Abstract
DNA barcoding based on a fragment of mitochondrial Cytochrome C Oxidase subunit 1 gene (COI) was applied to the two chironomids Clunio balticus Heimbach (690 base pairs) and C. ponticus Michailova (691 base pairs). The two species differed by one deletion in the nucleotide sequence Adenine. However, the 658-nucleotide long sequences of DNA from the mitochondrial Cytochrome C Oxidase subunit 1 gene (COI) of C. balticus and C. ponticus were identical upon comparison. Further, they compared with homologous sequences for C. marinus Holiday and C. tsushimensis Tokunaga from the Barcode of Life (BOLD) database and the results plotted as a weighted graph, where C. tsushimensis, C. marinus and C. balticus C. ponticus formed three almost equidistant groups. From this, we established that the genetic distance between the respective COI sequences of C. balticus and C. ponticus is minimal, indicating a close relationship between the species indicative of recent common origin. However, the comparative analysis between C. tsushimensis, C. marinus, C. balticus and C. ponticus showed a wider divergence in their respective nucleotide sequences. Overall, our results emphasized that the COI region does not work well as a DNA barcode to identify species within the Clunio genus. Either longer sequences or a multifaceted methodological approach, including morphology, cytogenetic and ecology is needed to distinguish some members of Clunio genus.
References
Caspers, H. (1951) Rhythmische Erscheinung in der Fortplanzung von Clunio marinus (Diptera, Chironomidae) und das Problem der lunaren Periodizität bei Organismen. Archiv fur Hydrobiologie, Supplement 18, 418–575.
Ekrem, T., Willassen, E & Stur, E. (2007) A comprehensive DNA sequence library is essential for identification with DNA barcodes. Molecular Phylogenetics and Evolution, 43, 530– 542.
https://doi.org/10.1016/j.ympev.2006.11.021
Folmer, O., Hoeh, W.R., Black, M.B. & Vrijenhoek, R.C. (1994) Conserved primers for PCR amplification of mitochondrial DNA from different invertebrate phyla. Molecular Marine Biology and Biotechnology, 3, 294–299.
Fuhrmann, N. & Kaiser, T. (2020) The importance of DNA barcode choice in biogeographic analyses a case study on marine midges of the genus Clunio. Genome, 8, 1–11.
https://doi.org/10.1139/gen-2019-0191
Hashimoto, H. (1975) Seasonal Emergence of Clunio aquilonius Tokunaga (Diptera, Chironomidae). Entomological Society of Japan, 43, 1, 49 – 54.
Hebert, P., Cywinska, A., Shelley, L. Ball, S.T. & de Waard, J.R. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society London B: Biological Sciences, 270, 313–321.
https://doi.org/10.1098/rspb.2002.2218
Heimbach, F. (1978) Sympatric species, Clunio marinus Hal. and C. balticus n. sp. (Dipt., Chironomidae), isolated by differences in diel emergence time. Oecologia, 32, 195–202.
https://doi.org/10.1007/BF00366071
Hubert, N., Hanner, R., Holm, E., Mandrak, N.E., Taylor, E., Burridge, M. & Watkinson, D. (2008) Identifying Canadian freshwater fishes through DNA barcodes. PLoS ONE, 3, e2490.
https://doi.org/10.1371/journal.pone.0002490
Jimbo, U., Kato, T. & Ito, M. (2011) Current progress in DNA barcoding and future implications for entomology. Entomological Science, 14 (2), 107–124.
https://doi.org/10.1111/j.1479-8298.2011.00449.x
Kaiser, T., Neumann, D., Heckel, D. & Berndonk, Th. (2010) Strong genetic differentiation and postglacial origin of populations in the marine midge Clunio marinus (Chironomidae, Diptera). Molecular Ecology, 19, 2845–2857.
https://doi.org/10.1111/j.1365-294X.2010.04706.x
Kaiser, T., Poehn, B., Szkiba, D., Preussner, M., Sedlazeck, F., Zrim, A., Neumann, T., Nguyen, L.-T., Betancourt, A., Hummel, Th.,Vogel, H., Dorner, S., Heyd, F., Haeseler, A. & Raible, K.T. (2016) The genomic basis of circadian and circa-lunar timing adaptations in a midge. Nature, 540 (7631), 69–73.
https://doi.org/10.1038/nature20151
Kress, W.J., Garca-Robledo, C., Uriarte, M. & Erickson, D.L. (2015) DNA barcodes for ecology, evolution and conservation. Trends in ecology & evolution, 30, 1, 25–35.
https://doi.org/10.1016/j.tree.2014.10.008
Lencioni, V., Marziali, L. & Rossaro, B. (2007) I Ditteri Chironomidi morfologia, tassonomia, ecologia, fisiologia e zoogeografia. Science Museum of Trento (Ed.), Quaderni del Museo Tridentino di Scienze Naturali. Vol. 1. Esperia, Lavis, Trento, pp. 1–172.
Li, W., Cowley, A., Uludag, M., Gur, T., McWilliam, H., Squizzato, S. & Lopez, R. (2015) The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Research, 43 (W1), 580–584.
https://doi.org/10.1093/nar/gkv279
Lin, X., Stur, E. & Ekrem, T. (2015) Exploring Genetic Divergence in a Species-Rich Insect Genus Using 2790 DNA. PLoS ONE, 10 (9), e0138993.
https://doi.org/10.1371/journal.pone.0138993
Makarchenko, E.A., Semenchenko, A.A. & Palatov, D.M. (2018) New data on taxonomy and systematics of the genus Diamesa Meigen (Diptera: Chironomidae: Diamesinae) from Tien Shan and Pamir mountains, with description of two new species. Journal of Limnology, 77, 50–58.
https://doi.org/10.4081/jlimnol.2018.1783
McWilliam, H., Li, W., Uludag, M., Squizzato, S., Park, Y.M., Buso, N. & Lopez, R. (2013) Analysis tool web services from the EMBL-EBI. Nucleic Acids Research, 41 (W1), 597–600.
https://doi.org/10.1093/nar/gkt376
Michailova, P. (1980) A review of the European species of genus Clunio Haliday, 1855 (Diptera, Chironomidae). Zoologischer Anzeiger, 205 (5/6), 417–432.
Montagna, M., Mereghetti, V., Lencioni, V. & Rossaro, B. (2016) Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae). PLoS ONE, 11, 1932–6203.
https://doi.org/10.1371/journal.pone.0159124
Moubayed-Breil, J. & Dominici, J.M. (2019) Clunio boudouresquei sp. n. and Thalassosmittia ballestai sp. n., two Tyrrhenian marine species occurring in Scandola Nature Reserve, West Corsica (Diptera, Chironomidae). Chironomus Journal of Chironomidae Researches, 32, 4–24.
Moubayed-Breil, J. & Michailova, P. (2020) Description of the adult and larva of Clunio balticus Heimbach, 1978 (Diptera, Chironomidae) from the seashore of Bergen (Norway). Zootaxa, 4822 (2), 209–220.
https://doi.org/10.11646/zootaxa.4822.2.4
Moubayed-Breil, J., Michailova, P. & Langton, P.H. (2020) Description of the adult and immature stages of Clunio ponticus Michailova, 1980 (Diptera, Chironomidae) from the Black Sea, Varna, Bulgaria. Chironomus Journal of Chironomidae Researches, 33, 31– 47.
https://doi.org/10.5324/cjcr.v0i33.3332
Nagy, T., Sonet, G., Glaw, F. & Vences, M. (2012) First large-scale DNA barcoding assessment of reptiles in the biodiversity hotspot of Madagascar, based on newly designed COI primers. PLoS ONE, 7 (3), e34506.
https://doi.org/10.1371/journal.pone.0034506
Neumann, D. (1976) Adaptation of chironomids to intertidal environments. Annual Review of Entomology, 21, 387–414.
https://doi.org/10.1146/annurev.en.21.010176.002131
Rach, J., Bergman,T., Paknia,O., DeSalle, R., Schierwate, B. & Hadrys, H. (2017) The marker choice: Unexpected resolving power of an unexplored CO1 region forlayered DNA barcoding approaches. PLoS ONE, 12 (4), e0174842.
https://doi.org/10.1371/journal.pone.0174842
Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K. & Li, W. (2011) Fast, scalable 1247 generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7, 539. [Epub 2011/10/13]
https://doi.org/10.1038/msb.2011.75
Strenzke, K. (1960) Metamorphose und verwadtschaftsbeziehungen der gattung Clunio Hal. (Dipt.). Terrestrische Chironomidae XXIV. Annales Zoologigi Societatis Zoologigae Botanicae Fennicae ‘Vasamo’, 22, 4, 1–30.
Tahir, H.M. & Akhtar, S. (2015) Services of DNA barcoding in different fields. Mitochondrial DNA, 27 (6) 4463–4474.
https://doi.org/10.3109/19401736.2015.1089572
Ward, R.D. (2009) DNA barcode divergence among species and genera of birds and fishes. Molecular Ecology Resources, 9, 1077–1085.
https://doi.org/10.1111/j.1755-0998.2009.02541.x