus ranged from a low of 11.4 ± 1.25 in C. matures to a high of 31.6 ± 2.62 in C. dorsalis (Table 3).
The flies of different species of Chironomus are identical in appearance but in minute details they differ greatly for achieving a distinct status of a species. This divergence may also be noted by cytological study at the chromosomal level because this species differs by morphological feature as a result of their genetic distinction (Gunderina et al., 2015;Martin, 1971).
It is therefore clear that divergence of the species or molecular evolution of Chironomid runs parallel along with the morphological features of the polytene chromosome organization.
The dendrogram based on the percentage of dissimilarity between different species examined indicated that G-H and C-B are closer to each other than other species (Fig. 11, Supplementary Figure 1).

Conclusions
A pair wise alignment of the 18S rRNA gene sequences of nine Chironomid species showed that the divergence of the species or molecular evolution of Chironomid runs parallel along with the morphological features of the polytene chromosome organization.