Mitogenomics of Psorodonotus ebneri Brunner von Wattenwyl, 1861 (Orthoptera: Tettigoniidae): Selection profile and patterns of intraspecific and interspecific divergence

Abstract

Advances in DNA sequencing technologies have made comparative mitogenome studies available. However, studies on intraspecific mitogenome variation are still rare. This study reports total mitogenome of Psorodonotus ebneri Brunner von Wattenwyl, 1861 (Orthoptera: Tettigoniidae), highlighting selection profile and intraspecific and interspecific divergence in protein-coding genes (PCGs). To this end, two datasets were analysed, one including five P. ebneri mitogenomes and the other additionally possessing a mitogenome of P. venosus (Fischer von Waldheim, 1839). Results showed that the mitogenome of P. ebneri consists of 37 genes and an AT-rich region, ordering as in Pancrustacean, constituting 15645-15668 base pairs. Among 38 gene borders, eight had intergenic sequences (IGS) and seven overlapping (OR). All tRNAs exhibited the typical clover-leaf structure except for trnS1. Pairwise distance ranges between 0.001-0.003 among sequences of P. ebneri and ~0.12 between P. ebneri and P. venosus. The dN/dS and neutrality index indicated purifying selection in PCGs. We arrived at the following conclusions: (i) P. ebneri mitogenomes show characteristics of Orthoptera and Pancrustacea, (ii) IGS and OR appear conserved in Tettigoniidae, (iii) low intraspecific variation is likely due to small population size, and (iv) most of the PCGs evolved under purifying selection suggesting no specific adaptation to montane habitats.

Keywords

• Mitogenomics
• Orthoptera
• selection
• Tettigoniidae

Project Number

Mohamed bin Zayed Species Conservation Fund, Project No: 192520509

Thanks

The next generation sequencing was funded by Mohamed bin Zayed Species Conservation Fund via project (Project No: 192520509) that granted to Dr. Battal Çıplak. We thank M. Yasemin Karakaş (Hatay Mustafa Kemal University) for her participation of this project and field and laboratory work. We thank our undergrad student A. Koray Koç (Akdeniz University) for his help during the visualizing the codon and RSCU data. The author contributions were as follows: O. Uluar; laboratory work, analysis, figure preparation and writing; M. Yartaş, analysis, documentation, and figure preparation; Ö. Yahyaoğlu; field and laboratory work; B. Çıplak; conception, funding, writing and design. All authors read and contributed to the manuscript. We acknowledge the use of ChatGPT (OpenAI, 2024) for grammar and language editing.

Psorodonotus ebneri Brunner von Wattenwyl, 1861 (Orthoptera, Tettigoniidae) mitogenomiği: Seçilim profili ve türiçi ve türler arası farklılaşma örüntüleri

Abstract

DNA dizileme teknolojilerindeki gelişmeler, karşılaştırmalı mitogenom çalışmalarını olası kılmıştır. Ancak, türiçi mitogenom varyasyonu üzerine yapılan çalışmalar hâlâ nadirdir. Bu çalışma, Psorodonotus ebneri Brunner von Wattenwyl, 1861 (Orthoptera: Tettigoniidae)’in total mitogenomunu rapor etmesinin yanısıra, türiçi ve türler arası farklılaşma oranı ve protein kodlayan genlerde (PKG) görülen seçilim profillerine odaklanmaktadır. Bu çalışmada, iki veri seti analiz edilmiştir: biri beş P. ebneri mitogenomunu, diğeri ise ek olarak P. venosus (Fischer von Waldheim, 1839)’u içermektedir. Sonuçlar, P. ebneri mitogenomunun 37 gen ve AT zengin bir bölgeden oluştuğunu, bunların Pancrustacea gen sırasına sahip olduğu ve toplamda 15645–15668 baz çifti uzunluğunda olduğunu göstermektedir. Otuzsekiz gen sınırının sekizinde genler arası bölgeler bulunurken, yedisinde genler örtüşmektedir. trnS1 dışındaki tüm tRNA’lar tipik yonca yaprağı yapısını göstermektedir. Beş P. ebneri dizisi arasındaki ikili uzaklıklar 0.001–0.003, P. ebneri ile P. venosus arasındaki ise ~0.12 olarak hesaplanmıştır. dN/dS oranı ve nötralite indeksi PKG’lerde saflaştırıcı seçilim profiline işaret etmektedir. Verilerden şu çıkarımlara varılmıştır: (i) P. ebneri mitogenomları Orthoptera ve Pancrustacea genel özelliklerini taşımaktadır, (ii) IGS ve OR örüntüleri Tettigoniidae’de korunmuş görünmektedir, (iii) düşük türiçi varyasyon olasılıkla populasyonun küçük olmasından kaynaklanmaktadır ve (iv) çoğu PKG, saflaştırıcı seçilim altında evrimleşmiştir ve dağlık habitatlara adaptasyonu gösteren bir işaret bulunmamıştır.

Keywords

• Mitogenomik
• Orthoptera
• seçilim
• Tettigoniidae

Project Number

Mohamed bin Zayed Species Conservation Fund, Project No: 192520509

References

1. M. Bernt, A. Donath, F. Jühling, F. Externbrink, C. Florentz, G. Fritzsch, J. Pütz, M. Middendorf, P.F. Stadler, MITOS: improved de novo metazoan mitochondrial genome annotation, Mol. Phylogenet. Evol., 69 (2013) 313–319.
2. S.L. Cameron, Insect mitochondrial genomics: A decade of progress, Annu. Rev. Entomol., 70 (2025) 83–101.
3. P.D. Hebert, A. Cywinska, S.L. Ball, J.R. deWaard, Biological identifications through DNA barcodes, Proc. R. Soc. B., 270 (2003) 313–321.
4. S. Hotaling, J.L. Kelley, P.B. Frandsen, Toward a genome sequence for every animal: Where are we now?, Proc. Natl. Acad. Sci., U.S.A., 118 (2021) e2109019118.
5. S.L. Cameron, Insect mitochondrial genomics: Implications for evolution and phylogeny, Annu. Rev. Entomol., 59 (2014) 95–117.
6. P.A. Morin, F.I. Archer, A.D. Foote, J. Vilstrup, E.E. Allen, P. Wade, J. Durban, K. Parsons, R. Pitman, L. Li, P. Bouffard, S.C.A. Nielsen, M. Rasmussen, E. Willerslev, M.T.P. Gilbert, T. Harkins, Complete mitochondrial genome phylogeographic analysis of killer whales (Orcinus orca) indicates multiple species, Genome Res., 20 (2010) 908–916.
7. P.S. Gehring, K.A. Tolley, F.S. Eckhardt, T.M. Townsend, T. Ziegler, F. Ratsoavina, F. Glaw, M. Vences, Hiding deep in the trees: Discovery of divergent mitochondrial lineages in Malagasy chameleons of the Calumma nasutum group, Ecol. Evol., 2 (2012) 1468–1479.
8. C. Aguilar, L.F. De Léon, J.R. Loaiza, W.O. McMillan, M.J. Miller, Extreme sequence divergence between mitochondrial genomes of two subspecies of White-breasted Wood-wren (Henicorhina leucosticta, Cabanis, 1847) from western and central Panamá, Mitochondrial DNA A, 27 (2016) 956–957.

9. M.R. Garvin, J.P. Bielawski, L.A. Sazanov, A.J. Gharrett, Review and meta-analysis of natural selection in mitochondrial complex I in metazoans, J. Zool. Syst. Evol. Res., 53 (2015) 1–17.
10. F. Alda, A.J. Adams, P. Chakrabarty, W.O. McMillan, Mitogenomic divergence between three pairs of putative geminate fishes from Panama, Mitochondrial DNA B, 3 (2018) 1–5.
11. J.T. Sun, P.Y. Jin, A.A. Hoffmann, X.Z. Duan, J. Dai, G. Hu, X.F. Xue, X.Y. Hong, Evolutionary divergence of mitochondrial genomes in two Tetranychus species distributed across different climates, Insect Mol. Biol., 27 (2018) 698–709.
12. M. Lubośny, B. Śmietanka, A. Przyłucka, A. Burzyński, Highly divergent mitogenomes of Geukensia demissa(Bivalvia, Mytilidae) with extreme AT content, J. Zool. Syst. Evol. Res., 58 (2020) 571–580.
13. Z. Wu, G. Waneka, D.B. Sloan, The tempo and mode of angiosperm mitochondrial genome divergence inferred from intraspecific variation in Arabidopsis thaliana, G3, 10 (2020) 1077–1086.
14. E.A. Beck, S. Bassham, W.A. Cresko, Extreme intraspecific divergence in mitochondrial haplotypes makes the threespine stickleback fish an emerging evolutionary mutant model for mito-nuclear interactions, Front. Genet., 13 (2022) 925786.
15. K. Himmelstrand, M. Brandström Durling, M. Karlsson, J. Stenlid, Å. Olson, Multiple rearrangements and low inter- and intra-species mitogenome sequence variation in the Heterobasidion annosum s.l. species complex, Front. Microbiol., 14 (2023) 1159811.
16. S. Zhou, X. Wang, L. Wang, X. Gao, T. Lyu, T. Xia, L. Shi, Y. Dong, X. Mei, Z. Zhang, H. Zhang, Different evolutionary trends of Galloanseres: Mitogenomics analysis, Animals, 14(10) (2024) 1437.
17. M.M. Cigliano, H. Braun, D.C. Eades, D. Otte, Orthoptera Species File [retrieval date: 12.01.2025], http://orthoptera.speciesfile.org/
18. N.C. Sheffield, H. Song, S.L. Cameron, M.F. Whiting, A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles, Mol. Biol. Evol., 25(11) (2008) 2499-2509.
19. C. Liu, J. Chang, C. Ma, L. Li, S. Zhou, Mitochondrial genomes of two Sinochlora species (Orthoptera): Novel genome rearrangements and recognition sequence of replication origin, BMC Genomics, 14 (2013) 1471-2164.
20. U. Karşı, B. Çıplak, Mitogenome of Anterastes babadaghi (Orthoptera: Tettigoniinae; Platycleidini): Frequent conserved overlapping regions within Tettigoniinae, Zootaxa, 4651(1) (2019) 173–190.
21. P.N. Öztürk, B. Çıplak, Phylomitogenomics of Phaneropteridae (Orthoptera): Combined data indicate a poorly conserved mitogenome, Int. J. Biol. Macromol., 132 (2019) 1318-1326.
22. D.E. Kim, P. Kim, H. Lee, N. Kim, D. Kim, M. Lee, Y. Ban, B. Jang, J. Park, Comprehensive analysis of the complete mitochondrial genome of Melanoplus differentialis (Acrididae: Melanoplinae) captured in Korea, Entomol. Res., 53(2) (2023) 66-81.
23. J. Dong, Y. Liu, M.K. Tan, R.A. Wahab, R. Nattier, P. Chifflet-Belle, T. Robillard, Museomics allows comparative analyses of mitochondrial genomes in the family Gryllidae (Insecta, Orthoptera) and confirms its phylogenetic relationships, PeerJ, 12 (2024) e17734.
24. S. Kaya, D.P. Chobanov, J. Skejo, K.-G. Heller, B. Çıplak, The Balkan Psorodonotus (Orthoptera: Tettigoniidae): Testing the existing taxa confirmed presence of three distinct species, Eur. J. Entomol., 112(3) (2015) 525-541.
25. S. Kaya, B. Çıplak, Budding speciation via peripheral isolation: The Psorodonotus venosus (Orthoptera, Tettigoniidae) species group example, Zool. Scripta, 45(5) (2016) 521-537.
26. S. Kaya, B. Çıplak, Phylogeography and taxonomy of the Psorodonotus caucasicus (Orthoptera, Tettigoniidae) group: Independent double invasion of the Balkans from the Caucasus, Syst. Entomol., 42(1) (2017) 118-133.
27. B. Çıplak, Ö. Yahyaoğlu, O. Uluar, Revisiting Pholidopterini (Orthoptera, Tettigoniidae): Rapid radiation causes homoplasy and phylogenetic instability, Zool. Scripta, 50 (2021) 225-240.
28. B. Çıplak, Ö. Yahyaoğlu, O. Uluar, Ö. Doğan, H.H. Başibüyük, E.M. Korkmaz, Phylogeography of Pholidopterini: Revising molecular clock calibration by Mid-Aegean Trench, Insect Syst. Evol., 53(5) (2022) 515-535.
29. Ö. Yahyaoğlu, O. Uluar, B. Çıplak, Priceless or worthless: Conserving Psorodonotus ebneri, one of the World’s 100 most threatened species, J. Insect Conserv., 29(2) (2025) 22.
30. U. Karşı, B. Çıplak, Complete mitogenome of Psorodonotus venosus (Orthoptera, Tettigoniidae; Tettigoniinae): Short intergenic spacers shorten the total genome, Zootaxa, 4614(3) (2019) 498-510.
31. J.E.M. Baillie, E.R. Butcher, Priceless or worthless? The world's most threatened species, Zoological Society of London, United Kingdom, 2012.
32. M.D. Brand, Uncoupling to survive? The role of mitochondrial inefficiency in ageing, Exp. Gerontol., 35 (2000) 811–820.
33. E. Afgan, D. Baker, B. Batut, M. Van Den Beek, D. Bouvier, M. Ech, J. Chilton, D. Clements, N. Coraor, B.A. Grüning, A. Guerler, J. Hillman-Jackson, S. Hiltemann, V. Jalili, H. Rasche, N. Soranzo, J. Goecks, J. Taylor, A. Nekrutenko, D. Blankenberg, The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update, Nucleic Acids Res., 46(W1) (2018) W537–W544.
34. B. Langmead, S.L. Salzberg, Fast gapped-read alignment with Bowtie 2, Nat. Methods, 9(4) (2012) 357–359.
35. M. Kearse, R. Moir, A. Wilson, S. Stones-Havas, M. Cheung, S. Sturrock, S. Buxton, A. Cooper, S. Markowitz, C. Duran, T. Thierer, B. Ashton, P. Meintjes, A. Drummond, Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data, Bioinformatics, 28(12) (2012) 1647–1649.
36. D. Laslett, B. Canbäck, ARWEN: A program to detect tRNA genes in metazoan mitochondrial nucleotide sequences, Bioinformatics, 24(2) (2008) 172–175.
37. K. Katoh, J. Rozewicki, K.D. Yamada, MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization, Brief. Bioinform., 20(4) (2019) 1160–1166.
38. S. Kumar, G. Stecher, K. Tamura, MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets, Mol. Biol. Evol., 33(7) (2016) 1870–1874.
39. The pandas development team, pandas-dev/pandas: Pandas (latest version), Zenodo, (2020).
40. C.R. Harris, K.J. Millman, S.J. van der Walt, R. Gommers, P. Virtanen, D. Cournapeau, E. Wieser, J. Taylor, S. Berg, N.J. Smith, R. Kern, M. Picus, S. Hoyer, M.H. van Kerkwijk, M. Brett, A. Haldane, J.F. del Río, M. Wiebe, P. Peterson, et al., Array programming with NumPy, Nature, 585(7825) (2020) 357–362.
41. Y. Shimoyama, pyCirclize: Circular visualization in Python, https://github.com/moshi4/pyCirclize, (2022).
42. Plotly Technologies Inc., Collaborative data science, Plotly Technologies Inc., https://plot.ly, (2015).
43. M. Vences, S. Patmanidis, V. Kharchev, S.S. Renner, Concatenator, a user-friendly program to concatenate DNA sequences, implementing graphical user interfaces for MAFFT and FastTree, Bioinform. Adv., 2(1) (2022).
44. K. Tamura, M. Nei, S. Kumar, Prospects for inferring very large phylogenies by using the neighbor-joining method, Proc. Natl. Acad. Sci., 101(30) (2004) 11030–11035.
45. J.H. McDonald, M. Kreitman, Adaptive protein evolution at the Adh locus in Drosophila, Nature, 351 (1991) 652–654.
46. P. Librado, J. Rozas, DnaSP v5: A software for comprehensive analysis of DNA polymorphism data, Bioinformatics, 25(11) (2009) 1451–1452.
47. H. Song, C. Amedegnato, M.M. Cigliano, L.D. Grandcolas, S.W. Heads, Y. Huang, D. Otte, M.F. Whiting, 300 million years of diversification: Elucidating the patterns of orthopteran evolution based on comprehensive taxon and gene sampling, Cladistics, 31(6) (2015) 621–651.
48. Z. Zhou, L. Zhao, N. Liu, H. Guo, B. Guan, J. Di, F. Shi, Towards a higher-level Ensifera phylogeny inferred from mitogenome sequences, Mol. Phylogenet. Evol., 108 (2017) 22–33.
49. I. Kim, S.Y. Cha, M.H. Yoon, J.S. Hwang, S.M. Lee, H.D. Sohn, B.R. Jin, The complete nucleotide sequence and gene organization of the mitochondrial genome of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae), Gene, 353(1) (2005) 155–168.
50. H.-L. Liu, S. Chen, Q.-D. Chen, D.-Q. Pu, Z.-T. Chen, Y.-Y. Liu, X. Liu, The first mitochondrial genomes of the family Haplodiplatyidae (Insecta: Dermaptera) reveal intraspecific variation and extensive gene rearrangement, Biol., 11(6) (2022) 807.
51. D.-X. Zhang, G.M. Hewitt, Insect mitochondrial control region: A review of its structure, evolution and usefulness in evolutionary studies, Biochem. Syst. Ecol., 25(2) (1997) 99–120.
52. J.L. Boore, Animal mitochondrial genomes, Nucleic Acids Res., 27(8) (1999) 1767–1780.
53. J. Yang, F. Ye, Y. Huang, Mitochondrial genomes of four katydids (Orthoptera: Phaneropteridae): New gene rearrangements and their phylogenetic implications, Gene, 575 (2016) 702–711.
54. J. Wang, Z. Qiu, H. Yuan, Y. Huang, The complete mitochondrial genomes of two Phaneroptera species (Orthoptera: Tettigoniidae) and comparative analysis of mitochondrial genomes in Orthoptera, Unpublished, (2017).
55. M.Y. Karakaş, O. Uluar, M. Yartaş, B. Çıplak, Mitophylogenetic patterns in Tettigoniidae: Insights from the complete mitogenome of Saga natoliae (Orthoptera), Zootaxa, 5569(3) (2025) 459–476.
56. M.Y. Karakaş, Ö. Yahyaoğlu, O. Uluar, M. Budak, B. Çıplak, Mitochondrial genome of Poecilimon cretensis (Orthoptera: Tettigoniidae: Phaneropterinae): Strong phylogenetic signals in gene overlapping regions, Zootaxa, 5263(1) (2023) 141–147.
57. Ö. Yahyaoğlu, C.T. Doğan, O. Uluar, M.Y. Karakaş, B. Çıplak, Mitogenome of Xya pfaendleri (Orthoptera: Caelifera): Its comparative description and phylogenetic position within Tridactylidea, Zootaxa, 5369(4) (2023) 576–584.
58. S.I. Cameron, M.F. Whiting, The complete mitochondrial genome of the tobacco hornworm, Manduca sexta (Insecta: Lepidoptera: Sphingidae), and an examination of mitochondrial gene variability within butterflies and moths, Gene, 408 (2008) 112–123.
59. B. Chen, M. Duan, S. Liu, et al., The complete mitochondrial genome and phylogenetic implications of Paradoxopsyllus custodis and Stenischia montanis yunlongensis, Sci. Rep., 14 (2024) 31555.
60. G.K. Karademir, S. Teber, C.C. Kulig, A.M. Toroslu, O. Ibis, A. Yıldırım, Complete mitochondrial genome analyses of Forcipomyia pulchrithorax (Diptera: Ceratopogonidae): Genome orientation and phylogenetic implications, Kafkas Univ. Vet. Fac. J., 30(2) (2024) 223–231.
61. E.M. Korkmaz, Ö. Doğan, M. Budak, H.H. Başıbüyük, Two nearly complete mitogenomes of wheat stem borers, Cephus pygmeus (L.) and Cephus sareptanus Dovnar-Zapolshij (Hymenoptera: Cephidae): An unusual elongation of the rrnS gene, Gene, 558 (2015) 254–264.
62. E.M. Korkmaz, M. Budak, M.N. Ördek, H.H. Başıbüyük, The complete mitogenomes of Calameuta filiformis (Eversmann, 1847) and Calameuta idolon (Rossi, 1794) (Hymenoptera: Cephidae): The remarkable features of the elongated A+T-rich region in Cephini, Gene, 576 (2016) 404–411.
63. J. Yang, Y. Wang, H. Yang, X. Huang, The complete mitochondrial genome of Brachytarsina amboinensis (Diptera: Hippoboscoidea: Streblidae) provides new insights into phylogenetic relationships of Hippoboscoidea, Bull. Entomol. Res., 114(6) (2024) 836–846.
64. R. Zardoya, A. Meyer, Phylogenetic performance of mitochondrial protein-coding genes in resolving relationships among vertebrates, Mol. Biol. Evol., 13(7) (1996) 933–942.
65. E. Mohandesan, R.R. Fitak, J. Corander, A. Yadamsuren, B. Chuluunbat, O. Abdelhadi, A. Raziq, P. Nagy, G. Stalder, C. Walzer, B. Faye, P.A. Burger, Mitogenome sequencing in the genus Camelus reveals evidence for purifying selection and long-term divergence between wild and domestic Bactrian camels, Sci. Rep., 7(1) (2017) 9970.
66. OpenAI, ChatGPT (December 3 version) [Large language model], https://chat.openai.com/chat, (2023).