Sevindik et al. 2024, Biologica Nyssana 15(2) 15 (2) December 2024: 53-60 DOI: 10.5281/zenodo.13902177 Phylogenetic relationships among Lamiaceae species from Aydin (Türkiye), based on rbcL sequences Original Article Emre Sevindik Aydın Adnan Menderes University, Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Cakmar, Aydın, Türkiye ph.d-emre@hotmail.com (corresponding author) Alaattin Efe Can Aydın Adnan Menderes University, Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Cakmar, Aydın, Türkiye Zekiye Özkara Aydın Adnan Menderes University, Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Cakmar, Aydın, Türkiye Yusuf Duşgun Isparta University of Applied Sciences, Faculty of Agriculture, Department of Agricultural Biotechnology, Isparta, Türkiye Şükrü Toksöz Aydın Adnan Menderes University, Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Cakmar, Aydın, Türkiye Hayrettin İlker Özdemir Aydın Adnan Menderes University, Koçarlı Vocational School, Laboratory Technology Program, Koçarlı/Aydın, Türkiye Received: July 20, 2024 Revised: September 19, 2024 Accepted: September 23, 2024 Abstract: In this study, phylogenetic analyses were performed using chloroplast rbcL sequences from Ajuga chamaepitys, Thymbra capitata, Lamium moschatum, Marrubium vulgare, Stachys cretica, Teucrium polium, and Vitex agnus-castus species distributed in Aydın province, Türkiye. After isolating DNA from the samples, PCR amplification was carried out using the rbcLaF and rbcLaR primers. Both forward and reverse sequences were edited using BioEdit 7.2.3, FinchTV 1.4.0, and Sequencher 5.4.6 software. The corrected sequences were converted to protein sequences, and phylogenetic analyses were conducted using MEGA 6.0 software. The rbcL proteins have been motif analysed and their 3D structure revealed. As a result, the phylogenetic tree constructed with sequences obtained from National Center for Biotechnology Information (NCBI) showed that Lamium and Marrubium species formed a clade, while Sideritis and Stachys species formed another one. Additionally, the species of Thymbra, Vitex, Ajuga, and Teucrium each formed distinct groups. In the analyses involving only these seven species, the genetic distance matrix revealed that the closest species were Ajuga chamaepitys and Vitex agnus- castus (0.009), while the most distantly related species were Teucrium polium and Thymbra capitata (0.043). Also, the nucleotide diversity was calculated to be π = 0.025278. Overall, the rbcL sequence results were instrumental in elucidating the phylogenetic relationships within the Lamiaceae family. Key words: Lamiaceae, phylogeny, rbcL, Türkiye Apstrakt: Filogenetski odnosi između vrsta iz porodice Lamiaceae iz Ajdina (Turska) na osnovu rbcL sekvenci U ovoj studiji su, korišćenjem hloroplastnih rbcL sekvenci Ajuga chamaepitys, Thymbra capitata, Lamium moschatum, Marrubium vulgare, Stachys cretica, Teucrium polium i Vitex agnus-castus, vrsta prisutnih u provinciji Ajdin, Turska, sprovedene filogenetske analize. Nakon izolacije DNK iz uzoraka, izvršena je PCR amplifikacija koristeći rbcLaF i rbcLaR prajmere. Obe sekvence (forward i reverse) uređene su korišćenjem softvera BioEdit 7.2.3, FinchTV 1.4.0 i Sequencher 5.4.6. Korigovane sekvence konvertovane su u proteinske sekvence, a filogenetske analize sprovedene korišćenjem softvera MEGA 6.0. rbcL proteini su analizirani na motive, a otkrivena je njihova 3D struktura. Kao rezultat, filogenetsko stablo konstruisano sa sekvencama dobijenim iz Nacionalnog centra za biotehnološke informacije (NCBI) pokazalo je da su Lamium i Marrubium formirale jednu, a Sideritis i Stachys drugu kladu. Dodatno, Thymbra, Vitex, Ajuga i Teucrium formirale su zasebne grupe. U analizama koje su uključivale samo navedenih sedam vrsta, matrica genetičke distance je pokazala da su najbliže vrste Ajuga chamaepitys i Vitex agnus-castus (0,009), dok su najudaljenije vrste bile Teucrium polium i Thymbra capitata (0,043). Takođe, izračunati diverzitet nukleotida iznosi π = 0,025278. Pokazano je da su rbcL sekvence ključne u razjašnjavanju filogenetskih odnosa unutar porodice Lamiaceae. Ključne reči: Lamiaceae, filogenija, rbcL, Turska Introduction Türkiye is located in three phytogeographic regions and is considered a globally important center of biodiversity (Başer & Kırımer, 2018; Yıldırım et al., 2024). The Lamiaceae family in Türkiye is © 2024 Sevindik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 53 represented by 787 taxa across 48 genera, including 608 species and 179 subspecies and varieties (Behçet & Cengiz, 2023). Notable genera within this family include Calamintha Mill., Mentha L., Salvia L., Satureja L., Scutellaria L., Stachys L., Plectranthus L’Hér., Hyptis Jacq., Teucrium L., Vitex L., Thymus L., and Nepeta L. (Mamadalieva et al., 2021; Kilinç et al., 2022). Many members of the Lamiaceae family are rich in essential oils, aromatic compounds, and secondary metabolites, making them significant in fields such as medicine, pharmaceuticals, cosmetics, and food production (Agostin et al. 2009; Hilooğlu et al. 2016; Bekut et al. 2018). Additionally, these species are known for their antimicrobial (Ricci et al., 2005), antioxidant (Özgen et al., 2006), anti- influenza (Protsenko et al., 2022), anticancer (Kilinç et al., 2022), and antimutagenic (Martínez-Rocha et al., 2008) properties. The angiosperm chloroplast (cpDNA) genome ranges from 107 to 218 kb and is organised as a circular DNA molecule with a highly conserved tetragonal structure. Most of the cpDNA sequence encodes protein-coding genes, transfer RNA (tRNA), and ribosomal RNA (rRNA) (Lorenzana & Rico, 2024). Chloroplast DNA sequences are effective and reliable for creating DNA barcodes for plants, helping to resolve confusion in morphological identification (Furan, 2024). In plants, the matK, ndhF, rbcL, rpoB, rpoC1, psbA-trnH, psbK-psbL, trnL-F and atpF-atpH regions of cpDNA sequences have been proposed as barcode gene regions (Manjarres-Hernández & Morillo-Coronado, 2023; Chen et al., 2024; Sevindik et al., 2024). Among these gene regions, rbcL is recognized as an universal barcode gene that is ideal for plant species discrimination studies due to its high amplification efficiency and low mutation rate. This low mutation rate makes the rbcL gene suitable for detailed studies of intraspecific genetic and phylogenetic diversity (Pere et al., 2023). In this study, cpDNA rbcL sequences were used for the phylogenetic analysis of several Lamiaceae species distributed in the Aydin province of Türkiye. Materials and Methods Plant sampling and DNA extraction technique Ajuga chamaepitys (L.) Schreb., Thymbra capitata (L.) Cav, Lamium moschatum Mill., Marrubium vulgare L., Stachys cretica L., Teucrium polium L., and Vitex agnus-castus L. species were collected from the South Campus of Aydın Adnan Menderes University, Faculty of Agriculture (37°45’35.85”N, 27°45’16.93”E) and brought to the laboratory for herbarium preparation. Herbarium numbers of the species are given in Tab. 1. Genomic DNA was isolated from the fresh green leaves using a commercial kit (GeneMark Cat No: DP022). Protocols and methods for polymerase chain reaction (PCR) and sequence analysis The information on the primer sequences, PCR protocol, and PCR components used in this study is provided in Tab. 2. The PCR protocol was carried out according to Sevindik et al. (2024). The polymerase chain reaction products were visualized using 1.0% agarose gel electrophoresis. The PCR results were sent to Triogen Biotechnology (Istanbul, Türkiye) for Sanger sequencing analysis. For the analysis of both forward and reverse sequences, contig sequences were generated using BioEdit 7.2.3 (Hall, 1999), FinchTV 1.4.0 and Sequencher 5.4.6 software. The resulting sequences were then blasted at NCBI to check their similarity levels. Species were uploaded to NCBI and accession numbers were obtained (Tab. 1). Phylogenetic and protein 3D structure analysis Sequences of some species from the Lamiaceae family, obtained from The National Center for Biotechnology Information 54 BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Phylogenetic relationships among Lamiaceae species from Aydin (Türkiye), based on rbcL sequences Table 1. Herbarium number, accession number, A+T, G+C (%) and total length (bp) of cpDNA rbcL sequences in Lamiaceae species Species Herbarium number Accession number A+T G+C Total Ajuga chamaepitys LKR#1600 PQ303556 55.8 44.2 536 Lamium moschatum LKR#1606 PQ303557 55.8 44.2 536 Marrubium vulgare LKR#1603 PQ303558 56.5 43.5 538 Stachys cretica LKR#1604 PQ303559 56 44 539 Teucrium polium LKR#1601 PQ303560 56.3 43.7 535 Thymbra capitata LKR#1612 PQ306792 56 44 536 Vitex agnus castus E. Sevindik 1203 PQ303555 56.2 43.8 535 Avg. 56 44 536.4 BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Phylogenetic relationships among Lamiaceae species from Aydin (Türkiye), based on rbcL sequences 55 (NCBI), were aligned using the MEGA 6.0 program (Tamura et al., 2013). A neighbour- joining (NJ) phylogenetic tree (Saitou & Nei, 1987) was constructed using the same software. The phylogenetic tree was evaluated using the bootstrap test on a resampling of 1000 replicates (Felsenstein, 1985). Moreover, the nucleotide composition, genetic distance matrix, and nucleotide diversity of the rbcL sequences were determined. The rbcL sequences were converted to amino acid sequences using the DNA-to-protein conversion tool in MEGA 6.0 software, and the amino acid content was analyzed using the same program. Furthermore, the protein sequences were modeled in 3D using SWISS-MODEL (https://swissmodel.expasy.org/ interactive). To analyze all domains and conserved protein motifs, Protein BLAST was employed in conjunction with MEME (http://memesuite.org/doc/ fasta-format.html) (Bailey et al., 2009). Results and discussion The rbcL sequences ranged from 535 to 539 bp, with an average A+T content of 56.0% and a G+C ratio of 44.0% (Tab. 1). According to the genetic distance matrix, the closest species were identified as Ajuga chamaepitys and Vitex agnus-castus (0.009), while the most distant species were Teucrium polium and Thymbra capitata (0.043) (Tab. 3). Tajima’s Neutrality Test indicated that the nucleotide diversity was π=0.025278. Additionally, transition/ transversion rates for purines and pyrimidines, as well as the general transition/transversion rates independent of any base group, were calculated for the studied species and presented in Tab. 4. It is shown that although the most important change is in pyrimidine bases, this situation is more balanced for purine bases than for pyrimidine bases. In this study, sequences of several species, including Table 2. Primers, PCR components, and PCR protocols Primer name, 5’-3’ sequences and references PCR components PCR Protocols rbcLa-F: 5’-ATGTCACCACAAACAGAGACTAAAGC-3’ (Levin, 2013) 1 μL genomic DNA 1 μL primer (forward), 1 μL primer (reverse), 5 μL master mix (PCR buffer, 2 Mm MgCl2, dNTP, 0.75 U Taq DNA polymerase) and 17 μL dH2O 95°C, 1 min; 35× (95°C, 30 s; 51°C, 30 s; 68°C, 1 min); 68°C, 5 min.rbcLa-R: 5’-GTAAAATCAAGTCCACCRCG-3’ (Kress et al., 2009) Table 3. Pairwise genetic distance matrix obtained from cpDNA rbcL sequences Species 1 2 3 4 5 6 7 Ajuga chamaepitys - Lamium moschatum 0.027 - Marrubium vulgare 0.021 0.013 - Stachys cretica 0.023 0.021 0.017 - Teucrium polium 0.017 0.039 0.035 0.033 - Thymbra capitata 0.029 0.033 0.035 0.037 0.043 - Vitex agnus castus 0.009 0.023 0.019 0.021 0.027 0.025 - Table 4. Maximum Composite Likelihood estimates of nucleotide substitution patterns A T C G A - 4.1 3.11 11.1 T 4.13 - 19.78 3.35 C 4.13 26.07 - 3.35 G 13.66 4.1 3.11 - Lamium purpureum L., Lamium maculatum (L.) L., Lamium album L., Lamium amplexicaule L., Vitex negundo var. incisa (Lam.) C.B.Clarke, Vitex zeyheri Sond. ex Schauer, Vitex trifolia L., Stachys byzantina K.Koch, Stachys germanica L., Stachys alpina L., Ajuga parviflora Benth., Ajuga bracteosa f. alba Gürke, Ajuga ciliata Bunge, Teucrium chamaedrys L., Teucrium scordium L., Teucrium scorodonia L., Marrubium aschersonii Magnus, 56 BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Phylogenetic relationships among Lamiaceae species from Aydin (Türkiye), based on rbcL sequences Marrubium peregrinum L., Marrubium incanum Desr. and Thymbra spicata L. were obtained from NCBI, and the neighbor-joining (NJ) phylogenetic tree was constructed. The phylogenetic analysis identified the following groups: Marrubium and Lamium species (bootstrap value 55%) Stachys and Sideritis species (bootstrap value 97%), Vitex species (bootstrap value 79%), Ajuga species (bootstrap value 78%), Teucrium species (bootstrap value 54%), and Thymbra spicata and Thymbra capitata species (bootstrap value 100%) (Fig. 1). (9.53%), while methionine was the least abundant (0.56%). Five conserved motifs within the rbcL protein sequences were identified using the MEME program (Fig. 3). All species contain motifs 1, 2, 3, 4 and 5, which are associated with protein structure and function. Some peptide motifs play important roles in protein protein-protein interactions (Filiz & Tombuloğlu, 2014). The 3D structure of rbcL proteins was modeled using the SWISS-MODEL program and is represented in Fig. 4. The three- dimensional structure of proteins facilitates drug Fig. 1. The Neighbour-Joining tree generated using cpDNA rbcL sequences of Lamiaceae species, with sequences retrieved from NCBI The rbcL-based, phylogenetic tree showed clear genus-level clustering. The average amino acid composition of rbcL proteins was analyzed using the MEGA 6.0 program and graphically represented in Fig. 2. Glycine was the most abundant amino acid design. It helps to understand protein function and active sites (Filiz & Koç, 2014). In their SDS- PAGE analysis, Ahmed and Al-Sodany (2019) identified Teucrium polium along with Micromeria imbricata and Salvia deserti, as well as Marrubium BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Phylogenetic relationships among Lamiaceae species from Aydin (Türkiye), based on rbcL sequences 57 vulgare with Otostegia fruticosa. In the RAPD and ISSR dendrogram, Teucrium polium and Marrubium vulgare were clustered together. Similarly, in their RAPD-PCR analysis, Topdemir et al. (2024) grouped Lamium, Stachys, and Sideritis species together, Teucrium and Ajuga species together, while Marrubium species formed a separate group. In our results, Marrubium, Lamium, Sideritis and Stachys were grouped together in a large group. This was supported by 81% of the bootstrap values. In the study by Ayaz et al. (2020), cpDNA rps14 sequence analysis placed Marrubium vulgare with Lamium album and Lamium amplexicaule. In our study, the NCBI obtained Lamium album sequence was also grouped with Marrubium species. Bendiksby et al. (2011) reported that the genus Lamium is not monophyletic. In the phylogenetic tree constructed with the trnL intron, the trnL-F spacer, the rps16 intron and the matK sequences, the Sideritis and Stachys species appeared together, indicating that Sideritis and Stachys are not monophyletic genera. Our study similarly found that Stachys and Sideritis species formed a single group. Also, according to Bendiksby et al. (2011), Marrubium species were Fig. 2. Average amino acid composition of rbcL proteins Fig. 3. Combined block diagrams of conserved protein motifs in rbcL proteins, as determined by MEME family. Acknowledgements. This research was supported by the TUBITAK 2022/2209- A (Project no.: 1919B012223491). References Agostini, F., Santos, C. A., Rossato, M., Márcia, R. 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Conclusion In this study, phylogenetic analysis based on the chloroplast rbcL sequence was performed on seven species (Ajuga chamaepitys, Thymbra capitata, Lamium moschatum, Marrubium vulgare, Stachys cretica, Teucrium polium, and Vitex agnus-castus). The genetic distance matrix revealed that the closest species were Ajuga chamaepitys and Vitex agnus-castus (0.009), while the most distantly related species were Teucrium polium and Thymbra capitata (0.043). Also, the nucleotide diversity was determined to be π=0.025278. In the neighbour- joining phylogenetic tree, Lamium and Marrubium species appeared together, and Sideritis and Stachys species appeared together. Thymbra, Vitex, Ajuga and Teucrium species were grouped at the genus level. This study suggested that cpDNA rbcL analyses could be used in phylogenetic analyses at the genus level. 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