Exploring Phylogenetic Relationships Among Some Bamboo Genera in Indonesia Using cpDNA trnL-trnF Intergenic Spacer Sequences: An In-silico Analysis

Muhammad Azli Ritonga; Syamsuardi; Nurainas; Tesri Maideliza; I Putu Gede P. Damayanto

doi:10.22146/jtbb.16146

Muhammad Azli Ritonga Doctoral Program, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Andalas. Jl. Unand, Kampus Limau Manis, Padang, 25163, West Sumatra, Indonesia https://orcid.org/0000-0003-0197-4475
Syamsuardi Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Andalas. Jl. Unand, Kampus Limau Manis, Padang, 25163, West Sumatra, Indonesia https://orcid.org/0000-0001-8351-6528
Nurainas Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Andalas. Jl. Unand, Kampus Limau Manis, Padang, 25163, West Sumatra, Indonesia https://orcid.org/0000-0003-1682-2976
Tesri Maideliza Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Andalas. Jl. Unand, Kampus Limau Manis, Padang, 25163, West Sumatra, Indonesia https://orcid.org/0009-0006-8331-7418
I Putu Gede P. Damayanto Herbarium Bogoriense, Research Center for Biosystematics and Evolution, National Research and Innovation Agency (BRIN), Indonesia. Jl. Raya Jakarta-Bogor, km 46, Cibinong, Bogor, 16911, West Java, Indonesia https://orcid.org/0000-0001-8740-0696

DOI: https://doi.org/10.22146/jtbb.16146

Keywords: Bambusoideae, DNA barcoding, In-silico, Phylogeny, Taxonomy

Abstract

Identifying and classifying bamboo genera remains challenging in Indonesia. DNA barcoding, using trnL-trnF intergenic spacer (IGS) region, offers a promising solution. This study evaluated the trnL-trnF IGS region’s ability to construct phylogeny relationships among various bamboo genera using in-silico analysis. An in-silico analysis of trnL-trnF IGS sequences from various Indonesian bamboo species (Bambusa, Chloothamnus, Dendrocalamus, Dinochloa, Gigantochloa, Neololeba, Schizostachyum, and Thyrsostachys) was performed using data from the National Center for Biotechnology Information. Phylogenetic analysis was conducted in Mega v.11 with Neighbor-Joining and Maximum Parsimony methods (bootstrap = 1000), using Widjajachloa as an outgroup. Clustering patterns were visualized with a heatmap. Additionally, morphological traits were examined for comparison with the phylogenetic results. Neighbor-Joining analysis failed to effectively differentiate bamboo genera. In contrast, Maximum Parsimony analysis identified three distinct clades: Dinochloa, Schizostachyum, and BDG+CNT (comprising Bambusa, Dendrocalamus, Gigantochloa, Chloothamnus, Neololeba, and Thyrsostachys). Dinochloa and Schizostachyum clades exhibited strong phylogenetic support (bootstrap = 90% and 87%, respectively), while the BDG+CNT clade showed limited resolution (bootstrap < 70%). Morphologically, Dinochloa are climbing bamboos differing from typical bamboos. Schizostachyum possess long, thin culms compared to the short, thick culms of the BDG+CNT clade. Heatmap analysis revealed a distinct clustering pattern for Schizostachyum compared to other genera. The trnL-trnF IGS region cannot fully distinguish all bamboo genera in the phylogenetic tree, although it has the potential to differentiate genera such as Dinochloa and Schizostachyum with relatively high bootstrap values, which aligns with their easily recognizable morphological characteristics.

References

Clark, L., 2006, Bamboo Biodiversity, viewed 13 June 2024, from https://www.eeob.iastate.edu/research/bamboo/index.html.

Clark, L.G., Londoño, X. & Ruiz-Sanchez, E., 2015. Bamboo Taxonomy and Habitat. In Bamboo the Plant and Its Uses. Switzerland. Cham: Springer, pp.1–30. doi: 10.1007/978-3-319-14133-6_1.

Damayanto, I.P.G.P., 2017. Climbing bamboos of Lesser Sunda Islands, Indonesia. Proceedings The SATREPS Conference, 1(1), pp.90–93.

Damayanto, I.P.G.P., 2018. Dinochloa malayana S. Dransf. (Poaceae: Bambusoideae), a new record for Indonesia. Reinwardtia, 17(1), pp.35–37. doi: 10.14203/reinwardtia.v17i1.3351.

Damayanto, I.P.G.P., 2024. Keanekaragaman Jenis Bambu (Poaceae-Bambusoideae) Kepulauan Sunda Kecil. IPB University.

Damayanto, I.P.G.P. & Fefirenta, A.D., 2021. Pola persebaran marga bambu di Indonesia. Prosiding Biologi Achieving the Sustainable Development Goals with Biodiversity in Confronting Climate Change, 7(1), pp.24‒41. doi: 10.24252/psb.v7i1.22233.

Damayanto, I.P.G.P. & Irsyam, A.S.D., 2022. Penelusuran spesimen herbarium dalam jaringan. Baca: Jurnal Dokumentasi dan Informasi, 43(1), pp.15–32. doi: 10.14203/j.baca.v43i1.848.

Damayanto, I.P.G.P., Dalimunthe, S.H. & Megawati, 2021. Dinochloa scandens (Poaceae-Bambusoideae): distribution, habitat preference, and notes on synonymy. Jurnal Biodjati, 6(2), pp.174–189. doi: 10.15575/ biodjati.v6i2.12485.

Drábková, L. et al., 2004. TrnL-trnF intergenic spacer and trnL intron define major clades within Luzula and Juncus (Juncaceae): importance of structural mutations. Journal of Molecular Evolution, 59(1), pp.1–10. doi: 10.1007/s00239-004-2598-7.

Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), pp.1792–1797. doi: 10.1093/nar/gkh340.

Farris, J.S., 1970. Methods for computing Wagner trees. Systematic Biology, 19(1), pp.83–92. doi: 10.1093/sysbio/19.1.83.

Felsenstein, J., 1973. Maximum likelihood and minimum-steps methods for estimating evolutionary trees from data on discrete characters. Systematic Biology, 22(3), pp.240–249. doi: 10.1093/sysbio/22.3.240.

Felsenstein, J., 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), pp.783–791. doi: 10.2307/2408678.

Felsenstein, J., 2004. Inferring Phylogenies, Sinauer Associates.

Fu, D.L., 2024. New names and new combinations of the genera of Bambusa, Dinochloa and Guadua (Bambusaceae). American Journal of Agriculture and Forestry, 12(3), pp.174–184. doi: 10.11648/j.ajaf.20241203.14.

Goh, W.L. et al., 2010. Phylogenetic relationships among Southeast Asian climbing bamboos (Poaceae: Bambusoideae) and the Bambusa complex. Biochemical Systematics and Ecology, 38(4), pp.764–773. doi: 10.1016/j.bse.2010.07.006.

Guha, P.K. et al., 2019. In silico identification of long non-coding RNA based simple sequence repeat markers and their application in diversity analysis in rice. Gene Reports, 16, 100418. doi: 10.1016/j.genrep.2019.100418.

Heatmapper., 2024, ‘Expression’, in Heatmapper viewed 17 July 2024, from http://www.heatmapper.ca/expression.

Ho, V.T. & Nguyen, M.P., 2020. An in silico approach for evaluation of rbcL and matK loci for DNA barcoding of Cucurbitaceae family. Biodiversitas Journal of Biological Diversity, 21(8), pp.3879–3885. doi: 10.13057/biodiv/d210858.

Hodkinson, T.R. et al., 2010. Phylogenetic analyses of plastid and nuclear DNA sequences indicate a rapid late Miocene radiation of the temperate bamboo tribe Arundinarieae (Poaceae, Bambusoideae). Plant Ecology & Diversity, 3(2), pp.109–120. doi: 10.1080/17550874.2010.521524.

Janzen, D.H., 1976. Why bamboos wait so long to flower. Annual Review of Ecology and Systematics, 7, pp.347–391. doi: 10.1146/annurev.es.07.110176.002023.

Katoh, K., Kuma, K. & Miyata, T., 2001. Genetic algorithm-based maximum-likelihood analysis for molecular phylogeny. Journal of Molecular Evolution, 53, pp.477–484. doi: 10.1007/s002390010238

Liu, J.X. et al., 2020. ddRAD analyses reveal a credible phylogenetic relationship of the four main genera of Bambusa-Dendrocalamus-Gigantochloa complex (Poaceae: Bambusoideae). Molecular Phylogenetics and Evolution, 146, 106758. doi: 10.1016/j.ympev.2020.106758.

Ludwig, W., Glöckner, F.O. & Yilmaz, P., 2011. 16-The use of rRNA gene sequence data in the classification and identification of prokaryotes. Methods in Microbiology, 38, pp.349–384. doi: 10.1016/b978-0-12-387730-7.00016-4.

McClure, F.A., 1966. The bamboos: A fresh perpective, Harvard University Press.

Muzakki, F.A., 2020. Keberagaman Bambu Marga Schizostachyum Nees di Jawa. IPB University.

Nei, M. & Kumar, S., 2000. Molecular evolution and phylogenetics, Oxford University Press.

Ngo, T.K.A., Tran, T.A.T. & Ho, V.T., 2024. In silico comparative analysis of the complete chloroplast genome sequences in the mulberry family (Moraceae). Korean Journal of Plant Taxonomy, 54(2), pp.110–120. doi: 10.11110/kjpt.2024.54.2.110.

Olmstead, R.G. & Palmer, J.D., 1994. Chloroplast DNA systematics: A review of methods and data analysis. American Journal of Botany, 81(9), pp.1205–1224. doi: 10.1002/j.1537-2197.1994.tb15615.x.

Pandey, S. & Bhargava, A., 2024. In-silico identification of simple sequence repeat (SSR) markers and phylogenetic analysis from chloroplast genomes of the genus Bambusa. Gene Reports, 37, 102048. doi: 10.1016/j.genrep.2024.102048.

Pratiwi, A. et al., 2023. In silico approach for DNA barcoding using phylogenetic analysis of Coelogyne spp. based on the matK, rpoC1, rbcL and nrDNA markers. Journal of Tropical Biodiversity and Biotechnology, 8(3), jtbb73130. doi: 10.22146/jtbb.73130.

Qiang, Z. et al., 2005. A preliminary analysis of phylogenetic relationships of Arundinaria and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-F intergenic spacer). Journal of Forestry Research, 16(1), pp.5–8. doi: 10.1007/BF02856844.

Ritonga, M.A., 2023. Keanekaragaman Jenis dan Etnobotani Bambu (Poaceae: Bambusoideae) di Pulau Weh, Provinsi Aceh, Indonesia. Andalas University.

Ritonga, M.A. et al., 2023. Bamboo diversity in Weh Island, Aceh, Indonesia. Biodiversitas Journal of Biological Diversity, 24(5), pp.2563–2576. doi: 10.13057/biodiv/d240508.

Ritonga, M.A. et al., 2024. A new record of Dinochloa malayana S.Dransf. (Poaceae, Bambusoideae) from Central Sumatra, Indonesia, Reveals the continuous distribution of Dinochloa in Western Malesia. Check List, 20(5), pp.1157–1163. doi: 10.15560/20.5.1157.

Ritonga, M.A. et al., 2025a. New species and distribution record of Schizostachyum (Poaceae, Bambusoideae) from Indonesia. Phytotaxa, 704(2), pp.165–177. doi: 10.11646/phytotaxa.704.2.4.

Ritonga, M.A. et al., 2025b. Rediscovery of Schizostachyum cornutum Widjaja (Poaceae, Bambusoideae), an endemic bamboo of West Sumatra, Indonesia, after more than three decades. Check List, 21(3), pp.627–634. doi: 10.15560/21.3.627.

Robiah, Y., Rosalina, D. & Damayanto, I.P.G.P., 2022. Bamboo diversity in the Maluku Islands, Indonesia. Jurnal Biodjati, 7(2), pp.292–308. doi: 10.15575/biodjati.v7i2.18713.

Sahu, V.K. et al., 2024. In-silico approaches for discrimination of Curcuma species and their closely related family using the novel technique of DNA Barcoding. Plant Science Today, 11(3), pp.368–376. doi: 10.14719/pst.3317.

Saitou, N. & Nei, M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), pp.406–425. doi: 10.1093/oxfordjournals.molbev.a040454.

Sakka, H. et al., 2013. Chloroplast DNA analysis in Tunisian date-palm cultivars (Phoenix dactylifera L.): Sequence variations and molecular evolution of trnL (UAA) intron and trnL (UAA) trnF (GAA) intergenic spacer. Scientia Horticulturae, 164, pp.256–269. doi: 10.1016/j.scienta.2013.09.038.

Sikdar, S. et al., 2018. An in silico approach for evaluation of rbcL and matK loci for DNA barcoding of Fabaceae family. International Journal of Chemical Studies, 6(6), pp.2446–2451.

Soreng, R.J. et al., 2017. A worldwide phylogenetic classification of the Poaceae (Gramineae) II: An update and a comparison of two 2015 classifications. Journal of Systematics and evolution, 55(4), pp.259–290. doi: 10.1111/jse.12262.

Sungkaew, S. et al., 2009a. Non-monophyly of the woody bamboos (Bambuseae; Poaceae): A multi-gene region phylogenetic analysis of Bambusoideae s.s. Journal of Plant Research, 122, pp.95–108. doi: 10.1007/s10265-008-0192-6.

Sungkaew, S. et al., 2009b. Relationships between Phuphanochloa (Bambuseae, Bambusoideae, Poaceae) and its related genera. 8th World Bamboo Congress Proceedings, 5, pp.55–63.

Taberlet, P. et al., 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology, 17, pp.1105–1109. doi: 10.1007/BF00037152.

Tamura, K., Nei, M. & Kumar, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences, 101(30), pp.11030–11035. doi: 10.1073/pnas.0404206101.

Tamura, K., Stecher, G. & Kumar, S., 2021. MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38(7), pp.3022–3027. doi: 10.1093/molbev/msab120.

Thiers, B.M., 2025, ‘Index Herbariorum’, in The New York Botanical Garden, viewed 13 June 2025, from https://sweetgum.nybg.org/science/ih/.

Widjaja, E.A., 2001. Identikit jenis-jenis bambu di Jawa, Pusat Penelitian dan Pengembangan Biologi-LIPI.

Widjaja, E.A., 2023. Fimbribambusa jokowii Widjaja (Poaceae: Bambusoideae), a new scrambling bamboo from Flores, Indonesia. Advances in Bamboo Science, 4, 100033. doi: 10.1016/j.bamboo.2023.100033.

Widjaja, E.A., Larasati, I. & Sukaesih, Y.Y., 2025. Critical notes on bamboos from Flores Island. Reinwardtia, 24(1), pp.41‒50. doi: 10.55981/reinwardtia.2025.596.

Wong, K.M. & Dransfield, S., 2016. Ruhooglandia and Widjajachloa, two new genera of Malesian bamboos (Poaceae: Bambusoideae) and their distinction from Nastus and Chloothamnus. Sandakania, 22, pp.1–9.

Wong, K.M., 2004. Bamboo the amazing grass, a guide to the diversity and study of bamboos in Southeast Asia, Malaysia: IPGRI & University of Malaya.

Xie, F.M. et al., 2020. Adaptation, evolution, and phylogenetic analyses of species in Chinese Allium section Pallasia and related species based on complete chloroplast genome sequences. BioMed Research International, 2020(1), 8542797. doi: 10.1155/2020/8542797.

Zhou, M.Y. et al., 2017. Towards a complete generic-level plastid phylogeny of the paleotropical woody bamboos (Poaceae: Bambusoideae). Taxon, 66(3), pp.539–553. doi: 10.12705/663.2.