Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Faculty of Natural Sciences Matej Bel University Banská Bystrica Slovakia

Institute for Environmental Studies Faculty of Science Charles University Prague Czech Republic

Institute of Ecology University of Innsbruck Innsbruck Austria

Institute of Hydrobiology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Institute of Zoology Slovak Academy of Sciences Bratislava Slovakia

Institute of Zoology University of Innsbruck Innsbruck Austria

Zobrazit více v PubMed

Sommer C, Malz P, Seehaus TC, Lippl S, Zemp M, Braun MH. Rapid glacier retreat and downwasting throughout the European Alps in the early 21st century. Nature Communications. 2020; 11: 3209. doi: 10.1038/s41467-020-16818-0 PubMed DOI PMC

Lencioni V, Stella E, Zanoni MG, Bellin A. On the delay between water temperature and invertebrate community response to warming climate. Science of the Total Environment. 2022; 837: 155759. doi: 10.1016/j.scitotenv.2022.155759 PubMed DOI

Lencioni V, Rossaro B. Microdistribution of chironomids (Diptera: Chironomidae) in Alpine streams: an autoecological perspective. Hydrobiologia. 2005; 533: 61–76. doi: 10.1007/s10750-004-2393-x DOI

Lencioni V, Maiolini B, Rossaro B. The kryal and rhithral chironomid community in the Care`Alto system (Italian central-eastern Alps). Verhandlungen der Internationale Vereinigung für Theoretische und Angewandte Limnologie. 2001; 27: 711–715. doi: 10.1080/03680770.1998.11901326 DOI

Lods-Crozet B, Lencioni V, Ólafsson JS, Snook DL, Velle G, Brittain JE, et al.. Chironomid (Diptera: Chironomidae) communities in six European glacier-fed streams. Freshwater Biology. 2001; 46: 1791–1809. doi: 10.1046/j.1365-2427.2001.00859.x DOI

Rossaro B. Chironomids and temperature. Aquatic Insects. 1991; 13: 87–98.

Eggermont H., Heiri O. The chironomid-temperature relationship: expression in nature and palaeoenvironmental implications. Biological Reviews. 2012; 87, 430–456. doi: 10.1111/j.1469-185X.2011.00206.x PubMed DOI

Ward JV. Ecology of alpine streams. Freshwater Biology. 1994; 32: 277–294. doi: 10.1111/j.1365-2427.1994.tb01126.x DOI

Füreder L. High alpine streams: cold habitats for insect larvae. In: Margesin R, Schinner F (eds): Cold-Adapted Organisms. Springer, Berlin, Heidelberg. 1999. doi: 10.1007/978-3-662-06285-2_10 DOI

Serra-Tosio B. Ecologìe et biogéographie des Diamesini d’Europe (Diptera, Chironomidae). Travaux Du Laboratorie D’ Hydrobiologie Et De Pisciculture De L’université De Grenoble. 1973; 63: 5–175.

Sæther OA, Andersen T. 7. The larvae of Diamesinae (Diptera: Chironomidae) of the Holarctic Region–Keys and diagnoses. In: Andersen T, Sæther OA, Cranston PS, Epler JH (eds.): Chironomidae of the Holarctic Region. Keys and diagnoses. Larvae. Insect Systematics & Evolution. 2013; Supplement 66: 145–183.

Ferrington LC Jr. Global diversity of non-biting midges (Chironomidae; Insecta-Diptera) in freshwater. Hydrobiologia. 2008; 595: 447–455. doi: 10.1007/s10750-007-9130-1 DOI

Burgherr P, Ward JV. Longitudinal and seasonal distribution patterns of the benthic fauna of an alpine glacial stream (Val Roseg, Swiss Alps). Freshwater Biology. 2001; 46: 1705–1721. doi: 10.1046/j.1365-2427.2001.00853.x DOI

Milner AM, Brittain JE, Castella E, Petts GE. Trends of macroinvertebrate community structure in glacier-fed rivers in relation to environmental conditions: a synthesis. Freshwater Biology. 2001; 46(12): 1833–1847. doi: 10.1046/j.1365-2427.2001.00861.x DOI

Rossaro B, Montagna M, Lencioni V. Environmental traits affect chironomid communities in glacial areas of the Southern Alps: evidence from a long‐lasting case study. Insect Conservation and Diversity. 2016; 9(3): 192–201. doi: 10.1111/icad.12157 DOI

Ashe P, O’Connor JP. A World Catalogue of Chironomidae (Diptera). Part 1. Buchonomyiinae, Chilenomyiinae, Podonominae, Aphroteniinae, Tanypodinae, Usambaromyiinae, Diamesinae, Prodiamesinae and Telmatogetoninae. Irish Biogeographical Society & National Museum of Ireland, Dublin. 2009; 445. ISBN: 9780955080647.

Spies M, Sæther OA. Chironomidae. Fauna Europaea: In: Beuk P, Pape T (Eds): Fauna Europaea: Diptera, Nematocera. Fauna Europaea version 2.6. 2013; Internet database: http://www.faunaeur.org (Accessed on 2023-06-29).

Langton PH, Pinder LCV. Keys to the adult male Chironomidae of Britain and Ireland. 2 Vols. Freshwater Biological Association, Scientific publication. 2007; 64: 1–239 + 1–168.

Langton PH, Visser H. Chironomidae exuviae. A key to pupal exuviae of the West Palaearctic Region. Interactive Identification System for the Europaean Limnofauna (IISEL), World Biodiversity Database, CD–ROM Series. 2003.

Rossaro B, Lencioni V. A key to larvae of Diamesa Meigen, 1835 (Diptera: Chironomidae), well known as adult males and pupae from Alps (Europe). Journal of Entomological and Acarological Research. 2015a; 47: 123–138. doi: 10.4081/jear.2015.5516 DOI

Rossaro B, Lencioni V. A key to larvae of species belonging to the genus Diamesa from Alps and Apennines (Italy). European Journal of Environmental Sciences. 2015b; 5(1): 62–79.

Montagna M, Mereghetti V, Lencioni V, Rossaro B. Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae). PLoS ONE. 2016a; 11(3): e0149673. doi: 10.1371/journal.pone.0149673 PubMed DOI PMC

Krasheninnikov AB, Makarchenko EA, Semenchenko AA, Gavrilo MV, Vshivkova KA. Morphological description and DNA barcoding of some Diamesinae (Diptera, Chironomidae) from the Severnaya Zemlya Archipelago and the Vaigach Island (Russian Arctic). Zootaxa. 2020; 4802(3): 587–600. doi: 10.11646/zootaxa.4802.3.13 PubMed DOI

Makarchenko EA, Semenchenko AA, Palatov DM. Fauna and taxonomy of Diamesinae (Diptera, Chironomidae) from the Caucasus, with a morphological description and DNA barcoding of new taxa and a discussion of diagnostic problems for Diamesa Meigen and Pseudodiamesa Goetghebuer. Zootaxa. 2023; 5271(2): 313–328. doi: 10.11646/zootaxa.5271.2.6 PubMed DOI

Anslan S, Tedersoo L. Performance of cytochrome c oxidase subunit I (COI), ribosomal DNA Large Subunit (LSU) and Internal Transcribed Spacer 2 (ITS2) in DNA barcoding of Collembola. European Journal of Soil Biology. 2015; 69: 1–7. doi: 10.1016/j.ejsobi.2015.04.001 DOI

Hebert PDN, Ratnasingham S, de Waard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society B. 2003; 270: 96–99. doi: 10.1098/rsbl.2003.0025 PubMed DOI PMC

Pentinsaari M, Salmela H, Mutanen M, Roslin T. Molecular evolution of a widely-adopted taxonomic marker (COI) across the animal tree of life. Scientific Reports. 2016; 6: 35275. doi: 10.1038/srep35275 PubMed DOI PMC

Ebong MAS, Petit E, Le Gall P, Chen PP, Nieser N, Guilbert E, et al.. Molecular Species Delimitation and Morphology of Aquatic and Sub-Aquatic Bugs (Heteroptera) in Cameroon. PLoS ONE. 2016; 11(5): e0154905. doi: 10.1371/journal.pone.0154905 PubMed DOI PMC

Laurito M, de Oliveira TMP, Almiron WR, Sallum MAM. COI barcode versus morphological identification of Culex (Culex) (Diptera: Culicidae) species: a case study using samples from Argentina and Brazil. Memorias do Instituto Oswaldo Cruz. 2013; 108. doi: 10.1590/0074-0276130457 PubMed DOI PMC

Park DS, Foottit R, Maw E, Hebert PDN. Barcoding Bugs: DNA-Based Identification of the True Bugs (Insecta: Hemiptera: Heteroptera). PLoS ONE. 2011; 6(4): e18749. doi: 10.1371/journal.pone.0018749 PubMed DOI PMC

Wang G, Li C, Guo X, Xing D, Dong Y, Wang Z, et al.. Identifying the Main Mosquito Species in China Based on DNA Barcoding. PLoS ONE. 2012; 7(10): e47051. doi: 10.1371/journal.pone.0047051 PubMed DOI PMC

Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS, Donoghue MJ. The ITS Region of Nuclear Ribosomal DNA: A Valuable Source of Evidence on Angiosperm Phylogeny. Annals of the Missouri Botanical Garden. 1995; 82(2):247–277. doi: 10.2307/2399880 DOI

Chen S, Yao H, Han J, Liu C, Song J, Shi L, et al.. Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PLoS ONE. 2010; 5(1): e8613. doi: 10.1371/journal.pone.0008613 PubMed DOI PMC

Schoch CL, Seifert KA, Huhndorf S, Schindel D. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences. 2012; 109(16). doi: 10.1073/pnas.1117018109 PubMed DOI PMC

Yao H, Song J, Liu C, Luo K, Han J, Li Y, et al.. Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. PLoS ONE. 2010; 5(10): e13102. doi: 10.1371/journal.pone.0013102 PubMed DOI PMC

Rajasegaran P, Koosakulnirand S, Tan KK, Khoo JJ, Suliman Y, Mansor MS, et al.. Multi-locus sequence analysis indicates potential cryptic speciation in the chigger mite Neoschoengastia gallinarum (Hatori, 1920) parasitizing birds in Asia. 2023; PREPRINT available at Research Square. doi: 10.21203/rs.3.rs-3682938/v1 PubMed DOI PMC

Bourke BP, Oliveira TP, Suesdek L, Bergo ES, Sallum MAM. A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae). Parasites Vectors. 2013; 6: 11. doi: 10.1186/1756-3305-6-111 PubMed DOI PMC

Brown WM, George M Jr, Wilson AC. Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Sciences of the United States of America. 1979; 76(4): 1967–1971. doi: 10.1073/pnas.76.4.1967 PubMed DOI PMC

Schon EA, DiMauro S, Hirano M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nature Reviews Genetics. 2012; 13(12): 878–90. doi: 10.1038/nrg3275 PubMed DOI PMC

Dvorak M, Dittmann IL, Pedrini-Martha V, Hamerlík L, Bitušík P, Stuchlik E, et al.. Energy status of chironomid larvae (Diptera: Chironomidae) from high alpine rivers (Tyrol, Austria). Comparative Biochemistry and Physiology, Part A. 2023; 284: 111477. doi: 10.1016/j.cbpa.2023.111477 PubMed DOI

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994; 3(5): 294–299. . PubMed

Newburn E, Krane D. Molecular Identification of Chironomid Species. In: Lipnick RL, Mason RP, Phillips ML, Pittman CU Jr (Eds): Chemicals in the Environment. American chemical society, Washington DC, USA. 2002: 363–382. doi: 10.1021/bk-2002-0806.ch021 DOI

Katoh K, Standley DM. MAFFT Multiple sequence alignment software version 7: Improvements in performance and usability. Molecular biology and evolution. 2013; 30(4): 772–780. doi: 10.1093/molbev/mst010 PubMed DOI PMC

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. doi: 10.1093/bioinformatics/btu033 PubMed DOI PMC

Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11. Molecular biology and evolution. 2021; 38(7): 3022–3027. doi: 10.1093/molbev/msab120 PubMed DOI PMC

Lencioni V, Rodriguez-Prieto A, Allegrucci G. Congruence between molecular and morphological systematics of Alpine non-biting midges (Chironomidae, Diamesinae). Zoologica Scripta. 2021; 50: 455–472. doi: 10.1111/zsc.12480 DOI

Zhang H, Bu W. Exploring Large-Scale Patterns of Genetic Variation in the COI Gene among Insecta: Implications for DNA Barcoding and Threshold-Based Species Delimitation Studies. Insects. 2022; 13(5): 425–436. doi: 10.3390/insects13050425 PubMed DOI PMC

Pankratova VY. Key to the larvae of the genera of the sub-family Orthocladiinae from Larvae and Pupae of midges of the sub-family Orthocladiinae of the fauna of the USSR. FBA Translations (New Series). 1970; 54: 51–5.

Rossaro B. Description of some unknown larvae of Diamesa genus and corrections to previous descriptions (Diptera, Chrironomidae). Archiv für Hydrobiologie. 1980; 90(3): 298–308. ISSN 0003-9136.

Ferrarese U, Rossaro B. Chironomidi 12., 1 (Diptera, Chironomidae: generalià, Diamesinae, Prodiamesinae). Guide per il riconoscimento delle specie animali delle acque interne italiane C.N.R. AQ/1/129. 1981; 12: 1–97.

Schmid PE. A key to the Chironomidae and their instars from Austrian Danube region streams and rivers. Part I. Diamesinae Prodiamesinae and Orthocladiinae. 1993.

Montagna M, Urbanelli S, Rossaro B. The species of the genus Diamesa (Diptera, Chironomidae) known to occur in Italian Alps and Apennines. Zootaxa. 2016b; 4193(2): 317–331. doi: 10.11646/zootaxa.4193.2.7 PubMed DOI

Kownacki A, Kownacka M. Diamesa martae sp. n. (Diptera: Chironomidae) and zoogerographical distribution of the species of Diamesa latitarsis group. Acta Hydrobiologica. 1980; 22/3: 313–326.

Kownacki A, Kownacka M. Diamesa starmachi sp. n. (Diptera, Chironomidae). Bulletin de l´Academie Polonaise des Sciences. 1970; 18/12: 777–780.

Bitušík P, Svitok M, Dragúňová M. The actual longitudinal zonation of the river Hron (Slovakia) based on chironomid assemblages (Diptera, Chironomidae). Acta Universitatis Carolinae Biologica. 2006; 50: 5–17.

Kownacki A. Ecology and biogeography of the Diamesa steinboecki group. In: Lellák J (ed): Proceedings of the 6th International Symposium Chironomidae, Prague, 17–20 August 1976. Acta Universitatis Carolinae. Biologica. 1978: 95–102.

Lencioni V. Survival strategies of freshwater insects in cold environments. Journal of limnology. 2004; 63(1s): 45–55. doi: 10.4081/jlimnol.2004.s1.45 DOI

Khamis K, Hannah DM, Brown LE, Tiberti R, Milner AM. The use of invertebrates as indicators of environmental change in alpine rivers and lakes. Science of the Total Environment. 2014; 493: 1242–1254. doi: 10.1016/j.scitotenv.2014.02.126 PubMed DOI

Bitušík P. A biological assessment of the Turiec river and its selected tributaries using chironomid pupal exuviae (Diptera, Chironomidae). Dipterologica bohemoslovaca. 1995; 7: 17–26.

Hroch M, Brabec K. Analysis of multiple-pressure pattern in rivers and its effects on the structure of macroinvertebrate communities. Limnologica. 2022; 97: 126027. doi: 10.1016/j.limno.2022.126027 DOI

Kownacki A. Znaczenie i ochrona Chironomidae (Diptera, Insecta) w ekosystemach wodnych Polski. Forum Faunistyczne. 2011; 1(1): 4–11).