When Morphology Meets Molecules: Barcoding Confirms an Ancient Separation of Phytoecia tigrina subspecies (Coleoptera, Cerambycidae)
Abstract
The molecular analysis of Phytoecia tigrina revealed substantial genetic differentiation (5.8–7.0 %) between populations on opposite sides of the Carpathian Arc. Two distinct haplogroups were identified: PhtZk (western, haplotype PhtZk-1) and PhtPo (eastern, haplotypes PhtPo-1 and PhtPo-2), including the type locality of Phytoecia tigrina podillica Zamoroka, Ruicănescu& Manci, 2024. These findings support the division of Ph. tigrina into two subspecies. The pronounced genetic divergence suggests that the separation between Ph. tigrina tigrina and Ph. tigrina podillica, occurred much earlier than previously assumed. Moreover, the coexistence of two highly distinct haplotypes within Ph. tigrina podillica populations suggests episodes of rapid range expansions and contractions during the Late Pleistocene and Holocene. Overall, our results provide valuable insights into the evolutionary processes driving intraspecific diversification in Ph. tigrina in response to cyclical climate fluctuations.
References
Anisimova, M. & Gascuel, O. 2006. Approximate likelihood ratio test for branches: A fast, accurate and powerful alternative. Systematic Biology, 55 (4), 539-552.
https://doi.org/10.1080/10635150600755453
Bacal, S., Burduja, D., Buşmachiu, G., Cebotari, C. & Merkl, O. 2020. Longhorn beetles in the entomological collections of the Republic of Moldova (Coleoptera: Cerambycidae). Folia Entomologica Hungarica, 81, 43-72.
https://doi.org/10.17112/FoliaEntHung.2020.81.43
Bouckaert, R., Vaughan, T.G., Barido-Sottani, J., Duchêne, S., Fourment, M., Gavryushkina, A. et al. 2019. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Computational Biology, 15 (4), e1006650.
https://doi.org/10.1371/journal.pcbi.1006650
Caba, F. & Dascălu, M. 2024. Dissecting natural hybridisation in longhorned beetles through an integrative approach: Further proof of reticulate evolution in Dorcadionini (Cerambycidae, Lamiinae). Zoologica Scripta, 53 (5), 665-687.
https://doi.org/10.1111/zsc.12664
Council Directive 92/43/EEC. 1992. On the conservation of natural habitats and of wild fauna and flora. Official Journal of the European Communities, L (206), 7-50.
Council of Europe. 2011. Revised Annex I of Resolution 6 of the Bern Convention listing the species requiring specific habitat conservation measures.
Crișan, A., Manci, C.-O., Ruicănescu, A. & Rákosy, L. 2017. Information about the biology, ecology and distribution of Pilemia tigrina (Mulsant, 1851) in Romania (Coleoptera: Cerambycidae). Entomologica Romanica, 21: 9-14.
https://doi.org/10.24193/entomolrom.21.2
Dascălu, M.-M., Caba, F.-G. & Fusu, L. 2021. DNA barcoding in Dorcadionini (Coleoptera, Cerambycidae) uncovers mitochondrial-morphological discordance and the hybridogenic origin of several subspecies. Organisms Diversity & Evolution, 22(1), 205-229.
https://doi.org/10.1007/s13127-021-00531-x
Drummond, A.J., Ho, S.Y.W., Phillips, M.J. & Rambaut, A. 2006. Relaxed phylogenetics and dating with confidence. PLoS Biology, 4 (5), e88.
https://doi.org/10.1371/journal.pbio.0040088
Eckert, C.G., Samis, K.E. & Lougheed, S.C. 2008. Genetic variation across species' geographical ranges: The central-marginal hypothesis and beyond. Molecular Ecology, 17 (5), 1170-1188.
https://doi.org/10.1111/j.1365-294X.2007.03659.x
European Commission. 2018. Report under Article 17 of the Habitats Directive (reporting period: 2013-2018). Pilemia tigrina assessments at EU biogeographical level.
Gascuel, O. 1997. BIONJ: An improved version of the NJ algorithm based on a simple model of sequence data. Molecular Biology and Evolution, 14 (7), 685-695.
https://doi.org/10.1093/oxfordjournals.molbev.a025808
Gernhard, T. 2008. The conditioned reconstructed process. Journal of Theoretical Biology, 253 (4), 769-778.
https://doi.org/10.1016/j.jtbi.2008.04.005
Georgiev, G. 2020. New records of longhorn beetles (Coleoptera: Cerambycidae) in entomological collections in Bulgaria. Forest Science, 56 (1), 87-99.
Gouy, M., Tannier, E., Comte, N. & Parsons, D.P. 2021. Seaview Version 5: A multiplatform software for multiple sequence alignment, molecular phylogenetic analyses, and tree reconciliation. Methods in Molecular Biology, 2231, 241-260.
https://doi.org/10.1007/978-1-0716-1036-7_15
Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology, 59 (3), 307-321.
https://doi.org/10.1093/sysbio/syq010
Hargreaves, A. L. & Eckert, C. G. 2013. Evolution of dispersal and mating systems along geographic gradients: Implications for shifting ranges. Functional Ecology, 28 (1), 5-21.
https://doi.org/10.1111/1365-2435.12170
Ilić, N. & Ćurčić, S. 2015. A checklist of longhorn beetles (Coleoptera: Cerambycidae) of Serbia. Zootaxa, 4026 (1), 1-97.
https://doi.org/10.11646/zootaxa.4026.1.1
Jukes, T. H. & Cantor, C. R. 1969. Evolution of Protein Molecules. In: Munro, H. N., ed. Mammalian Protein Metabolism. Academic Press, New York, 21-132.
https://doi.org/10.1016/B978-1-4832-3211-9.50009-7
Karpiński, L., Gorring, P. & Cognato, A. I. 2023. DNA vs. morphology in delineating species boundaries of endemic Mongolian Eodorcadion taxa (Coleoptera: Cerambycidae). Diversity, 15 (5), 662.
https://doi.org/10.3390/d15050662
Karpiński, L., Gorring, P., Enkhnasan, D. & Cognato, A. I. 2024. First support for phylogenetically segregated ecotypes and delineating thresholds for inter‐ and intraspecific ranks in phytophagous Central Asian beetles (Coleoptera). Zoologica Scripta, 54 (2), 144-162.
https://doi.org/10.1111/zsc.12702
Korlević, P., McAlister, E., Mayho, M., Makunin, A., Flicek, P. & Lawniczak, M. K. N. 2021. A minimally morphologically destructive approach for DNA retrieval and whole-genome shotgun sequencing of pinned historic Dipteran vector species. Genome Biology and Evolution, 13 (10).
https://doi.org/10.1093/gbe/evab226
Makunin, A., Korlević, P., Park, N., Goodwin, S., Waterhouse, R. M., von Wyschetzki, K., Jacob, C. G., Davies, R., Kwiatkowski, D., St. Laurent, B., Ayala, D. & Lawniczak, M. K. N. 2022. A targeted amplicon sequencing panel to simultaneously identify mosquito species and Plasmodium presence across the entire Anopheles genus. Molecular Ecology Resources, 22 (1), 28-44.
https://doi.org/10.1111/1755-0998.13436
Rambaut, A. 2014. FigTree v1.4.2, a graphical viewer of phylogenetic trees. Available at: http://tree.bio.ed.ac.uk/software/figtree/
Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. 2018. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67 (5), 901-904.
https://doi.org/10.1093/sysbio/syy032
Scherrenberg, M. D. W., Berends, C. J., & van de Wal, R. S. W. 2025. CO2 and summer insolation as drivers for the Mid-Pleistocene Transition. Climate of the Past, 21 (6), 1061-1077.
https://doi.org/10.5194/cp-21-1061-2025
Srivathsan, A., Lee, L., Katoh, K., Hartop, E., Kutty, S. N., Wong, J., Yeo, D. & Meier, R. 2021. ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC Biology, 19 (1).
https://doi.org/10.1186/s12915-021-01141-x
Srivathsan, A., Feng, V., Suárez, D., Emerson, B. & Meier, R. 2024. ONTbarcoder 2.0: Rapid species discovery and identification with real-time barcoding facilitated by Oxford Nanopore R10.4. Cladistics, 40 (2), 192-203.
https://doi.org/10.1111/cla.12566
Tezcan, S., Karsavuran, Y., Pehlivan, E. &Özdikmen, H. 2020. Catalogue of longhorned beetles of LEMT (Lodos Entomological Museum, Turkey) (Coleoptera: Cerambycidae) Part II: Lamiinae and Dorcadioninae. Munis Entomology & Zoology, 15(1), 145-170.
Tóth, I.Z., Csathó, A.I., Buşmachiu, G. & Merkl, O. 2016. Pilemia tigrina: New and corrected records from the Republic of Moldova, Hungary and Romania (Coleoptera: Cerambycidae). Folia Entomologica Hungarica, 77, 33-40.
https://doi.org/10.17112/FoliaEntHung.2016.77.33
Turner, T. 2013. Faculty Opinions recommendation of Macroevolutionary speciation rates are decoupled from the evolution of intrinsic reproductive isolation in Drosophila and birds. [dataset]. In: Faculty Opinions - Post-Publication Peer Review of the Biomedical Literature. H1 Connect.
https://doi.org/10.3410/f.718098614.793485011
Vitali, F. 2015. Saperdacaroli n. sp., a new fossil species from Early Eocene of Colorado, with taxonomic remarks on its extinct congeners (Coleoptera, Cerambycidae). Les Cahiers Magellanes, Nouvelle Série, 17, 20-28.
Zamoroka, A. M., Semaniuk, D. V., Shparyk, V. Yu. & Mykytyn, T. V. 2019. Taxonomic position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae) based on molecular and morphological data. Vestnik Zoologii, 53 (3), 209-226.
https://doi.org/10.2478/vzoo-2019-0021
Zamoroka, A. 2022 a. Phytoecia in Ukraine (Coleoptera: Cerambycidae). In: Myroniuk, I.S., Roshko, V.H., Hasynets, Ya.S. et al. eds. Abstracts of International Scientific Conference (Ukraine, Uzhhorod, 30 September-02 October, 2022). Uzhhorod: "Hoverla", 9-10.
Zamoroka, A. 2022 b. The longhorn beetles included in the Red Data Book of Ukraine, the fourth edition - data analysis. In: Yanytskyi, T.P. et al. (eds.) Current Issues in the Study of the Entomofauna of the Western Region of Ukraine: Abstracts of the Scientific and Practical Conference "XVI Lviv Entomological School" (Lviv, October 25, 2022). Lviv: State Natural History Museum of the NAS of Ukraine, 19-21 [In Ukrainian].
Zamoroka, A. M. 2022 c. The longhorn beetles (Coleoptera, Cerambycidae) of Ukraine: Results of two centuries of research. Biosystem Diversity, 30 (1), 46-74.
https://doi.org/10.15421/012206
Zamoroka, A. M. 2022 d. Molecular revision of Rhagiinisensu lato (Coleoptera, Cerambycidae): Paraphyly, intricate evolution and novel taxonomy. Biosystem Diversity, 30 (3), 295-309.
https://doi.org/10.15421/012232
Zamoroka, A. M., Tròcoli, S., Shparyk, V. Yu. & Semaniuk, D. V. 2022. Polyphyly of the genus Stenurella (Coleoptera, Cerambycidae): Consensus of morphological and molecular data. Biosystem Diversity, 30 (2), 119-136.
https://doi.org/10.15421/012212
Zamoroka, A. M. 2023. New additions to the fauna of the longhorn beetles in Ukraine with a new record of rare, poorly known and invasive species. Baltic Journal of Coleopterology, 23 (2), 159-188.
Zamoroka, A. M., Ruicănescu, A. & Manci, C.-O. 2024. East and West of the Carpathian Arc: Evidence of postglacial ecological and morphological divergence of Phytoecia tigrina metapopulations (Coleoptera, Cerambycidae). Biosystem Diversity, 32 (1), 12-29.
