Abstract
ITS2 is often suggested as a potential marker for evolutionary studies and species barcoding. However, there are many lineages have not been studied. This study focuses on ITS2 in Polyneoptera at the order and species levels. ITS2 sequences representing six polyneopteran orders and 15 species in the genus Anterastes are studied. We arrived at the following conclusions: (i) ITS2 is highly variable and contains little phylogenetic information in Polyneoptera, (ii) the shortest length and the highest GC content of ITS2 is found in Orthoptera among insects, (iii) the secondary structure exhibits general characteristics of eukaryotes especially in helices II and III, and with no order-specific architecture, (iv) ITS2 is highly conserved at the species level, both in linear sequences and secondary structures, (v) helices I, IA, II, IIA and III almost invariable in nucleotide sequence shared by all species in the genus. At the generic level, the most conspicuous result is the variable pattern in ITS2. It is highly conserved in helical sequences, but highly variable in non/peri-helical regions which we considered to be mutation islands. These frequently mutated regions contain a significant amount of molecular homoplasy, thus, the utility of ITS2 in phylogenetic analyses and species barcoding is low, at least in Polyneoptera.
References
Álvarez, I. & Wendel, J.F. (2003) Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution, 29, 417–434.
https://doi.org/10.1016/S1055-7903(03)00208-2
Ankenbrand, M.J., Keller, A., Wolf, M., Schultz, J. & Forster, F. (2015) ITS2 database V: Twice as much. Molecular Biology and Evolution, 32, 3030–3032.
https://doi.org/10.1093/molbev/msv174
Bargues, M.D., Klisiowicz, D.R., Panzera, F., Noireau, F., Marcilla, A., Perez, R., Rojas, M.G., O’Connor, J.E., Gonzalez-Candelas, F., Galvão, C., Jurberg, J., Carcavallo, R.U., Dujardin, J.P. & Mas-Coma, S. (2006) Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size. Infection, Genetics and Evolution, 6, 46–62.
https://doi.org/10.1016/j.meegid.2005.01.006
Batovska, J., Cogan, N.O.I., Lynch, S.E. & Blacket, M.J. (2017) Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2 . G3: Genes|Genomes|Genetics, 7, 19–29.
https://doi.org/10.1534/g3.116.036145
Budak, M., Güler, M., Korkmaz, E.M., Hastaoðlu Örgen, S. & Baþibüyük, H.H. (2016) The characterisation and taxonomic utility of ITS2 in Tenthredopsis Costa, 1859 (Tenthredinidae: Hymenoptera) with some new records from Turkey. Biochemical Systematics and Ecology, 66, 76–85.
https://doi.org/10.1016/j.bse.2016.03.008
Caisová, L. & Melkonian, M. (2014) Evolution of helix formation in the ribosomal internal transcribed spacer 2 (ITS2) and its significance for RNA secondary structures. Journal of Molecular Evolution, 78, 324–337.
https://doi.org/10.1007/s00239-014-9625-0
Chobanov, D.P., Kaya, S., Grzywacz, B., Warchałowska-Śliwa, E. & Çıplak, B. (2016) The Anatolio-Balkan phylogeographic fault: a snapshot from the genus Isophya (Orthoptera, Tettigoniidae). Zoologica Scripta, 46 (2), 165–179.
https://doi.org/10.1111/zsc.12194
Çıplak, B. (2004) Systematics, phylogeny and biogeography of Anterastes (Orthoptera, Tettigoniidae, Tettigoniinae): Evolution within a refugium. Zoologica Scripta, 33, 19–44.
https://doi.org/10.1111/j.1463-6409.2004.00131.x
Çıplak, B., Kaya, S. & Gündüz, I. (2010) Phylogeography of Anterastes serbicus species group (Orthoptera, Tettigoniidae): Phylogroups correlate with mountain belts, but not with the morphospecies. Journal of Orthoptera Research, 19, 89–100.
https://doi.org/10.1665/034.019.0115
Çıplak, B., Kaya, S., Boztepe, Z. & Gündüz, I. (2015) Mountainous genus Anterastes (Orthoptera, Tettigoniidae): Autochthonous survival across several glacial ages via vertical range shifts. Zoologica Scripta, 44, 534–549.
https://doi.org/10.1111/zsc.12118
Coleman, A.W. (2003) ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends in Genetics, 19 (7), 370–375.
https://doi.org/10.1016/S0168-9525(03)00118-5
Coleman, A.W. (2007) Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucleic Acids Research, 35 (10), 3322–3329.
https://doi.org/10.1093/nar/gkm233
Coleman, A.W. & Van Oppen, M.J.H. (2008) Secondary structure of the rRNA ITS2 region reveals key evolutionary patterns in acroporid corals. Journal of Molecular Evolution, 67, 389–396.
https://doi.org/10.1007/s00239-008-9160-y
Coleman, A.W. (2009) Is there a molecular key to the level of “biological species” in eukaryotes? A DNA guide. Molecular Phylogenetics and Evolution, 50 (1), 197–203.
https://doi.org/10.1016/j.ympev.2008.10.008
Coleman, A.W. (2015) Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure. Trends in Genetics, 31 (3), 157–163.
https://doi.org/10.1016/j.tig.2015.01.002
Cote, C.A. (2001) Role of the ITS2-proximal stem and evidence for indirect recognition of processing sites in pre-rRNA processing in yeast. Nucleic Acids Research, 29, 2106–2116.
https://doi.org/10.1093/nar/29.10.2106
Ding, S., Li, W., Wang, Y., Cameron, S.L., Murányi, D. & Yang, D. (2019) The phylogeny and evolutionary timescale of stoneflies (Insecta: Plecoptera) inferred from mitochondrial genomes. Molecular Phylogenetics and Evolution, 135, 123–135.
https://doi.org/10.1016/j.ympev.2019.03.005
Excoffier, L. & Lischer, H.E.L. (2010) Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources, 10, 564–567.
https://doi.org/10.1111/j.1755-0998.2010.02847.x
Ganley, A.R.D. & Kobayashi, T. (2007) Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data. Genome Research, 17, 184–191.
https://doi.org/10.1101/gr.5457707
Gomez-Zurita, J. (2000) Sequence, secondary structure and phylogenetic analyses of the ribosomal internal transcribed spacer (ITS2) in the timarche leaf beetles (coleoptera: chrisomelidae). Insect Molecular Biology, 9, 591–604.
https://doi.org/10.1046/j.1365-2583.2000.00223.x
Hadjiolova, K.V, Normann, A., Cavaille, J., Soupene, E., Mazan, S., Hadjiolov, A.A. & Bachellerie, J. (1994) Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells. Molecular and Cellular Biology, 14, 4044–4056.
https://doi.org/10.1128/MCB.14.6.4044
Hershkovitz, M.A. & Zimmer, E.A. (1996) Conservation patterns in angiosperm rDNA ITS2 sequences. Nucleic Acids Research, 24, 2857–2867.
https://doi.org/10.1093/nar/24.15.2857
Ishiwata, K., Sasaki, G., Ogawa, J., Miyata, T. & Su, Z.H. (2011) Phylogenetic relationships among insect orders based on three nuclear protein-coding gene sequences. Molecular Phylogenetics and Evolution, 58, 169–180.
https://doi.org/10.1016/j.ympev.2010.11.001
Kasuga, T., White, T.J. & Taylor, J.W. (2002) Letter to the Editor Estimation of Nucleotide Substitution Rates in Eurotiomycete Fungi. Molecular Biology and Evolution, 19, 2318–2324.
https://doi.org/10.1093/oxfordjournals.molbev.a004056
Kaya, S., Boztepe, Z. & Çıplak, B. (2013) Phylogeography of Troglophilus (Orthoptera: Troglophilinae) based on Anatolian members of the genus: Radiation of an old lineage following the Messinian. Biological Journal of the Linnean Society, 108, 335–348.
https://doi.org/10.1111/j.1095-8312.2012.02025.x
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P. & Drummond, A. (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647–1649.
https://doi.org/10.1093/bioinformatics/bts199
Keller, A., Förster, F., Müller, T., Dandekar, T., Schultz, J. & Wolf, M. (2010) Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees. Biology Direct, 5, 4.
https://doi.org/10.1186/1745-6150-5-4
Kitthawee, S. (2013) ITS2 sequence variations among members of Diachasmimorpha longicaudata complex (Hymenoptera: Braconidae) in Thailand. Journal of Asia-Pacific Entomology, 16, 173–179.
https://doi.org/10.1016/j.aspen.2013.01.002
Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35, 1547–1549.
https://doi.org/10.1093/molbev/msy096
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., Mcgettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947–2948.
https://doi.org/10.1093/bioinformatics/btm404
LaRue, B., Gaudreau, C., Bagre, H.O. & Charpentier, G. (2009) Generalized structure and evolution of ITS1 and ITS2 rDNA in black flies (Diptera: Simuliidae). Molecular Phylogenetics and Evolution, 53, 749–757.
https://doi.org/10.1016/j.ympev.2009.07.032
Librado, P. & Rozas, J. (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452.
https://doi.org/10.1093/bioinformatics/btp187
Mallatt, J. & Giribet, G. (2006) Further use of nearly complete 28S and 18S rRNA genes to classify Ecdysozoa: 37 more arthropods and a kinorhynch. Molecular Phylogenetics and Evolution, 40, 772–794.
https://doi.org/10.1016/j.ympev.2006.04.021
Marinho, M.A.T., Azeredo-Espin, A.M.L. & Zanchin, N.I.T. (2013) Structural characterization of the internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA) cluster in calyptratae (Diptera: Schizophora) and its implications for molecular phylogenetic analyses. Journal of Molecular Evolution, 76, 158–171.
https://doi.org/10.1007/s00239-013-9548-1
Marinho, M.A.T., Junqueira, A.C.M. & Azeredo-Espin, A.M.L. (2011) Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae). Genetica, 139, 1189–1207.
https://doi.org/10.1007/s10709-011-9621-x
Mukha, D.V., Sidorenko, A.P., Lazebnaya, I.V., Wiegmann, B.M. & Schal, C. (2000) Analysis of intraspecies polymorphism in the ribosomal DNA cluster of the cockroach Blattella germanica. Insect Molecular Biology, 9, 217–222.
https://doi.org/10.1046/j.1365-2583.2000.00175.x
Müller, T., Philippi, N., Dandekar, T., Schultz, J. & Wolf, M. (2007) Distinguishing species. RNA, 13, 1469–1472.
https://doi.org/10.1261/rna.617107
Nosil, P., Crespi, B.J. & Sandoval, C.P. (2002) Host-plant adaptation drives the parallel evolution of reproductive isolation. Nature, 417 (6887), 440–443.
https://doi.org/10.1038/417440a
Oh, H.K., Yoon, H.J., Lee, J.Y., Park, J.S. & Kim, I. (2013) Population Genetic Structure of the Bumblebee, Bombus ignitus (Hymenoptera: Apidae), Based on Mitochondrial COI Gene and Nuclear Ribosomal ITS2 Sequences. International Journal of Industrial Entomology, 27 (1), 142–158.
https://doi.org/10.7852/ijie.2013.27.1.142
Paredes-Esquivel, C.C. & Townson, H. (2014) Functional constraints and evolutionary dynamics of the repeats in the rDNA internal transcribed spacer 2 of members of the Anopheles barbirostris group. Parasites & Vectors, 7, 106.
https://doi.org/10.1186/1756-3305-7-106
Percy, D.M., Page, R.D.M. & Cronk, Q.C.B. (2004) Plant-insect interactions: Double-dating associated insect and plant lineages reveals asynchronous radiations. Systematic Biology, 53, 120–127.
https://doi.org/10.1080/10635150490264996
R Core Team, (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Available from: http://www.R-project.org/ (accessed 10 December 2018)
Ruhl, M.W., Wolf, M. & Jenkins, T.M. (2010) Molecular Phylogenetics and Evolution Compensatory base changes illuminate morphologically difficult taxonomy. Molecular Phylogenetics and Evolution, 54, 664–669.
https://doi.org/10.1016/j.ympev.2009.07.036
Sasaki, G., Ishiwata, K., Machida., R., Miyata, T. & Su, Z. (2013) Molecular phylogenetic analyses support the monophyly of Hexapoda and suggest the paraphyly of Entognatha, BMC Evolutionary Biology, 13, 236.
https://doi.org/10.1186/1471-2148-13-236
Schliep, K.P. (2011) phangorn: Phylogenetic analysis in R. Bioinformatics, 27, 592–593.
https://doi.org/10.1093/bioinformatics/btq706
Schultz, J., Maisel, S., Gerlach, D., Muller, T., Wolf, M. & Müller, T. (2005) A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA, 11, 361–364.
https://doi.org/10.1261/rna.7204505
Song, N., Li, H., Song, F. & Cai, W. (2016) Molecular phylogeny of Polyneoptera (Insecta) inferred from expanded mitogenomic data. Scientific Reports, 6, 1–10.
https://doi.org/10.1038/srep36175
Song, Z.K., Wang, X.Z. & Liang, G.Q. (2008) Molecular evolution and phylogenetic utility of the internal transcribed spacer 2 (ITS2) in calyptratae (Diptera: Brachycera). Journal of Molecular Evolution, 67, 448–464.
https://doi.org/10.1007/s00239-008-9144-y
Swofford, D.L. (2002) PAUP: Phylogenetic Analysis Using Parsimony. Version 4.0b10. Computer program distributed by Illinois Natural History Survey, Champaign, Illinois. [program]
Sword, G.A., Senior, L.B., Gaskin, J.F. & Joern, A. (2007) Double trouble for grasshopper molecular systematics: Intra-individual heterogeneity of both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer ribosomal DNA sequences in Hesperotettix viridis (Orthoptera: Acrididae). Systematic Entomology, 32, 420–428.
https://doi.org/10.1111/j.1365-3113.2007.00385.x
Trizzino, M., Audisio, P., Antonini, G., De Biase, A. & Mancini, E. (2009) Comparative analysis of sequences and secondary structures of the rRNA internal transcribed spacer 2 (ITS2) in pollen beetles of the subfamily Meligethinae (Coleoptera, Nitidulidae): Potential use of slippage-derived sequences in molecular systematics. Molecular Phylogenetics and Evolution, 51, 215–226.
https://doi.org/10.1016/j.ympev.2008.11.004
Ullrich, B., Reinhold, K., Niehuis, O. & Misof, B. (2010) Secondary structure and phylogenetic analysis of the internal transcribed spacers 1 and 2 of bush crickets (Orthoptera: Tettigoniidae: Barbitistini). Journal of Zoological Systematics and Evolutionary Research, 48, 219–228.
https://doi.org/10.1111/j.1439-0469.2009.00553.x
Vu, D., Groenewald, M., de Vries, M., Gehrmann, T., Stielow, B., Eberhardt, U., Al-Hatmi, A., Groenewald, J.Z., Cardinali, G., Houbraken, J., Boekhout, T., Crous, P.W., Robert, V. & Verkley, G.J.M. (2019) Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in Mycology, 92, 135–154.
https://doi.org/10.1016/j.simyco.2018.05.001
Wang, Y., Engel, M.S., Rafael, J.A., Dang, K., Wu, H., Wang, Y., Xie, Q. & Bu, W. (2013) A Unique Box in 28S rRNA Is Shared by the Enigmatic Insect Order Zoraptera and Dictyoptera. PLoS ONE, 8 (1), e53679.
https://doi.org/10.1371/journal.pone.0053679
Wiemers, M., Keller, A. & Wolf, M. (2009) ITS2 secondary structure improves phylogeny estimation in a radiation of blue butterflies of the subgenus Agrodiaetus (Lepidoptera: Lycaenidae: Polyommatus ). BMC Evolutionary Biology, 9, 300.
https://doi.org/10.1186/1471-2148-9-300
Wipfler, B., Letsch, H., Frandsen, P.B., Kapli, P., Mayer, C., Bartel, D., Buckley, T.R., Donath, A., Edgerly-Rooks, J.S., Fujita. M., Liu, S., Machida, R., Mashimo, Y., Misof, B., Niehuis, O., Peters, R.S., Petersen, M., Podsiadlowski, .L, Schütte, K., Shimizu, S., Uchifune, T., Wilbrandt, J., Yan, E., Zhou, X. & Simon, S. (2019) Evolutionary history of Polyneoptera and its implications for our understanding of early winged insects. Proceedings of the National Academy of Sciences USA, 116, 3024–3029.
https://doi.org/10.1073/pnas.1817794116
Wolf, M., Chen, S., Song, J., Ankenbrand, M. & Müller, T. (2013) Compensatory Base Changes in ITS2 Secondary Structures Correlate with the Biological Species Concept Despite Intragenomic Variability in ITS2 Sequences—A Proof of Concept. PLoS ONE, 8 (6), e66726.
https://doi.org/10.1371/journal.pone.0066726
Wolf, M., Koetschan, C. & Müller, T. (2014) ITS2, 18S, 16S or any other RNA - simply aligning sequences and their individual secondary structures simultaneously by an automatic approach. Gene, 546, 145–149.
https://doi.org/10.1016/j.gene.2014.05.065
Yang, Y., Wu, Z., Xu, H., Zheng, X. & Lu, Z. (2017) Structural characterization and applications of ITS2 from rice leaffolders Cnaphalocrocis medinalis and Marasmia patnalis (Lepidoptera: Pyralidae). Journal of Asia-Pacific Entomology, 20, 313–318.
https://doi.org/10.1016/j.aspen.2016.12.017
Yao, H., Song, J., Liu, C., Luo, K., Han, J., Li, Y., Pang, X., Xu, H., Zhu, Y., Xiao, P. & Chen, S. (2010) Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS ONE, 5 (10), e13102.
https://doi.org/10.1371/journal.pone.0013102
Young, I. & Coleman, A.W. (2004) The advantages of the ITS2 region of the nuclear rDNA cistron for analysis of phylogenetic relationships of insects: A Drosophila example. Molecular Phylogenetics and Evolution, 30, 236–242.
https://doi.org/10.1016/S1055-7903(03)00178-7
Zomuanpuii, R., Ringngheti, L., Brindha, S., Gurusubramanian, G. & Kumar, S.N. (2013) ITS2 characterization and Anopheles species identification of the subgenus Cellia. Acta Tropica, 125, 309–319.
https://doi.org/10.1016/j.actatropica.2012.12.001
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31, 3406–3415.
https://doi.org/10.1093/nar/gkg595
Zuker, M., Jaeger, J.A. & Turner, D.H. (1991) A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison. Nucleic Acids Research, 19, 2707–2714.