Comparative Analysis of Codon Usage Bias in Chloroplast Genomes of eight Argentina Species of Rosaceae and Their Phylogeny

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Codon usage bias (CUB), an important genomic feature has not been systematically investigated in this genus, limiting our understanding of its chloroplast evolution and adaptive mechanisms. Results In this study, we sequenced and annotated the complete chloroplast genomes of three key Argentina species ( A. stenophylla , A. anserina , and A. phanerophlebia ) and reconstructed a phylogeny using 21 taxa (18 Argentina and three Potentilla outgroups). We further conducted a comparative analysis of synonymous codon usage bias in eight representative species. The chloroplast genomes exhibited a typical quadripartite structure with high conservation in size (155,148–155,972 bp), gene order, and content (129 genes). Phylogenetic analysis strongly supported the monophyly of Argentina and identified four major clades. Codon usage analysis revealed a weak overall bias and a consistent preference for A/T-ending codons. Neutrality, ENC-, and PR2-plots indicated that natural selection plays a dominant role in shaping CUB, overriding mutational pressure. We identified 16 optimal codons, only one of which (GGA, encoding Gly) was shared across all species, suggesting lineage-specific codon adaptation. Conclusions This study provides the first comprehensive insight into codon usage patterns in Argentina chloroplast genomes, enhancing our understanding of phylogenetic relationships and molecular evolution in this genus, with implications for future genetic research and codon optimization strategies. Chloroplast Genome Codon Usage Bias Argentina Phylogeny Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The genus Argentina Hill (Rosaceae: Rosoideae, Potentilleae), which comprises approximately 75 species primarily distributed across the Sino-Himalayan region and Malay Archipelago, represents a taxonomically complex group with significant ethnobotanical value [ 1 – 3 ]. Species such as A. anserina (L.) Rydb. produce starch-rich rhizomes traditionally used for nutritional supplementation and anemia treatment, while A. lineata (Trevis.) Soják and A. leuconota (D.Don) Soják are known for their hemostatic and detoxifying properties, respectively (Flora Reipublicae Popularis Sinicae, http://frps.eflora.cn/ ) [ 1 ]. Despite these functional attributes, molecular studies aimed at resolving the phylogeny of the genus have been hindered by taxonomic ambiguities resulting from morphological convergence with allied genera such as Potentilla and Sibbaldia . Initial generic delineation by Rydberg [ 4 ], based on stylar insertion, was later refined by Soják [ 2 ] using stipule morphology. However, molecular analyses employing chloroplast markers ( matK , trnL-F ) and nuclear loci (ITS) have produced conflicting topologies regarding the monophyly of the genus and the transfer of Sibbaldia micropetala to A. micropetala [ 5 , 6 ]. These inconsistencies highlight the need for phylogenomic approaches utilizing whole chloroplast genomes to clarify evolutionary relationships within Potentilleae [ 7 ]. Chloroplast genomes, characterized by their conserved quadripartite structure—consisting of Large and Small Single-Copy regions (LSC, SSC) flanked by Inverted Repeats (IRs)—and moderate nucleotide substitution rates, provide a robust foundation for phylogenetic reconstruction [ 8 , 9 ]. Beyond structural conservation, synonymous codon usage bias (SCUB) in chloroplast protein-coding genes serves as a key indicator of molecular evolution, reflecting the interplay of mutational pressure, natural selection, and genetic drift [ 9 ]. Key metrics such as Relative Synonymous Codon Usage (RSCU), which quantifies amino acid-specific codon preferences [ 10 ], the Effective Number of Codons (ENC), measuring deviation from random codon usage [ 11 ], and neutrality plots (GC₁₂ vs. GC₃), which discriminate between mutation-selection equilibrium [ 12 ], reveal lineage-specific adaptations in translational efficiency and nucleotide composition. Although SCUB analysis has been applied in some Rosaceae species [ 13 , 14 ], systematic studies within Potentilleae, particularly in Argentina , are lacking. This presents an opportunity to elucidate codon optimization mechanisms driving chloroplast evolution in this understudied clade. To address these gaps, this study integrates chloroplast genomics with comparative codon usage analysis. We sequenced and annotated the complete chloroplast genomes of three taxonomically pivotal Argentina species— A. stenophylla , A. anserina , and A. phanerophlebia —and reconstructed a phylogeny using 21 taxa, including 18 Argentina species and three Potentilla outgroups. Based on this phylogenetic framework, we conducted a comparative SCUB analysis of representative species from the major clades. This work provides new insights into the evolutionary mechanisms shaping organellar genome function in Argentina . Results Characteristics of the Chloroplast Genomes of Three Argentina Species The complete chloroplast genomes of three Argentina species were assembled and annotated. Their sizes were similar, ranging from 155,148 bp ( A. anserina ) to 155,972 bp ( A. stenophylla ) (Additional file 1: Table S1 ). Each genome exhibited the typical quadripartite structure of chloroplast DNA, consisting of a large single-copy region (LSC: 85,068–85,619 bp), a small single-copy region (SSC: 18,461–18,766 bp), and a pair of inverted repeat regions (IRs: 25,702–25,992 bp) (Additional file 1: Table S1 ; Fig. 1 ). The overall GC content ranged from 36.74–37.15%. A total of 129 genes were identified in each plastome, including 84 protein-coding genes (PCGs), 37 tRNA genes, and 8 rRNA genes (Additional file 2: Table S2 ). These genes were categorized into four functional groups: self-replication, photosynthesis, other functions, and genes of unknown function. The protein-coding genes are primarily involved in photosynthesis, photosynthetic phosphorylation, and genetic expression (Fig. 1 ; Additional file 2: Table S2 ). The order and arrangement of genes were highly conserved across the three species, indicating structural stability of the chloroplast genomes in Argentina . Phylogenetic analyses We reconstructed the phylogeny of Argentina using complete chloroplast genome sequences from 18 species, with three Potentilla species as outgroups (Fig. 2 ). The results strongly supported the monophyly of the genus Argentina (PP = 1, BS = 100). Within the genus, four distinct clades were identified. A. anserina formed a basal sister taxon to A. smithiana (PP = 1, BS = 100), and these two species were most closely related to A. micropetala . A. phanerophlebia constituted a separate branch. The remaining species were divided into two major clades: six Argentina species formed one well-supported branch, while A. stenophylla clustered closely with A. microphylla , A. stenophylla var. emergens , A. taliensis , A. festiva , A. lineata , A. polyphylla , and A. parvula . For subsequent codon usage analysis, two representative species from each clade were selected, resulting in a total of eight species: A. anserina , A. smithiana , A. phanerophlebia , A. micropetala , A. cardotiana , A. leuconota , A. stenophylla , and A. parvula . Analysis of Codon Usage Bias Parameters We analyzed 53 protein-coding sequences from the chloroplast genomes of the eight Argentina species (Additional file 3: Table S3 ; Fig. 2 ). The overall GC content (GCall) was similar among species, ranging between 37.80% and 38.00%. The GC content at each codon position was below 47%, with the trend GC1 (45.98–46.15%) > GC2 (37.78–37.92%) > GC3 (29.43–29.92%). Nucleotide composition analysis at the third codon position revealed a marked bias: T3 (46.93–47.25%) > A3 (42.86–43.17%) > G3 (18.04–18.34%) > C3 (16.66–16.97%), indicating a pronounced preference for A and T endings. The Effective Number of Codons (ENC) values ranged from 35 to 60 across all species (Additional file 3: Table S3 ; Fig. 2 ), suggesting weak codon usage bias. Correlation analysis revealed that GCall was significantly correlated with GC1, GC2, and GC3 (P < 0.01; correlation coefficients: 0.46–0.82). GC1 and GC2 were also significantly correlated (P < 0.05), but no significant correlation was observed between GC1 and GC3 (Fig. 3 ), indicating that mutational bias was most pronounced at the third codon position. ENC showed a significant positive correlation with GC3 in all species (P < 0.01) and a negative correlation with GC2 in A. cardotiana . No significant correlation was detected between ENC and GC1 or GCall (Fig. 3 ). The Codon Adaptation Index (CAI) values all exceeded 0.40, further supporting low codon bias and moderate gene expression levels. CAI was significantly correlated with GC1 in all species, but not with GCall in A. micropetala , A. parvula , or A. phanerophlebia (Fig. 3 ). Optimal Codon Analysis A total of 29 codons with RSCU > 1 were identified across the eight Argentina species, indicating high-frequency usage (Additional file 4: Table S4 ). Among these, UUA (Leu) exhibited the highest RSCU value, followed by GCU (Ala). Notably, 55.2% of these preferred codons ended in U, 41.4% in A, and only one (UUG, Leu) ended in G, further supporting a pronounced bias toward A/U-ending codons in the chloroplast genomes of Argentina . This pattern suggests a shared codon usage strategy among species within the genus. Based on established criteria (RSCU > 1 and ΔRSCU ≥ 0.08), 16 codons were identified as optimal (Fig. 4 ). However, the number of optimal codons varied considerably among species: A. anserina and A. cardotiana each contained 12; A. smithiana and A. stenophylla possessed 6 and 7; A. leuconota and A. parvula had 5; A. micropetala ; while A. phanerophlebia contained 4 and 2, respectively. Remarkably, only one codon, GGA (Gly), was common to all eight species, underscoring substantial interspecific divergence in codon use preference. Clustering analysis based on RSCU values revealed relationships distinct from those in the phylogenetic tree. A. stenophylla formed the earliest diverging branch, followed by A. anserina and A. smithiana . A. cardotiana and A. leuconota clustered together as a sister clade to another group comprising A. micropetala and A. parvula (Fig. 4 ). This discordance suggested that codon usage bias in Argentina may be influenced by lineage-specific evolutionary pressures beyond those reflected in phylogenetic structure. Neutrality Plot Analysis Neutrality analysis, which examines the relationship between GC12 and GC3, serves to evaluate the relative contributions of mutation pressure and natural selection to codon usage bias. In the genus Argentina , GC12 values ranged from 0.32 to 0.54, while GC3 values varied between 0.18 and 0.37 (Fig. 5 ). The correlation between GC12 and GC3 was weak and non-significant, with correlation coefficients ranging from 0.0289 to 0.0421, indicating distinct evolutionary constraints acting on these nucleotide positions. Regression analysis further revealed that mutation pressure accounted for only 24.76–29.59% of the variation in codon usage, whereas natural selection explained 70.41–75.24%. These results demonstrate that natural selection played a dominant role in shaping codon preference in the chloroplast genomes of Argentina . ENC-Plot Analysis To further assess the influence of compositional constraints on codon usage, ENC-plot analysis was conducted. The majority of genes across all eight species fell below the expected ENC curve (Fig. 6 ), indicating that codon usage bias is influenced by both mutation pressure (particularly at the third codon position) and natural selection. The consistent distribution pattern among species reinforced the conclusion that natural selection exerted a stronger influence than mutational bias, corroborating the findings from the neutrality plot analysis (Fig. 5 ). PR2-Plot Analysis According to the PR2, under pure mutational pressure, the frequencies of (A and T) and (G and C) at the third codon position are expected to be equal. However, deviations from this equilibrium reflect the influence of natural selection. In the eight Argentina species, gene points were asymmetrically distributed across the four quadrants, with a notable concentration in the lower right region (T > A and G > C) (Fig. 7 ). This biased distribution indicates a preferential use of T and G at the third codon position and further supports the predominant role of natural selection in shaping codon usage patterns. Discussion Chloroplast genomes have been widely employed in plant phylogenetic studies through various approaches, including restriction fragment/site comparisons, structural rearrangements, and sequence variation analyses [ 8 ]. Previous studies indicated that chloroplast genomes of Argentina species are highly conserved in terms of genome structure, sequence, gene order, and content. Consequently, structural homoplasy limits their utility as phylogenetic markers; however, several highly divergent regions have been identified that offer resolution at deeper nodes [ 8 ]. In this study, we newly sequenced and assembled the chloroplast genomes of three Argentina species and conducted a comprehensive phylogenetic analysis incorporating 18 species within the genus. Our results robustly support the monophyly of Argentina and its division into four major clades, consistent with recent phylogenomic studies based on complete chloroplast genomes [ 15 ]. These findings confirm that chloroplast genomic data provide a reliable phylogenetic framework for resolving deep evolutionary relationships within Argentina . Synonymous mutations have traditionally been considered “silent,” as they do not alter the encoded amino acid. However, growing evidence indicates that such mutations can profoundly influence gene expression, protein function, and organismal fitness through mechanisms such as modulation of mRNA stability, translation efficiency, and co-translational folding. For instance, 75.9% of synonymous mutations in yeast significantly reduce fitness [ 16 ]. In this study, we systematically analyzed synonymous codon usage in the chloroplast genomes of eight Argentina species. We identified a weak codon usage bias and a pronounced preference for A/T-ending codons, particularly at the third position—a pattern commonly observed across land plants [ 13 , 17 ]. Furthermore, correlation analyses indicated that gene expression levels (inferred via CAI) may be associated with GC content at the first codon position. Optimal codons are known to enhance translational efficiency through mechanisms such as matching tRNA abundance, accelerating elongation rates, and facilitating proper protein folding [ 18 , 19 ]. A recent study in cucumber demonstrated that a synonymous mutation in the ACS2 gene affects translation efficiency via m6A RNA modification and altered mRNA secondary structure, ultimately influencing fruit length [ 20 ]. However, the functional impacts of codon usage may be modulated by positional effects, tissue specificity, and global cellular constraints [ 21 – 24 ]. Notably, optimal codon identities varied considerably among the Argentina species studied, suggesting divergent evolutionary trajectories or species-specific regulatory adaptations. Our analyses—including neutrality plots, ENC-plots, and PR2 analysis—consistently indicated that natural selection plays a dominant role in shaping codon usage bias in the chloroplast genomes of Argentina , outweighing the influence of mutation pressure. This finding aligns with previous reports in Caragana [ 17 ] and Lilium [ 25 ]. Species of Argentina often inhabit high-altitude environments (above 3,000 m), and their adaptations—such as compact morphology, pubescent leaves, and signatures of positive selection in chloroplast genes [ 15 , 26 ]—may be reflected in codon usage patterns. It is well established that natural selection can favor specific synonymous codons to optimize translation efficiency and accuracy, often in accordance with genomic GC content [ 27 ]. We speculate that selection may favor A/U-ended codons to match the host’s tRNA pool, thereby enhancing translational efficiency. Future studies quantifying tRNA abundance and calculating tRNA adaptation indices could provide deeper insights into the mechanisms of translational selection. Ultimately, understanding species-specific codon preferences may inform strategies for transgene optimization and synthetic biology applications in these taxa. Conclusion This study presents the first comprehensive analysis of codon usage bias in the chloroplast genomes of Argentina species, integrated with a robust phylogenomic framework. Our results confirm the monophyletic status of the genus and reveal four well-supported clades, consistent with recent phylogenetic studies. The chloroplast genomes are highly conserved in structure and gene content, yet exhibit significant synonymous codon usage variation. The overall weak codon bias and predominant A/T-ending preference reflect adaptive strategies likely influenced by natural selection aimed at optimizing translational efficiency. The identification of species-specific optimal codons underscores the diversity in regulatory mechanisms among Argentina species. These findings not only elucidate the evolutionary dynamics of chloroplast genomes in Argentina but also provide a foundation for future functional studies and genetic engineering applications, such as enhancing transgene expression through codon optimization. Further research into tRNA abundance and adaptation indices will be essential to fully unravel the mechanisms of translational selection in this genus. Materials and Methods Plant Materials, DNA Extraction, Sequencing, and Assembly Fresh leaves from three Argentina species ( A. stenophylla , A. anserina , and A. phanerophlebia ) were collected from various provinces in China and rapidly dried using silica gel. Species identification was confirmed by Prof. Shu-Dong Zhang, and voucher specimens were deposited at the Herbarium of Liupanshui Normal University. Total genomic DNA was extracted using a modified CTAB method. DNA libraries were constructed and subjected to paired-end sequencing (PE150) on the Illumina NovaSeq platform. High-quality reads were used for de novo assembly of chloroplast genomes with SPAdes v3.15 [ 28 ]. The resulting contigs were visualized and circularized using Bandage [ 29 ]. Complete chloroplast genomes were annotated with the Plastid Genome Annotator (PGA) [ 30 ], using reference genomes of related Argentina species. Genome maps were generated using OGDRAW [ 31 ]. Phylogenetic analysis of Argentina A total of 21 cp genomes, comprising 18 Argentina species and three Potentilla species as outgroups (see Table S1 ), were included in the phylogenetic analysis. Whole chloroplast genome sequences were aligned using MAFFT v6.833 [ 32 ]. Phylogenetic trees were reconstructed using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods, following previously established protocols [ 33 ]. Codon Usage Analysis Protein-coding sequences (CDS) longer than 300 bp were extracted from the chloroplast genomes of eight Argentina species. Only sequences with standard start (ATG) and stop (TAA, TAG, TGA) codons were retained. Nucleotide composition indices, including GC content at the first, second, and third codon positions (GC1, GC2, GC3) and overall GC content (GCall), were computed using EMBOSS-cusp ( https://www.bioinformatics.nl/cgi-bin/emboss/cusp ). Relative Synonymous Codon Usage (RSCU) and the Effective Number of Codons (ENC) were calculated using CodonW 1.4.2 [ 11 , 34 ]. Codons with RSCU > 1 were considered preferentially used, and an ENC value ≤ 35 was indicative of significant codon usage bias [ 35 – 37 ]. Sources of Codon Usage Bias Neutrality plots (GC12 vs. GC3) were constructed to evaluate the influence of mutation pressure versus natural selection [ 38 ]. A regression slope near 0 suggests dominant natural selection, whereas a slope near 1 indicates mutation-dominated bias [ 39 ]. ENC plots were generated by comparing observed ENC values against expected values under neutral evolution (ENCexp = 2 + GC3 + 29/[GC3² + (1 − GC3)²]) [ 40 ]. Points distributed near the expected curve imply mutation-driven bias, whereas deviations below the curve suggest selective constraints [ 41 ]. Parity Rule 2 (PR2) analysis [ 42 ] was applied to examine strand-specific bias using the parameters A/(A + T) and G/(G + C) at the third codon position. Optimal Codon Identification Genes were ranked based on their ENC values, with the top and bottom 10% defined as high- and low-expression sets, respectively. The difference in RSCU ΔRSCU = RSCU high − RSCU low ) was computed for each codon. Optimal codons were identified as those with RSCU > 1 and ΔRSCU ≥ 0.08 [ 43 ]. RSCU Clustering RSCU values from the eight Argentina species were subjected to hierarchical clustering analysis using Euclidean distance. Abbreviations CDS coding DNA sequence cp chloroplast CUB codon usage bias ENC the effective number of codons IR inverted repeat LSC large single-copy PCGs protein-coding genes RSCU relative synonymous codon usage SSC small single-copy Declarations Clinical trial number Not applicable Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets analyzed during the current study are available from the NCBI database under accession number PX057651 ( A. stenophylla ), OR863700 ( A. phanerophlebia ) and PX057649 ( A. anserina ). Competing interests The authors declare that they have no competing interests. Funding This research was supported by the Open Subject from Key Laboratory of Qinghai-Tibetan Plateau Biotechnology of Ministry of Education (2023-SYS-04), Science and Technology Project of Liupanshui, grant number 52020-2019-05-05, 52020[2022]PT-20, and 52020-2023-0-2-18. Author Contributions Conceptualization, S-D.Z.; methodology, L-Z.L., G-N. Z. and J.T.; formal analysis, G-N. Z., L-Z.L. and S-D.Z.; writing—original draft preparation, G-N. Z, L-Z.L. and S-D.Z.; writing—review and editing, S-D.Z. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable References Feng T, Moore MJ, Sun Y, Meng A, Chu H, Li J, et al. A new species of Argentina (Rosaceae, Potentilleae) from Southeast Tibet, with reference to the taxonomic status of the genus. Plant Syst Evol. 2015;301(3):911–21. J. S: Argentina Hill, a genus distinct from Potentilla (Rosaceae). Thaiszia J Bot. 2010; 20:91–7. Kechaykin AA, Shmakov AI. 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Supplementary Files TableS1.docx Additional files Additional file 1: Table S1 The information of eight Argentina species cp genomes TableS2.docx Additional file 2: Table S2 The gene information of three Argentina species cp genomes TableS3.docx Additional file 3: Table S3 Codon usage bias related parameters of eight Argentina species TableS4.xlsx Additional file 4: Table S4 RSCU values of each codon among eight species TableS5.xlsx Additional file 5: Table S5 The information of species used for phylogenetic analysis in this study Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7501003","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":514630714,"identity":"eaaeed6f-b4d4-495c-85b9-3b84ef8cabac","order_by":0,"name":"Guang-Nan Zhang","email":"","orcid":"","institution":"Academy of Agriculture and Forestry Sciences of Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Guang-Nan","middleName":"","lastName":"Zhang","suffix":""},{"id":514630717,"identity":"9c54f7b8-ff8e-405a-bf84-2a7be01e6cd8","order_by":1,"name":"Li-Zhen Ling","email":"","orcid":"","institution":"Liupanshui Normal University","correspondingAuthor":false,"prefix":"","firstName":"Li-Zhen","middleName":"","lastName":"Ling","suffix":""},{"id":514630718,"identity":"62376b71-9b0b-4ee7-ac5c-104bb505ff66","order_by":2,"name":"Jie Tian","email":"","orcid":"","institution":"Academy of Agriculture and Forestry Sciences of Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Tian","suffix":""},{"id":514630720,"identity":"52c01e81-d4b1-471f-96ef-5a099c3e5764","order_by":3,"name":"Shu-Dong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYFCCBDYQKcfeAKIMLIjXYsxzAKxFgngtiT1gLQxEaJFvT3724OeO2vQe9h7TDT8KJBj427sT8GoxOPPM3LD3zPHcHp5jaTd7gA6TOHN2A34tEjlsErxtx3L3SyQfu8ED1GIgkYtfi/yMHDbJv23H0nkkEttu/iFGC8ONHDZp3raaBB6gLbeJsgXoFzNp2bYDhiC/3JYxkOAh6BdQiEm+bauT52HvMbv55o+NHH97LwGHQcBhOIuHGOUgUEeswlEwCkbBKBiJAACSCUUaAPKlAwAAAABJRU5ErkJggg==","orcid":"","institution":"Liupanshui Normal University","correspondingAuthor":true,"prefix":"","firstName":"Shu-Dong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-08-31 13:53:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7501003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7501003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91708466,"identity":"550bee9b-77df-43b5-8935-6b9c67fb86cb","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":260046,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome map of threet \u003cem\u003eArgentina \u003c/em\u003especies. The grey inside circle indicates the GC level of every genomic position.\u003c/p\u003e\n\u003cp\u003eGenes inside in the outer circle of genomic map are transcribed clockwise and vice versa. The different functional gene categories are shown\u003c/p\u003e\n\u003cp\u003ein the different colors\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/18dddf800f04f0b63e74673f.jpg"},{"id":91708464,"identity":"93f7df60-50af-43d4-83c1-7cef9153fb39","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147742,"visible":true,"origin":"","legend":"\u003cp\u003eCodon usage bias related parameters of eight \u003cem\u003eArgentina\u003c/em\u003especies\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/4097d0ee21997f1334a7acbb.jpg"},{"id":91709281,"identity":"e3a782a7-d537-4635-86b9-bc421bf1a84d","added_by":"auto","created_at":"2025-09-19 12:13:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":261429,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between parameters of each gene of eight \u003cem\u003eArgentina\u003c/em\u003e species\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/76b832e93adaecb7eaa75a7f.jpg"},{"id":91708467,"identity":"b562bb30-2a6d-49e8-a7b1-e2fa02c70fe2","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199060,"visible":true,"origin":"","legend":"\u003cp\u003eClustering analysis of RSCU values in eight \u003cem\u003eArgentina \u003c/em\u003especies\u003c/p\u003e\n\u003cp\u003eNote: The codons with the star indicate the optimal codons.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/bb790b0a0837a1622987f139.jpg"},{"id":91709284,"identity":"b2b3f24d-97f9-4944-b1eb-0713bbaa4c8b","added_by":"auto","created_at":"2025-09-19 12:13:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152309,"visible":true,"origin":"","legend":"\u003cp\u003eNeutrality plot of eight \u003cem\u003eArgentina\u003c/em\u003especies\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/a197aaf3b8d3acbface3581d.jpg"},{"id":91708475,"identity":"f006a404-dd7e-4d03-99b1-2b2d2afbfb5b","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":117842,"visible":true,"origin":"","legend":"\u003cp\u003eENC-plot of eight \u003cem\u003eArgentina\u003c/em\u003e species\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/e3880d99643152638d5d4373.jpg"},{"id":91708474,"identity":"1918799c-5420-40e6-aa31-c27f76d94201","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":108649,"visible":true,"origin":"","legend":"\u003cp\u003ePR2-plot of eight \u003cem\u003eArgentina\u003c/em\u003e species\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/d8b5a7c92f65cc1191c21fd0.jpg"},{"id":96363119,"identity":"f1865b29-ff27-4659-89ed-8f294a456f9d","added_by":"auto","created_at":"2025-11-20 10:04:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1986089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/4523d4f7-608f-4830-9325-ec9b401ef0d1.pdf"},{"id":91709282,"identity":"cf3f5ac8-1623-4d39-9fe5-d2d7c83f57a9","added_by":"auto","created_at":"2025-09-19 12:13:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional files\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional file 1: Table S1 The information of eight \u003cem\u003eArgentina\u003c/em\u003e species cp genomes\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/aa826a9adb18e7de7e55dbca.docx"},{"id":91709705,"identity":"ea051655-ba96-4b7c-bf5d-563f4780ed0d","added_by":"auto","created_at":"2025-09-19 12:21:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15020,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2: Table S2 The gene information of three\u003cem\u003e Argentina\u003c/em\u003e species cp genomes\u003c/p\u003e","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/a5d09af917d9058972a12a74.docx"},{"id":91708471,"identity":"f7777a92-edda-406f-a8e9-12361a38878a","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14640,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 3: Table S3 Codon usage bias related parameters of eight \u003cem\u003eArgentina \u003c/em\u003especies\u003c/p\u003e","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/d28a6123068219cfe93a1e7a.docx"},{"id":91709706,"identity":"f45ccae7-29b0-45b0-8bfa-b588462b9af6","added_by":"auto","created_at":"2025-09-19 12:21:57","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13423,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 4: Table S4 RSCU values of each codon among eight species\u003c/p\u003e","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/127aac4c483f89d16432d4b8.xlsx"},{"id":91708472,"identity":"cf148d55-0458-4634-8d1e-71c20314c633","added_by":"auto","created_at":"2025-09-19 12:05:57","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10556,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 5: Table S5 The information of species used for phylogenetic analysis in this study\u003c/p\u003e","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7501003/v1/98c291200039f8c44bd51818.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Analysis of Codon Usage Bias in Chloroplast Genomes of eight Argentina Species of Rosaceae and Their Phylogeny","fulltext":[{"header":"Background","content":"\u003cp\u003eThe genus \u003cem\u003eArgentina\u003c/em\u003e Hill (Rosaceae: Rosoideae, Potentilleae), which comprises approximately 75 species primarily distributed across the Sino-Himalayan region and Malay Archipelago, represents a taxonomically complex group with significant ethnobotanical value [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Species such as \u003cem\u003eA. anserina\u003c/em\u003e (L.) Rydb. produce starch-rich rhizomes traditionally used for nutritional supplementation and anemia treatment, while \u003cem\u003eA. lineata\u003c/em\u003e (Trevis.) Soj\u0026aacute;k and \u003cem\u003eA. leuconota\u003c/em\u003e (D.Don) Soj\u0026aacute;k are known for their hemostatic and detoxifying properties, respectively (Flora Reipublicae Popularis Sinicae, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://frps.eflora.cn/\u003c/span\u003e\u003cspan address=\"http://frps.eflora.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite these functional attributes, molecular studies aimed at resolving the phylogeny of the genus have been hindered by taxonomic ambiguities resulting from morphological convergence with allied genera such as \u003cem\u003ePotentilla\u003c/em\u003e and \u003cem\u003eSibbaldia\u003c/em\u003e. Initial generic delineation by Rydberg [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], based on stylar insertion, was later refined by Soj\u0026aacute;k [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] using stipule morphology. However, molecular analyses employing chloroplast markers (\u003cem\u003ematK\u003c/em\u003e, \u003cem\u003etrnL-F\u003c/em\u003e) and nuclear loci (ITS) have produced conflicting topologies regarding the monophyly of the genus and the transfer of \u003cem\u003eSibbaldia micropetala\u003c/em\u003e to \u003cem\u003eA. micropetala\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These inconsistencies highlight the need for phylogenomic approaches utilizing whole chloroplast genomes to clarify evolutionary relationships within Potentilleae [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChloroplast genomes, characterized by their conserved quadripartite structure\u0026mdash;consisting of Large and Small Single-Copy regions (LSC, SSC) flanked by Inverted Repeats (IRs)\u0026mdash;and moderate nucleotide substitution rates, provide a robust foundation for phylogenetic reconstruction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Beyond structural conservation, synonymous codon usage bias (SCUB) in chloroplast protein-coding genes serves as a key indicator of molecular evolution, reflecting the interplay of mutational pressure, natural selection, and genetic drift [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Key metrics such as Relative Synonymous Codon Usage (RSCU), which quantifies amino acid-specific codon preferences [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], the Effective Number of Codons (ENC), measuring deviation from random codon usage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and neutrality plots (GC₁₂ vs. GC₃), which discriminate between mutation-selection equilibrium [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], reveal lineage-specific adaptations in translational efficiency and nucleotide composition. Although SCUB analysis has been applied in some Rosaceae species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], systematic studies within Potentilleae, particularly in \u003cem\u003eArgentina\u003c/em\u003e, are lacking. This presents an opportunity to elucidate codon optimization mechanisms driving chloroplast evolution in this understudied clade.\u003c/p\u003e\u003cp\u003eTo address these gaps, this study integrates chloroplast genomics with comparative codon usage analysis. We sequenced and annotated the complete chloroplast genomes of three taxonomically pivotal \u003cem\u003eArgentina\u003c/em\u003e species\u0026mdash;\u003cem\u003eA. stenophylla\u003c/em\u003e, \u003cem\u003eA. anserina\u003c/em\u003e, and \u003cem\u003eA. phanerophlebia\u003c/em\u003e\u0026mdash;and reconstructed a phylogeny using 21 taxa, including 18 \u003cem\u003eArgentina\u003c/em\u003e species and three \u003cem\u003ePotentilla\u003c/em\u003e outgroups. Based on this phylogenetic framework, we conducted a comparative SCUB analysis of representative species from the major clades. This work provides new insights into the evolutionary mechanisms shaping organellar genome function in \u003cem\u003eArgentina\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCharacteristics of the Chloroplast Genomes of Three\u003c/b\u003e \u003cb\u003eArgentina\u003c/b\u003e \u003cb\u003eSpecies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe complete chloroplast genomes of three \u003cem\u003eArgentina\u003c/em\u003e species were assembled and annotated. Their sizes were similar, ranging from 155,148 bp (\u003cem\u003eA. anserina\u003c/em\u003e) to 155,972 bp (\u003cem\u003eA. stenophylla\u003c/em\u003e) (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each genome exhibited the typical quadripartite structure of chloroplast DNA, consisting of a large single-copy region (LSC: 85,068\u0026ndash;85,619 bp), a small single-copy region (SSC: 18,461\u0026ndash;18,766 bp), and a pair of inverted repeat regions (IRs: 25,702\u0026ndash;25,992 bp) (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The overall GC content ranged from 36.74\u0026ndash;37.15%. A total of 129 genes were identified in each plastome, including 84 protein-coding genes (PCGs), 37 tRNA genes, and 8 rRNA genes (Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These genes were categorized into four functional groups: self-replication, photosynthesis, other functions, and genes of unknown function. The protein-coding genes are primarily involved in photosynthesis, photosynthetic phosphorylation, and genetic expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The order and arrangement of genes were highly conserved across the three species, indicating structural stability of the chloroplast genomes in \u003cem\u003eArgentina\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analyses\u003c/h2\u003e\u003cp\u003eWe reconstructed the phylogeny of \u003cem\u003eArgentina\u003c/em\u003e using complete chloroplast genome sequences from 18 species, with three \u003cem\u003ePotentilla\u003c/em\u003e species as outgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results strongly supported the monophyly of the genus \u003cem\u003eArgentina\u003c/em\u003e (PP\u0026thinsp;=\u0026thinsp;1, BS\u0026thinsp;=\u0026thinsp;100). Within the genus, four distinct clades were identified. \u003cem\u003eA. anserina\u003c/em\u003e formed a basal sister taxon to \u003cem\u003eA. smithiana\u003c/em\u003e (PP\u0026thinsp;=\u0026thinsp;1, BS\u0026thinsp;=\u0026thinsp;100), and these two species were most closely related to \u003cem\u003eA. micropetala\u003c/em\u003e. \u003cem\u003eA. phanerophlebia\u003c/em\u003e constituted a separate branch. The remaining species were divided into two major clades: six \u003cem\u003eArgentina\u003c/em\u003e species formed one well-supported branch, while \u003cem\u003eA. stenophylla\u003c/em\u003e clustered closely with \u003cem\u003eA. microphylla\u003c/em\u003e, \u003cem\u003eA. stenophylla\u003c/em\u003e var. \u003cem\u003eemergens\u003c/em\u003e, \u003cem\u003eA. taliensis\u003c/em\u003e, \u003cem\u003eA. festiva\u003c/em\u003e, \u003cem\u003eA. lineata\u003c/em\u003e, \u003cem\u003eA. polyphylla\u003c/em\u003e, and \u003cem\u003eA. parvula\u003c/em\u003e. For subsequent codon usage analysis, two representative species from each clade were selected, resulting in a total of eight species: \u003cem\u003eA. anserina\u003c/em\u003e, \u003cem\u003eA. smithiana\u003c/em\u003e, \u003cem\u003eA. phanerophlebia\u003c/em\u003e, \u003cem\u003eA. micropetala\u003c/em\u003e, \u003cem\u003eA. cardotiana\u003c/em\u003e, \u003cem\u003eA. leuconota\u003c/em\u003e, \u003cem\u003eA. stenophylla\u003c/em\u003e, and \u003cem\u003eA. parvula\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnalysis of Codon Usage Bias Parameters\u003c/h3\u003e\n\u003cp\u003eWe analyzed 53 protein-coding sequences from the chloroplast genomes of the eight \u003cem\u003eArgentina\u003c/em\u003e species (Additional file 3: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The overall GC content (GCall) was similar among species, ranging between 37.80% and 38.00%. The GC content at each codon position was below 47%, with the trend GC1 (45.98\u0026ndash;46.15%)\u0026thinsp;\u0026gt;\u0026thinsp;GC2 (37.78\u0026ndash;37.92%)\u0026thinsp;\u0026gt;\u0026thinsp;GC3 (29.43\u0026ndash;29.92%). Nucleotide composition analysis at the third codon position revealed a marked bias: T3 (46.93\u0026ndash;47.25%)\u0026thinsp;\u0026gt;\u0026thinsp;A3 (42.86\u0026ndash;43.17%)\u0026thinsp;\u0026gt;\u0026thinsp;G3 (18.04\u0026ndash;18.34%)\u0026thinsp;\u0026gt;\u0026thinsp;C3 (16.66\u0026ndash;16.97%), indicating a pronounced preference for A and T endings. The Effective Number of Codons (ENC) values ranged from 35 to 60 across all species (Additional file 3: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting weak codon usage bias.\u003c/p\u003e\u003cp\u003eCorrelation analysis revealed that GCall was significantly correlated with GC1, GC2, and GC3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; correlation coefficients: 0.46\u0026ndash;0.82). GC1 and GC2 were also significantly correlated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but no significant correlation was observed between GC1 and GC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that mutational bias was most pronounced at the third codon position. ENC showed a significant positive correlation with GC3 in all species (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a negative correlation with GC2 in \u003cem\u003eA. cardotiana\u003c/em\u003e. No significant correlation was detected between ENC and GC1 or GCall (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The Codon Adaptation Index (CAI) values all exceeded 0.40, further supporting low codon bias and moderate gene expression levels. CAI was significantly correlated with GC1 in all species, but not with GCall in \u003cem\u003eA. micropetala\u003c/em\u003e, \u003cem\u003eA. parvula\u003c/em\u003e, or \u003cem\u003eA. phanerophlebia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eOptimal Codon Analysis\u003c/h3\u003e\n\u003cp\u003eA total of 29 codons with RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 were identified across the eight \u003cem\u003eArgentina\u003c/em\u003e species, indicating high-frequency usage (Additional file 4: Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Among these, UUA (Leu) exhibited the highest RSCU value, followed by GCU (Ala). Notably, 55.2% of these preferred codons ended in U, 41.4% in A, and only one (UUG, Leu) ended in G, further supporting a pronounced bias toward A/U-ending codons in the chloroplast genomes of \u003cem\u003eArgentina\u003c/em\u003e. This pattern suggests a shared codon usage strategy among species within the genus.\u003c/p\u003e\u003cp\u003eBased on established criteria (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 and ΔRSCU\u0026thinsp;\u0026ge;\u0026thinsp;0.08), 16 codons were identified as optimal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, the number of optimal codons varied considerably among species: \u003cem\u003eA. anserina\u003c/em\u003e and \u003cem\u003eA. cardotiana\u003c/em\u003e each contained 12; \u003cem\u003eA. smithiana\u003c/em\u003e and \u003cem\u003eA. stenophylla\u003c/em\u003e possessed 6 and 7; \u003cem\u003eA. leuconota\u003c/em\u003e and \u003cem\u003eA. parvula\u003c/em\u003e had 5; \u003cem\u003eA. micropetala\u003c/em\u003e; while \u003cem\u003eA. phanerophlebia\u003c/em\u003e contained 4 and 2, respectively. Remarkably, only one codon, GGA (Gly), was common to all eight species, underscoring substantial interspecific divergence in codon use preference.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eClustering analysis based on RSCU values revealed relationships distinct from those in the phylogenetic tree. \u003cem\u003eA. stenophylla\u003c/em\u003e formed the earliest diverging branch, followed by \u003cem\u003eA. anserina\u003c/em\u003e and \u003cem\u003eA. smithiana\u003c/em\u003e. \u003cem\u003eA. cardotiana\u003c/em\u003e and \u003cem\u003eA. leuconota\u003c/em\u003e clustered together as a sister clade to another group comprising \u003cem\u003eA. micropetala\u003c/em\u003e and \u003cem\u003eA. parvula\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This discordance suggested that codon usage bias in \u003cem\u003eArgentina\u003c/em\u003e may be influenced by lineage-specific evolutionary pressures beyond those reflected in phylogenetic structure.\u003c/p\u003e\n\u003ch3\u003eNeutrality Plot Analysis\u003c/h3\u003e\n\u003cp\u003eNeutrality analysis, which examines the relationship between GC12 and GC3, serves to evaluate the relative contributions of mutation pressure and natural selection to codon usage bias. In the genus \u003cem\u003eArgentina\u003c/em\u003e, GC12 values ranged from 0.32 to 0.54, while GC3 values varied between 0.18 and 0.37 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The correlation between GC12 and GC3 was weak and non-significant, with correlation coefficients ranging from 0.0289 to 0.0421, indicating distinct evolutionary constraints acting on these nucleotide positions. Regression analysis further revealed that mutation pressure accounted for only 24.76\u0026ndash;29.59% of the variation in codon usage, whereas natural selection explained 70.41\u0026ndash;75.24%. These results demonstrate that natural selection played a dominant role in shaping codon preference in the chloroplast genomes of \u003cem\u003eArgentina\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eENC-Plot Analysis\u003c/h3\u003e\n\u003cp\u003eTo further assess the influence of compositional constraints on codon usage, ENC-plot analysis was conducted. The majority of genes across all eight species fell below the expected ENC curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that codon usage bias is influenced by both mutation pressure (particularly at the third codon position) and natural selection. The consistent distribution pattern among species reinforced the conclusion that natural selection exerted a stronger influence than mutational bias, corroborating the findings from the neutrality plot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePR2-Plot Analysis\u003c/h2\u003e\u003cp\u003eAccording to the PR2, under pure mutational pressure, the frequencies of (A and T) and (G and C) at the third codon position are expected to be equal. However, deviations from this equilibrium reflect the influence of natural selection. In the eight \u003cem\u003eArgentina\u003c/em\u003e species, gene points were asymmetrically distributed across the four quadrants, with a notable concentration in the lower right region (T\u0026thinsp;\u0026gt;\u0026thinsp;A and G\u0026thinsp;\u0026gt;\u0026thinsp;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This biased distribution indicates a preferential use of T and G at the third codon position and further supports the predominant role of natural selection in shaping codon usage patterns.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChloroplast genomes have been widely employed in plant phylogenetic studies through various approaches, including restriction fragment/site comparisons, structural rearrangements, and sequence variation analyses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies indicated that chloroplast genomes of \u003cem\u003eArgentina\u003c/em\u003e species are highly conserved in terms of genome structure, sequence, gene order, and content. Consequently, structural homoplasy limits their utility as phylogenetic markers; however, several highly divergent regions have been identified that offer resolution at deeper nodes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this study, we newly sequenced and assembled the chloroplast genomes of three \u003cem\u003eArgentina\u003c/em\u003e species and conducted a comprehensive phylogenetic analysis incorporating 18 species within the genus. Our results robustly support the monophyly of \u003cem\u003eArgentina\u003c/em\u003e and its division into four major clades, consistent with recent phylogenomic studies based on complete chloroplast genomes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings confirm that chloroplast genomic data provide a reliable phylogenetic framework for resolving deep evolutionary relationships within \u003cem\u003eArgentina\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eSynonymous mutations have traditionally been considered \u0026ldquo;silent,\u0026rdquo; as they do not alter the encoded amino acid. However, growing evidence indicates that such mutations can profoundly influence gene expression, protein function, and organismal fitness through mechanisms such as modulation of mRNA stability, translation efficiency, and co-translational folding. For instance, 75.9% of synonymous mutations in yeast significantly reduce fitness [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, we systematically analyzed synonymous codon usage in the chloroplast genomes of eight \u003cem\u003eArgentina\u003c/em\u003e species. We identified a weak codon usage bias and a pronounced preference for A/T-ending codons, particularly at the third position\u0026mdash;a pattern commonly observed across land plants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, correlation analyses indicated that gene expression levels (inferred via CAI) may be associated with GC content at the first codon position. Optimal codons are known to enhance translational efficiency through mechanisms such as matching tRNA abundance, accelerating elongation rates, and facilitating proper protein folding [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A recent study in cucumber demonstrated that a synonymous mutation in the ACS2 gene affects translation efficiency via m6A RNA modification and altered mRNA secondary structure, ultimately influencing fruit length [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the functional impacts of codon usage may be modulated by positional effects, tissue specificity, and global cellular constraints [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Notably, optimal codon identities varied considerably among the \u003cem\u003eArgentina\u003c/em\u003e species studied, suggesting divergent evolutionary trajectories or species-specific regulatory adaptations.\u003c/p\u003e\u003cp\u003eOur analyses\u0026mdash;including neutrality plots, ENC-plots, and PR2 analysis\u0026mdash;consistently indicated that natural selection plays a dominant role in shaping codon usage bias in the chloroplast genomes of \u003cem\u003eArgentina\u003c/em\u003e, outweighing the influence of mutation pressure. This finding aligns with previous reports in \u003cem\u003eCaragana\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and \u003cem\u003eLilium\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Species of \u003cem\u003eArgentina\u003c/em\u003e often inhabit high-altitude environments (above 3,000 m), and their adaptations\u0026mdash;such as compact morphology, pubescent leaves, and signatures of positive selection in chloroplast genes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u0026mdash;may be reflected in codon usage patterns. It is well established that natural selection can favor specific synonymous codons to optimize translation efficiency and accuracy, often in accordance with genomic GC content [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We speculate that selection may favor A/U-ended codons to match the host\u0026rsquo;s tRNA pool, thereby enhancing translational efficiency. Future studies quantifying tRNA abundance and calculating tRNA adaptation indices could provide deeper insights into the mechanisms of translational selection. Ultimately, understanding species-specific codon preferences may inform strategies for transgene optimization and synthetic biology applications in these taxa.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents the first comprehensive analysis of codon usage bias in the chloroplast genomes of \u003cem\u003eArgentina\u003c/em\u003e species, integrated with a robust phylogenomic framework. Our results confirm the monophyletic status of the genus and reveal four well-supported clades, consistent with recent phylogenetic studies. The chloroplast genomes are highly conserved in structure and gene content, yet exhibit significant synonymous codon usage variation. The overall weak codon bias and predominant A/T-ending preference reflect adaptive strategies likely influenced by natural selection aimed at optimizing translational efficiency. The identification of species-specific optimal codons underscores the diversity in regulatory mechanisms among \u003cem\u003eArgentina\u003c/em\u003e species. These findings not only elucidate the evolutionary dynamics of chloroplast genomes in \u003cem\u003eArgentina\u003c/em\u003e but also provide a foundation for future functional studies and genetic engineering applications, such as enhancing transgene expression through codon optimization. Further research into tRNA abundance and adaptation indices will be essential to fully unravel the mechanisms of translational selection in this genus.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePlant Materials, DNA Extraction, Sequencing, and Assembly\u003c/h2\u003e\u003cp\u003eFresh leaves from three \u003cem\u003eArgentina\u003c/em\u003e species (\u003cem\u003eA. stenophylla\u003c/em\u003e, \u003cem\u003eA. anserina\u003c/em\u003e, and \u003cem\u003eA. phanerophlebia\u003c/em\u003e) were collected from various provinces in China and rapidly dried using silica gel. Species identification was confirmed by Prof. Shu-Dong Zhang, and voucher specimens were deposited at the Herbarium of Liupanshui Normal University. Total genomic DNA was extracted using a modified CTAB method. DNA libraries were constructed and subjected to paired-end sequencing (PE150) on the Illumina NovaSeq platform.\u003c/p\u003e\u003cp\u003eHigh-quality reads were used for de novo assembly of chloroplast genomes with SPAdes v3.15 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The resulting contigs were visualized and circularized using Bandage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Complete chloroplast genomes were annotated with the Plastid Genome Annotator (PGA) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], using reference genomes of related Argentina species. Genome maps were generated using OGDRAW [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis of\u003c/b\u003e \u003cb\u003eArgentina\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 21 cp genomes, comprising 18 \u003cem\u003eArgentina\u003c/em\u003e species and three \u003cem\u003ePotentilla\u003c/em\u003e species as outgroups (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), were included in the phylogenetic analysis. Whole chloroplast genome sequences were aligned using MAFFT v6.833 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Phylogenetic trees were reconstructed using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods, following previously established protocols [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCodon Usage Analysis\u003c/h2\u003e\u003cp\u003eProtein-coding sequences (CDS) longer than 300 bp were extracted from the chloroplast genomes of eight \u003cem\u003eArgentina\u003c/em\u003e species. Only sequences with standard start (ATG) and stop (TAA, TAG, TGA) codons were retained. Nucleotide composition indices, including GC content at the first, second, and third codon positions (GC1, GC2, GC3) and overall GC content (GCall), were computed using EMBOSS-cusp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.nl/cgi-bin/emboss/cusp\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.nl/cgi-bin/emboss/cusp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Relative Synonymous Codon Usage (RSCU) and the Effective Number of Codons (ENC) were calculated using CodonW 1.4.2 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Codons with RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered preferentially used, and an ENC value\u0026thinsp;\u0026le;\u0026thinsp;35 was indicative of significant codon usage bias [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSources of Codon Usage Bias\u003c/h2\u003e\u003cp\u003eNeutrality plots (GC12 vs. GC3) were constructed to evaluate the influence of mutation pressure versus natural selection [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A regression slope near 0 suggests dominant natural selection, whereas a slope near 1 indicates mutation-dominated bias [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eENC plots were generated by comparing observed ENC values against expected values under neutral evolution (ENCexp\u0026thinsp;=\u0026thinsp;2\u0026thinsp;+\u0026thinsp;GC3\u0026thinsp;+\u0026thinsp;29/[GC3\u0026sup2; + (1\u0026thinsp;\u0026minus;\u0026thinsp;GC3)\u0026sup2;]) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Points distributed near the expected curve imply mutation-driven bias, whereas deviations below the curve suggest selective constraints [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eParity Rule 2 (PR2) analysis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] was applied to examine strand-specific bias using the parameters A/(A\u0026thinsp;+\u0026thinsp;T) and G/(G\u0026thinsp;+\u0026thinsp;C) at the third codon position.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eOptimal Codon Identification\u003c/h2\u003e\u003cp\u003eGenes were ranked based on their ENC values, with the top and bottom 10% defined as high- and low-expression sets, respectively. The difference in RSCU ΔRSCU\u0026thinsp;=\u0026thinsp;RSCU\u003csub\u003ehigh\u003c/sub\u003e \u0026minus; RSCU\u003csub\u003elow\u003c/sub\u003e) was computed for each codon. Optimal codons were identified as those with RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 and ΔRSCU\u0026thinsp;\u0026ge;\u0026thinsp;0.08 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eRSCU Clustering\u003c/h2\u003e\u003cp\u003eRSCU values from the eight \u003cem\u003eArgentina\u003c/em\u003e species were subjected to hierarchical clustering analysis using Euclidean distance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCDS coding DNA sequence\u003c/p\u003e\n\u003cp\u003ecp chloroplast\u003c/p\u003e\n\u003cp\u003eCUB codon usage bias\u003c/p\u003e\n\u003cp\u003eENC the effective number of codons\u003c/p\u003e\n\u003cp\u003eIR inverted repeat\u003c/p\u003e\n\u003cp\u003eLSC large single-copy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCGs protein-coding genes\u003c/p\u003e\n\u003cp\u003eRSCU relative synonymous codon usage\u003c/p\u003e\n\u003cp\u003eSSC small single-copy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available from the NCBI database under accession number PX057651 (\u003cem\u003eA. stenophylla\u003c/em\u003e), OR863700 (\u003cem\u003eA. phanerophlebia\u003c/em\u003e) and PX057649 (\u003cem\u003eA. anserina\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Open Subject from Key Laboratory of Qinghai-Tibetan Plateau Biotechnology of Ministry of Education (2023-SYS-04), Science and Technology Project of Liupanshui, grant number 52020-2019-05-05, 52020[2022]PT-20, and 52020-2023-0-2-18.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S-D.Z.; methodology, L-Z.L., G-N. Z. and J.T.; formal analysis, G-N. Z., L-Z.L. and S-D.Z.; writing\u0026mdash;original draft preparation, G-N. Z, L-Z.L. and S-D.Z.; writing\u0026mdash;review and editing, S-D.Z. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFeng T, Moore MJ, Sun Y, Meng A, Chu H, Li J, et al. A new species of Argentina (Rosaceae, Potentilleae) from Southeast Tibet, with reference to the taxonomic status of the genus. Plant Syst Evol. 2015;301(3):911\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJ. S: \u003cem\u003eArgentina\u003c/em\u003e Hill, a genus distinct from \u003cem\u003ePotentilla\u003c/em\u003e (Rosaceae). 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PLoS ONE. 2015;10(6):e0129223.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chloroplast Genome, Codon Usage Bias, Argentina, Phylogeny","lastPublishedDoi":"10.21203/rs.3.rs-7501003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7501003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe genus \u003cem\u003eArgentina\u003c/em\u003e (Rosaceae) represents a taxonomically complex group with significant ethnobotanical value, yet the morphological convergence among genus remains poorly resolved. Codon usage bias (CUB), an important genomic feature has not been systematically investigated in this genus, limiting our understanding of its chloroplast evolution and adaptive mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, we sequenced and annotated the complete chloroplast genomes of three key \u003cem\u003eArgentina\u003c/em\u003e species (\u003cem\u003eA. stenophylla\u003c/em\u003e, \u003cem\u003eA. anserina\u003c/em\u003e, and \u003cem\u003eA. phanerophlebia\u003c/em\u003e) and reconstructed a phylogeny using 21 taxa (18 \u003cem\u003eArgentina\u003c/em\u003e and three \u003cem\u003ePotentilla\u003c/em\u003e outgroups). We further conducted a comparative analysis of synonymous codon usage bias in eight representative species. The chloroplast genomes exhibited a typical quadripartite structure with high conservation in size (155,148\u0026ndash;155,972 bp), gene order, and content (129 genes). Phylogenetic analysis strongly supported the monophyly of \u003cem\u003eArgentina\u003c/em\u003e and identified four major clades. Codon usage analysis revealed a weak overall bias and a consistent preference for A/T-ending codons. Neutrality, ENC-, and PR2-plots indicated that natural selection plays a dominant role in shaping CUB, overriding mutational pressure. We identified 16 optimal codons, only one of which (GGA, encoding Gly) was shared across all species, suggesting lineage-specific codon adaptation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study provides the first comprehensive insight into codon usage patterns in \u003cem\u003eArgentina\u003c/em\u003e chloroplast genomes, enhancing our understanding of phylogenetic relationships and molecular evolution in this genus, with implications for future genetic research and codon optimization strategies.\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of Codon Usage Bias in Chloroplast Genomes of eight Argentina Species of Rosaceae and Their Phylogeny","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 12:05:52","doi":"10.21203/rs.3.rs-7501003/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9f54590-811e-4627-9d76-0bc8694b582a","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T16:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-19 12:05:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7501003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7501003","identity":"rs-7501003","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}