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Comparative analyses of COI and ITS2 in the molecular identification of Chrysolina Motschulsky, 1860 (Coleoptera: Chrysomelidae) in Türkiye

Yıl 2025, Cilt: 49 Sayı: 4, 413 - 436, 06.01.2026
https://doi.org/10.16970/entoted.1791117

Öz

This study investigated the effectiveness of two molecular markers—the cytochrome c oxidase subunit I (COI) gene and the internal transcribed spacer II (ITS2) region—in delineating species boundaries in insects. While COI is widely used in insect taxonomy to define species boundaries, ITS2 has been less frequently tested. A total of 25 individuals from the genus Chrysolina (Coleoptera: Chrysomelidae), distributed across Türkiye, were analysed. Molecular analyses were conducted in 2017 at the Molecular Systematics Laboratory of the Department of Zoology, Faculty of Science, Ege University, and data analyses were completed in 2025. Using a Bayesian approach, they examined phylogenetic tree topology and species monophyly based on each marker and combined datasets. This marks the first comparative study of COI and ITS2 within Chrysolina species in Türkiye. Results showed that both markers supported morphological distinctions at the molecular level. All species formed monophyletic groups in trees constructed from COI, ITS2, and combined datasets. These findings suggest that the ITS2 may be as effective as the COI in evaluating genetic diversity and identifying species. The study highlights the potential of ITS2 as a reliable molecular marker in insect taxonomy and enhances understanding of species boundaries within the genus Chrysolina.

Destekleyen Kurum

TÜBİTAK 1001 (Project Number 113Z753) Ege University Scientific Research Projects Coordinatorship through Project Number 17FEN032

Proje Numarası

TÜBİTAK 1001 (Project Number 113Z753) Ege University Scientific Research Projects Coordinatorship through Project Number 17FEN032

Teşekkür

This publication is based on research conducted for the master’s thesis of the corresponding author. This work was partially supported by TÜBİTAK 1001 (Project Number 113Z753). Additionally, the authors gratefully acknowledges the financial support provided by Ege University Scientific Research Projects Coordinatorship through project number 17FEN032. I am grateful to Prof. Dr. Mahir Budak (Sivas Cumhuriyet University, Sivas), Prof. Dr. Ertan Mahir Korkmaz (Sivas Cumhuriyet University, Sivas) and Prof. Dr. Nurşen Alpagut Keskin (Ege University, Izmir) for their invaluable contributions to this research. The authors wish to express their gratitude to Prof. Dr. İsmail Şen (Isparta University, Isparta) for his invaluable contributions and expertise in the morphological identification of the species.

Kaynakça

  • Alvarez, J. M. & M. A. Hoy, 2002. Evaluation of the ribosomal ITS2 DNA sequences in closely related populations of the parasitoid Ageniaspis (Hymenoptera: Encyrtidae). Annals of the Entomological Society of America, 95 (2): 250-256.
  • Arnheim, N., 1983. “Concerted Evolution of Multigene Families in Evolution of Genes and Proteins, 38-61”. In: Evolution of Genes and Proteins (Eds. M. Nei & R. K. Koehn). Sinauer, Sunderland, Massachusetts, 346 pp.
  • Ashfaq, A., M. Erlandson & L. Braun, 2005. Hyper parasitism by Mesochorus spp (Hymenoptera: Ichneumonidae) in Peristenus sp. (Hymenoptera: Braconidae) and development of PCR primers for hyperparasitoid detection. Biological Control, 32 (3): 371-377.
  • Ball, S. L., P. D. N. Hebert, S. K. Burian & J. M. Webb, 2005. Biological identifications of mayflies (Ephemeroptera) using DNA barcodes. Journal of the North American Benthological Society, 24 (3): 508-524.
  • Becerra, J. X., 2004. Molecular systematics of Blepharida beetles (Chrysomelidae: Alticinae) and relatives. Molecular Phylogenetics and Evolution, 30 (1): 107-117.
  • Ben-David, T., S. Melamed, U. Gerson & S. Morin, 2007. ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae). Experimental and Applied Acarology, 41 (3): 169-181.
  • Bezděk, J. & L. Sekerka, 2024. Catalogue of Palaearctic Coleoptera: Chrysomeloidea II (Orsodacnidae, Megalopodidae, Chrysomelidae). Brill, Leiden, Netherlands, 750 pp.
  • Bieńkowski, A. O., 2019. “Chrysolina of the World – 2019 (Coleoptera: Chrysomelidae) Taxonomic Review”. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, 919 pp.
  • Bouckaert, R., T. G. Vaughan, J. Barido-Sottani, S. Duchêne, M. Fourment, A. Gavryushkina, J. Heled, G. Jones, D. Kühnert, N. D. Maio, M. Matschiner, F. K. Mendes, N. F. Müller, H. A. Ogilvie, L. Plessis, A. Popinga, A. Rambaut, D. Rasmussen, I. Siveroni, M. A. Suchard, C. H. Wu, D. Xie, C. Zhang, T. Stadler & A. J. Drummond, 2019. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Computational Biology, 15 (4): e1006650.
  • Budak, M., M. Güler, E. M. Korkmaz, S. Hastaoğlu Orgen & H. H. Başıbüyük, 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 (1): 76-85.
  • Calzolari, M., N. Bellin, M. Dottori, D. Torri, M. D. Luca, V. Rossi, G. Magoga & M. Montagna, 2024. Integrated taxonomy to advance species delimitation of the Anopheles maculipennis complex. Scientific Reports, 14 (1): 30914.
  • Campbell, B., J. D. Steffen-Campbell & J. H. Werren, 1993. Phlogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences. Insect Molecular Biology, 2 (4): 225-237.
  • Chen, S. L., H. Yao, J. P. Han, C. Liu & J. Y. Song, 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 5 (1): e8613 (1-8).
  • Dinno, A., 2018. Paran: Horn's test of principal components/factors. (Web page: https://CRAN.R-project.org/package=paran) (Date accessed: April 2025).
  • Edel, V., 1998. “Polymerase Chain Reaction in Mycology: An Overview, 1-20”. In: Applications of PCR in Mycology (Eds. P. D. Bridge), CAB International, Wallingford, New York, 357 pp.
  • Ezard, T., T. Fujisawa & T. Barraclough, 2021. Splits: Species limits by threshold statistics. (Web page: https://R-Forge.Rproject.org/projects/splits/) (Date accessed: April 2025).
  • Farris, J. S., M. Kallersjo, A. G. Kluge & C. Bult, 1994. Testing significance of incongruence. Cladistics, 10 (3): 315-319.
  • Fujisawa, T. & T. G. Barraclough, 2013. Delimiting species using single-locus data and the generalized mixed yule coalescent approach: a revised method and evaluation on simulated data sets. Systematic Biology, 62 (5):707–724.
  • Garin, C. F., C. Juan & E. Petitpierre, 1999. Mitochondrial DNA phylogeny and the evolution of host-plant use in palearctic Chrysolina (Coleoptera, Chrysomelidae) leaf beetles. Journal of Molecular Evolution, 48 (4): 435-444.
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  • Germain, J. F., C. Chatot, I. Meusnier, E. Artige, J. Y. Rasplus & A. Cruaud, 2013. Molecular identification of Epitrix potato flea beetles (Coleoptera: Chrysomelidae) in Europe and North America. Bulletin of Entomological Research, 103 (3): 354-362.
  • Hajibabaei, M., D. H. Janzen, J. M. Burns, W. Hallwachs & P. D. N. Hebert, 2006. DNA barcodes distinguish species of tropical Lepidoptera. The Proceedings of the National Academy of Sciences, 103 (4): 968-971.
  • Hebert, P. D. N., A. Cywinska, S. L. Ball & J. R. DeWaard, 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B: Biological Sciences, 270 (1512): 313-321.
  • Hebert, P. D. N., E. H. Penton, J. M. Burns, D. H. Janzen & W. Hallwachs, 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. The Proceedings of the National Academy of Sciences, 101 (41): 14812-14817.
  • Hebert, P. D. N., S. Ratnasingham & J. R. De Waard, 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London Series B: Biological Sciences, 270 (1): 96-99.
  • Ji, Y. J., D. X. Zhang & L. J. He, 2003. Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates. Molecular Ecology Notes, 3 (4): 581-585.
  • Jurado-Rivera, J. A. & E. Petitpierre, 2015. New contributions to the molecular systematics and the evolution of host-plant associations in the genus Chrysolina (Coleoptera, Chrysomelidae, Chrysomelinae). ZooKeys, 547 (547): 165-192.
  • Kasap, H., 1988. A list of some Chrysomelinae (Col., Chrysomelidae) from Turkey. Part I. Leptinotarsa, Crosita and Chrysomela (= Chrysolina), Türkiye Entomoloji Dergisi, 12 (1): 23-31.
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Türkiye'de dağılış gösteren Chrysolina Motschulsky, 1860 (Coleoptera: Chrysomelidae) cinsine dahil türlerin moleküler tanımlamasında COI ve ITS2 belirteçlerinin karşılaştırmalı analizleri

Yıl 2025, Cilt: 49 Sayı: 4, 413 - 436, 06.01.2026
https://doi.org/10.16970/entoted.1791117

Öz

Bu çalışma, böceklerde tür sınırlarını belirlemede iki moleküler belirtecin—sitokrom c oksidaz alt birimi I (COI) geni ve iç transkribe edilmiş aralık II (ITS2) bölgesinin—etkinliğini araştırmıştır. COI geni, böcek taksonomisinde tür sınırlarını belirlemede yaygın olarak kullanılmasına rağmen, ITS2 bölgesi bu amaçla daha az test edilmiştir. Türkiye genelinden toplanan Chrysolina cinsine (Coleoptera: Chrysomelidae) dahil 25 birey analiz edilmiştir. Moleküler analizler 2017 yılında Ege Üniversitesi Fen Fakültesi Zooloji Bölümü Moleküler Sistematik Laboratuvarı’nda gerçekleştirilmiş, veri analizleri ise 2025 yılında tamamlanmıştır. Her bir belirteç ve birleşik veri seti temelinde filogenetik ağaçların topolojisi ve türlerin monofiletikliği Bayesian yaklaşımıyla değerlendirilmiştir. Bu çalışma, Türkiye’de Chrysolina türleri için COI ve ITS2 belirteçlerinin ilk karşılaştırmalı analizini sunmaktadır. Elde edilen sonuçlar, her iki belirtecin de morfolojik ayrımları moleküler düzeyde desteklediğini ortaya koymuştur. COI, ITS2 ve birleşik veri setleriyle oluşturulan filogenetik ağaçlarda tüm türler monofiletik gruplar oluşturmuştur. Bulgular, ITS2 bölgesinin genetik çeşitliliğin değerlendirilmesi ve tür tanımlanmasında COI kadar etkili olabileceğini göstermektedir. Bu çalışma, ITS2’nin böcek taksonomisinde güvenilir bir moleküler belirteç olarak potansiyelini vurgulamakta ve Chrysolina cinsi içindeki tür sınırlarının anlaşılmasına katkı sağlamaktadır.

Destekleyen Kurum

TÜBİTAK 1001 (Project Number 113Z753) Ege University Scientific Research Projects Coordinatorship through Project Number 17FEN032

Proje Numarası

TÜBİTAK 1001 (Project Number 113Z753) Ege University Scientific Research Projects Coordinatorship through Project Number 17FEN032

Teşekkür

This publication is based on research conducted for the master’s thesis of the corresponding author. This work was partially supported by TÜBİTAK 1001 (Project Number 113Z753). Additionally, the authors gratefully acknowledges the financial support provided by Ege University Scientific Research Projects Coordinatorship through project number 17FEN032. I am grateful to Prof. Dr. Mahir Budak (Sivas Cumhuriyet University, Sivas), Prof. Dr. Ertan Mahir Korkmaz (Sivas Cumhuriyet University, Sivas) and Prof. Dr. Nurşen Alpagut Keskin (Ege University, Izmir) for their invaluable contributions to this research. The authors wish to express their gratitude to Prof. Dr. İsmail Şen (Isparta University, Isparta) for his invaluable contributions and expertise in the morphological identification of the species.

Kaynakça

  • Alvarez, J. M. & M. A. Hoy, 2002. Evaluation of the ribosomal ITS2 DNA sequences in closely related populations of the parasitoid Ageniaspis (Hymenoptera: Encyrtidae). Annals of the Entomological Society of America, 95 (2): 250-256.
  • Arnheim, N., 1983. “Concerted Evolution of Multigene Families in Evolution of Genes and Proteins, 38-61”. In: Evolution of Genes and Proteins (Eds. M. Nei & R. K. Koehn). Sinauer, Sunderland, Massachusetts, 346 pp.
  • Ashfaq, A., M. Erlandson & L. Braun, 2005. Hyper parasitism by Mesochorus spp (Hymenoptera: Ichneumonidae) in Peristenus sp. (Hymenoptera: Braconidae) and development of PCR primers for hyperparasitoid detection. Biological Control, 32 (3): 371-377.
  • Ball, S. L., P. D. N. Hebert, S. K. Burian & J. M. Webb, 2005. Biological identifications of mayflies (Ephemeroptera) using DNA barcodes. Journal of the North American Benthological Society, 24 (3): 508-524.
  • Becerra, J. X., 2004. Molecular systematics of Blepharida beetles (Chrysomelidae: Alticinae) and relatives. Molecular Phylogenetics and Evolution, 30 (1): 107-117.
  • Ben-David, T., S. Melamed, U. Gerson & S. Morin, 2007. ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae). Experimental and Applied Acarology, 41 (3): 169-181.
  • Bezděk, J. & L. Sekerka, 2024. Catalogue of Palaearctic Coleoptera: Chrysomeloidea II (Orsodacnidae, Megalopodidae, Chrysomelidae). Brill, Leiden, Netherlands, 750 pp.
  • Bieńkowski, A. O., 2019. “Chrysolina of the World – 2019 (Coleoptera: Chrysomelidae) Taxonomic Review”. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, 919 pp.
  • Bouckaert, R., T. G. Vaughan, J. Barido-Sottani, S. Duchêne, M. Fourment, A. Gavryushkina, J. Heled, G. Jones, D. Kühnert, N. D. Maio, M. Matschiner, F. K. Mendes, N. F. Müller, H. A. Ogilvie, L. Plessis, A. Popinga, A. Rambaut, D. Rasmussen, I. Siveroni, M. A. Suchard, C. H. Wu, D. Xie, C. Zhang, T. Stadler & A. J. Drummond, 2019. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Computational Biology, 15 (4): e1006650.
  • Budak, M., M. Güler, E. M. Korkmaz, S. Hastaoğlu Orgen & H. H. Başıbüyük, 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 (1): 76-85.
  • Calzolari, M., N. Bellin, M. Dottori, D. Torri, M. D. Luca, V. Rossi, G. Magoga & M. Montagna, 2024. Integrated taxonomy to advance species delimitation of the Anopheles maculipennis complex. Scientific Reports, 14 (1): 30914.
  • Campbell, B., J. D. Steffen-Campbell & J. H. Werren, 1993. Phlogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences. Insect Molecular Biology, 2 (4): 225-237.
  • Chen, S. L., H. Yao, J. P. Han, C. Liu & J. Y. Song, 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 5 (1): e8613 (1-8).
  • Dinno, A., 2018. Paran: Horn's test of principal components/factors. (Web page: https://CRAN.R-project.org/package=paran) (Date accessed: April 2025).
  • Edel, V., 1998. “Polymerase Chain Reaction in Mycology: An Overview, 1-20”. In: Applications of PCR in Mycology (Eds. P. D. Bridge), CAB International, Wallingford, New York, 357 pp.
  • Ezard, T., T. Fujisawa & T. Barraclough, 2021. Splits: Species limits by threshold statistics. (Web page: https://R-Forge.Rproject.org/projects/splits/) (Date accessed: April 2025).
  • Farris, J. S., M. Kallersjo, A. G. Kluge & C. Bult, 1994. Testing significance of incongruence. Cladistics, 10 (3): 315-319.
  • Fujisawa, T. & T. G. Barraclough, 2013. Delimiting species using single-locus data and the generalized mixed yule coalescent approach: a revised method and evaluation on simulated data sets. Systematic Biology, 62 (5):707–724.
  • Garin, C. F., C. Juan & E. Petitpierre, 1999. Mitochondrial DNA phylogeny and the evolution of host-plant use in palearctic Chrysolina (Coleoptera, Chrysomelidae) leaf beetles. Journal of Molecular Evolution, 48 (4): 435-444.
  • Geneious, 2025. Geneious, 10.2.6. (Web page: https://www.geneious.com) (Date accessed: March 2025).
  • Germain, J. F., C. Chatot, I. Meusnier, E. Artige, J. Y. Rasplus & A. Cruaud, 2013. Molecular identification of Epitrix potato flea beetles (Coleoptera: Chrysomelidae) in Europe and North America. Bulletin of Entomological Research, 103 (3): 354-362.
  • Hajibabaei, M., D. H. Janzen, J. M. Burns, W. Hallwachs & P. D. N. Hebert, 2006. DNA barcodes distinguish species of tropical Lepidoptera. The Proceedings of the National Academy of Sciences, 103 (4): 968-971.
  • Hebert, P. D. N., A. Cywinska, S. L. Ball & J. R. DeWaard, 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B: Biological Sciences, 270 (1512): 313-321.
  • Hebert, P. D. N., E. H. Penton, J. M. Burns, D. H. Janzen & W. Hallwachs, 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. The Proceedings of the National Academy of Sciences, 101 (41): 14812-14817.
  • Hebert, P. D. N., S. Ratnasingham & J. R. De Waard, 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London Series B: Biological Sciences, 270 (1): 96-99.
  • Ji, Y. J., D. X. Zhang & L. J. He, 2003. Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates. Molecular Ecology Notes, 3 (4): 581-585.
  • Jurado-Rivera, J. A. & E. Petitpierre, 2015. New contributions to the molecular systematics and the evolution of host-plant associations in the genus Chrysolina (Coleoptera, Chrysomelidae, Chrysomelinae). ZooKeys, 547 (547): 165-192.
  • Kasap, H., 1988. A list of some Chrysomelinae (Col., Chrysomelidae) from Turkey. Part I. Leptinotarsa, Crosita and Chrysomela (= Chrysolina), Türkiye Entomoloji Dergisi, 12 (1): 23-31.
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  • Li, Y. W., X. Zhou, G. Feng, H. Y. Hu & L. M. Niu, 2010. COI and ITS2 sequences delimit species, reveal cryptic taxa and host specificity of fig-associated Sycophila (Hymenoptera, Eurytomidae). Molecular Ecology Resources, 10 (1): 31-40.
  • Löbl, I. & R. A. B. Leschen, 2005. Demography of coleopterists and their thoughts on DNA barcoding and the phylocode, with commentary. Coleopterists Bulletin, 59 (3): 284-292.
  • Lopatin, I., 1977. Leaf Beetles (Chrysomelidae) of Middle Asia and Kazakhistan. Nauka, Leningrad, Russia, 268 pp.
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  • Warchalowski, A., 2010a. “The Palaearctic Chrysomelidae Identification Keys, Volume 1”. Natura Optima Dux Foundation (Eds. D. Iwan). Warszawska Drukarnia Naukowa, Warszawa, 629 pp.
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Toplam 61 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Entomoloji
Bölüm Araştırma Makalesi
Yazarlar

Duygu Kan 0000-0003-2894-0371

Bekir Keskin 0000-0002-6662-8736

Proje Numarası TÜBİTAK 1001 (Project Number 113Z753) Ege University Scientific Research Projects Coordinatorship through Project Number 17FEN032
Gönderilme Tarihi 26 Eylül 2025
Kabul Tarihi 28 Aralık 2025
Yayımlanma Tarihi 6 Ocak 2026
Yayımlandığı Sayı Yıl 2025 Cilt: 49 Sayı: 4

Kaynak Göster

APA Kan, D., & Keskin, B. (2026). Comparative analyses of COI and ITS2 in the molecular identification of Chrysolina Motschulsky, 1860 (Coleoptera: Chrysomelidae) in Türkiye. Turkish Journal of Entomology, 49(4), 413-436. https://doi.org/10.16970/entoted.1791117