Characterization of the complete chloroplast genome of Agave sp. (Asparagales: Asparagaceae: Agavoideae) and its phylogenetic analysis

Characterization of the complete chloroplast genome of Agave sp. (Asparagales: Asparagaceae: Agavoideae) and its phylogenetic analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization of the complete chloroplast genome of Agave sp. (Asparagales: Asparagaceae: Agavoideae) and its phylogenetic analysis Xinya Ma, Xinghui Xu, Chaoyang Luo, Zhenyu Xiong, Juan Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8552740/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Genus Agave is one of the most diverse and complex group of the family Asparagaceae, originates from the Americas (with Mexico as its native range) and holds significant economic value as a widely distributed crop. However, the systematic phylogenetic relationship of this group is still controversial. In this study, we successfully sequenced, assembled, and annotated the chloroplast genome of Agave sp. The result showed: 1) The chloroplast genome of Agave sp. was 157,489bp in length with a GC content of 38.57%. It consisted of a large single-copy region (LSC, 86,018 bp), a pair of inverted repeat regions (IR, 26,612bp), and a small single-copy region (SSC, 18,247bp). In addition, the chloroplast genome of Agave sp. contains a total of 113 unique genes, including 79 protein-coding genes (PCGs), 30 tRNAs, and 4 rRNAs. 2) phylogenetic analysis of Asparagaceae indicate that the genera Yucca and Hosta each form a distinct monophyletic clade, clustering independently on separate branches. Agave sp. shows a close evolutionary relationship with Agave attenuata , forming a sister group. Furthermore, our phylogenetic tree provides support for the taxonomic classification of Agave sensu lato . This study enriches the genomic data of Agave and offers crucial reference information for subsequent research on the evolutionary history, taxonomic classification, and resource utilization of the Asparagaceae family. Asparagaceae Agave sp. Chloroplast genome phylogenetic analysis Agave sensu lato Figures Figure 1 Figure 2 Figure 3 Introduction Agave originates from the arid and semi-arid regions of the Americas and has diversified under different environmental conditions. The Agave genus has about 210 species, of which roughly 160 are found in Mexico, (Eguiarte et al., 2021 ; González Trillo et al., 2024). Agave is widely cultivated around the world and extensively used for producing food, beverages, fibers, and medicines. In addition, due to its strong drought resistance and resource-use efficiency, it has become a potential group for addressing global climate change (Eguiarte et al., 2021 ; González Trillo et al., 2024). Generally, Agave shows potential to compete with or complement traditional bioenergy crops, providing a direction for the development of semi-arid lands (Torres et al., 2015; González Trillo et al., 2024). The classification and identification of Agave have long faced challenges. Widespread morphological variation caused by arid and semi-arid environments, natural hybridization, and convergent evolution together make it difficult to define species. Genetic and morphological differences within populations directly affect the quality fluctuations of Agave products (Torres et al., 2015). In previous studies, the core chloroplast fragments rbcL and ndhF were used to explore the phylogeny of the Agave genus , suggesting that Agave sensu stricto is not a monophyletic group. Manfreda , Polianthes , and Prochnyanthes are parallel lineages to Agave sensu stricto , but due to the limited variable sites of the molecular markers, the precise relationships among the groups could not be clearly determined (Bogler et al., 2006 ). Good-Avila et al ( 2006 ) integrated nuclear gene ITS and chloroplast fragments for a combined analysis, proposing the hypothesis of two divergence events in Agave sensu lato (clade Agave sensu stricto , Polianthes , Manfreda , and Prochnyanthes , which together represent 30% of the genus). However, due to insufficient resolution of the chloroplast data, the branch support was relatively low. Later, by further expanding the sampling of matk and nuclear genes, they not only confirmed the paraphyly of Agave sensu stricto but also found that this classification characteristic was not directly associated with the evolution of pollination syndromes (Flores-Abreu et al,.2019). By using the highly variable nuclear gene ITS sequences to construct a phylogenetic tree, the broadly defined Agave genus was successfully divided into five well-supported clades, basically clarifying the paraphyletic nature of the narrowly defined groups, while also pointing out the obvious limitations of traditional chloroplast fragment analysis (Jiménez-Barron O et al., 2020 ). However, relying solely on studies of nuclear ITS and traditional chloroplast fragments still makes it difficult to verify whether there is a conflict in the co-evolution of the nuclear and plastid genomes. Single-gene markers cannot completely avoid issues such as multi-copy incongruence and interference from random mutations, so the phylogenetic relationships of this group still need to be further investigated. The chloroplast genome is characterized by structural conservation, high copy number, and a slow evolutionary rate, and it contains regions with different evolutionary rates, such as coding and non-coding regions (Hang et al., 2025 ; Li et al., 2024 ). It can provide abundant molecular trait information, compensating for the limitations of a single marker or fragment. The complete sequence of the chloroplast genome has been widely used in plant phylogenetic studies (Wu et al., 2021 ; Ma et al., 2025 ; Yang et al., 2022 ). Previous studies of the complete chloroplast genomes have widely used in genus Agave , which supported the monophyly of Agave sensu stricto from a maternal inheritance perspective (González-Trillo et al., 2024; Yang et al., 2021 ; Xu et al., 2022 ). So far, nearly 1,278 chloroplast genomes of Asparagaceae are stored in NCBI (National Center for Biotechnology Information), but only 12 of them belong to Agave sensu lato . This severely restricts the in-depth study of this group. Therefore, this study aims to obtain the complete chloroplast genome sequence of Agave sp. discuss the phylogenetic analysis. This study will provide fundamental data support for future research on chloroplast-related evolution, ecology, and resource utilization. Material and methods Collection of plant material The fresh leaves were collected in Dali University, Dali, Yunnan Province, China (25°40′25″N 100°09′22″E) in March 2025. (specimen number: IEHBR-2025001) DNA extraction and chloroplast genome sequencing and assembly Genomic DNA was extracted from leaf samples using the kit according to the instructions provided by Tiangen. The quality and quantity of DNA were evaluated by agarose gel electrophoresis, NanoDrop 2000, and Qubit 3.0 fluorometer. DNA was prepared for sequencing using NEB kits, and PCR fragment amplification was performed. The resulting libraries were purified and quality-assessed using the Agilent 5400 system, and their concentrations were determined. High-throughput sequencing (paired-end 150 bp) was conducted on the Illumina HiSeq X system at BGI Genomics (Shaanxi, China) (Illumina, San Diego, CA). Gene annotation was performed using Geneious software, with the Agave durangensis genome as a reference. Annotations were manually reviewed to ensure accuracy, including the start and stop codons of protein-coding genes and the boundaries of introns and exons. A circular map of the Seravschanicus chloroplast genome was obtained using OGDRAW. Codon usage characteristics were analyzed with CodonW software, systematically examining the structure and features of the Agave sp. chloroplast genome, and comprehensively assessing structural characteristics, codon usage, and relative synonymous codon usage (RSCU) (Sharp PM et al., 1987). Finally, the chloroplast genome was submitted to GenBank with the accession number. Phylogenetic analysis To further explore the position of Agave sp. and phylogenetic relationships family Asparagaceae, this study constructed a phylogenetic tree using the complete chloroplast genome. The analysis covered 42 species of the Asparagaceae family, Asparagus cochinchinensis and Zephyranthes mesochloa used as outgroups (Table 1 ). Sequence acquisition were performed using PhyloSuite v1.2.2 (Zhang et al., 2020 ). Preliminary alignments were conducted with MAFFT v7.505 (Katoh and Standley, 2013 ) and the alignment results were optimized using MACSE v2.07 (Ranwez et al., 2018 ). ModelFinder (Kalyaanamoorthy et al., 2017 ) was used to determine the optimal model based on the Bayesian Information Criterion (BIC), which was found to be TVM. Maximum likelihood (ML) phylogenetic analysis was carried out in IQ-TREE v1.6.12 (Nguyen et al., 2015) with the branch support tested using 1000 ultrafast bootstrap replicates. Visualization and annotation of the phylogenetic tree were performed with the online tool iTOL v6 (Letunic and Bork, 2021), and images were optimized using Adobe Photoshop 2023 to meet publication requirements. Table 1 Basic characteristics of chloroplast genomes in 42 species for phylogenetic analysis. species GenBank NO species GenBank NO Agave attenuata NC_032696.1 Hosta minor NC_035999.1 Agave amaniensis NC_058311.1 Hosta plantaginea NC_053555.1 Agave americana NC_032053.1 Hosta sieboldiana NC_060662.1 Agave angustifolia NC_059876.1 Hosta tsushimensis NC_060664.1 Agave cantula NC_070107.1 Hosta tsushimensis var. NC_060663.1 Agave durangensis NC_072313.1 Hosta ventricosa NC_032706.1 Agave fourcroydes NC_059874.1 Hosta venusta NC_046895.1 Agave hybrid cultivar NC_045534.1 Hosta yingeri MZ919315.1 Agave sisalana NC_059875.1 Manfreda virginica NC_032707.1 Beschorneria septentrionalis NC_032699.1 Polianthes sp. KX931464.1 Camassia scilloides NC_032700.1 Schoenolirion croceum NC_032710.1 Chlorogalum pomeridianum NC_032701.1 Yucca brevifolia MW281833.1 Hesperaloe campanulata NC_032702.1 Yucca brevifolia x Yucca jaegeriana MW281856.1 Hesperaloe parviflora NC_032703.1 Yucca filamentosa NC_032712.1 Hesperocallis undulata NC_032704.1 Yucca gloriosa NC_086555.1 Hesperoyucca whipplei NC_032705.1 Yucca jaegeriana MW281819.1 Hosta capitata MZ919305.1 Yucca queretaroensis NC_032713.1 Hosta clausa NC_046896.1 Yucca schidigera NC_032714.1 Hosta clausa var. OL628765.1 Yucca treculeana NC_061319.1 Hosta jonesii MZ919311.1 Zephyranthes mesochloa ( outgroup ) NC_057554.1 Hosta longipes NC_070392.1 Asparagus cochinchinensis ( outgroup ) NC_060472.1 Table 2 Functional classification of genes in the chloroplast genome of Agave sp. Categoryfor gene Group of genes Name of genes Self-replication Large subunit of ribosome rpl20 , rpl22 , rpl32 , rpl23 ( X2 ), rpl14 , rpl33 , rpl16 , rpl36 , rpl2 ( X2 ) Small subunit of ribosome rps11 , rps12 ( X3 ), rps14 , rps15 , rps16 , rps3 , rps18 , rps4 , rps19 ( X2 ), rps7 ( X2 ), rps8 DNA dependent RNA polymerase rpoA , rpoB , rpoC1 , rpoC2 rRNA gene rrn5 ( X2 ), rrn4.5( X2), rrn16 ( X2 ), rrn23 ( X2 ) tRNA gene trnR-UCU , trnE-UUC , trnT-GGU , trnS-GGA , trnI-CAU ( X2 ), trnV-GAC ( X2 ), trnR-ACG ( X2 ), trnL-UAA , trnG-GCC , trnD-GUC , trnY-GUA , trnP-UGG , trnM-CAU , trnL-CAA(X2) , trnS-GCU , trnW-CCA , trnF-GAA , trnT-UGU , trnS-UGA , trnV-UAC , trnG-UCC , trnL-UAG , trnI-GAU ( X2 ), trnH-GUG ( X2 ), trnfM-CAU , trnQ-UUG , trnN-GUU ( X2 ), trnK-UUU , trnA-UGC ( X2 ), trnC-GCA Gene for photosynthesis Subunits of photosystem I psaA , psaB , psaC , psaI , psaJ Subunits of photosystem II psbL , psbZ , psbM , psbN , psbA , psbB , psbC , psbD , psbE , psbF , psbT , psbH , psbI , psbJ , psbK Subunits of NADH-dehydrogenase ndhG , ndhH , ndhI , ndhJ , ndhK , ndhA , ndhB ( X2 ), ndhC , ndhD , ndhE , ndhF Subunits of cytochrome b/f complex petL , petN , petA , petB , petD , petG Subunit for ATP synthase atpI , atpA , atpB , atpE , atpF , atpH Large subunit of rubisco rbcL Other genes Translational initiation factor infA Maturase matK Protease clpP Envelope membrane protein cemA Subunit of Acetyl-carboxylase accD C-type cytochrome synthesis gene ccsA Open reading frames(ORF, ycf ) ycf1 , ycf2 ( X2 ), ycf3 , ycf4 Results and discussion Chloroplast genome features The Agave sp. cp genome is 157,489 bp in size and contains two IR regions (IRa and IRb; each 26,612 bp), separating the LSC (86,018 bp) and SSC (18,247 bp) regions, forming the quadripartite structure characteristic of angiosperms. It contains 113 unique genes, including 4 ribosomal rRNAs, 79 CDS, and 30 tRNA genes. We found 17 duplicated genes in the IR regions, including 4 rRNAs, 5 CDS, and 8 tRNA genes. The overall guanine-cytosine (GC) content of the chloroplast genome is 38.57%, with the GC content of the LSC, IR, and SSC regions being 35.9%, 43.1%, and 31.8%, respectively. Analysis of GC content by different gene types showed that rRNAs have a GC content of 55.4%, tRNAs 53.1%, and coding sequences 38.2%. The total genome size, the size of each region, and gene content are consistent with those reported for Agave durangensis , Agave sisalana , and Agave amaniensis (González-Trillo et al., 2024; Yang et al., 2021 ; Xu et al., 2022 ). Relative synonymous codon usage The codon preference of the chloroplast genome is directly related to the genome's AT/GC content and can reflect the AT-rich characteristics of the Agave chloroplast genome. The chloroplast genome of Agave sp. contains 79 protein-coding genes (PCGs). Relative synonymous codon usage (RSCU) was calculated, and the results (Fig. 2 ) are shown. These genes are encoded by 19,582 codons and exhibit highly conserved codon usage bias (CUB). Leucine (Leu) is the most frequently used amino acid, appearing 1,963 times, while cysteine (Cys) is the least common, occurring 215 times. RSCU analysis shows that AGU has the highest RSCU value at 3.43, while UCG has the lowest at 0.10. Among these codons, 29 have RSCU values greater than 1, and 27 have values less than 1. Additionally, the codon for methionine (Met), AUG, and the codon for tryptophan (Trp), UGG, both have an RSCU value of 1 (Wang et al., 2023 ). Phylogenetic reconstruction The analysis of the complete chloroplast genome sequence constructed a maximum likelihood phylogenetic tree with strong support. Phylogenetic analysis indicates that this tree has four major clades, each with a bootstrap value of 100%. The genera Yucca and Hosta cluster together on a separate branch, representing a relatively independent evolutionary line within the Asparagaceae, forming a typical monophyletic group, which was consistent with previous morphological and molecular studies (Bogler et al., 2006 ; Xu et al., 2022 ). Camassia scilloides and Chlorogalum pomeridianum are sister species, forming a paraphyletic relationship with Hesperocallis undulata , Hesperoyucca whipplei , Schoenolirion croceum , and the Hesperaloe genus , which aligns with previous research findings (Yang et al., 2021 ). Agave sp. is closely related to Agave attenuata , forming a sister group. The traditionally defined Agave sensu stricto is not monophyletic and diverged over a short period. Agave sensu lato is paraphyletic, including Polianthes and Manfreda virginica . This is consistent with phylogeny from nuclear ITS gene (Jiménez-Barron et al., 2020 ). The phylogenetic tree clearly illustrates the evolutionary branching relationships of the genera Agave , Yucca , Hosta , and other closely related groups within the Asparagaceae. Conclusion This study presents the complete chloroplast genome sequence of Agave sp., with a cp genome size of 157,489 bp, featuring a typical quadripartite structure and 113 unique genes, similar to other agave species. Our chloroplast phylogenetic analysis of 42 species supports the paraphyly within the broad genus Agave while Yucca , Agave sensu lato , and Hosta are well-supported monophyletic groups. Our results provide a valuable insight into chloroplast evolution within the family Asparagaceae. Declarations Competing interests The authors declare no competing interests. Author Contribution Xinya Ma: Conceptualization, Methodology, Investigation, Visualization, Writing – original draft, Writing – review & editing.Xionghui Xu: Methodology, Investigation.Chaoyang Luo: Conceptualization.Zhenyu Xiong: Methodology, Visualization.Juan Zhang: Visualization.Yuan Mu: Conceptualization, Methodology, Investigation, Project administration, Supervision, Funding acquisition, Writing – original draft, Writing – review & editing. Data Availability The dataset generated and analyzed in this study is available in the NCBI-Nucleotide database. For the biological samples included in this study (with the study-generated identifier: PX665995), the relevant accessions are: NC_032696.1, NC_058311.1, NC_032053.1, NC_059876.1, NC_070107.1, NC_072313.1, NC_059874.1, NC_045534.1, NC_059875.1, NC_032699.1, NC_032700.1, NC_032701.1, NC_032702.1, NC_032703.1, NC_032704.1, NC_032705.1, MZ919305.1, NC_046896.1, OL628765.1, MZ919311.1, NC_070392.1, NC_035999.1, NC_053555.1, NC_060662.1, NC_060664.1, NC_060663.1, NC_032706.1, NC_046895.1, MZ919315.1, NC_032707.1, KX931464.1, NC_032710.1, MW281833.1, MW281856.1, NC_032712.1, NC_086555.1, MW281819.1, NC_032713.1, NC_032714.1, NC_061319.1, NC_057554.1, NC_060472.1 References Eguiarte LE, Jiménez Barrón OA, Aguirre-Planter E et al (2021) Evolutionary ecology of Agave: distribution patterns, phylogeny, and coevolution (an homage to Howard S. Gentry). 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Mol Biol Evol (1): 268–274 Letunic I, Bork P (2024) Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 52(W1):W78–W82 Wang Y, Jiang D, Guo K et al (2023) Comparative analysis of codon usage patterns in chloroplast genomes of ten Epimedium species. BMC Genom Data 24(1):3 Additional Declarations No competing interests reported. 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. We do this by developing innovative software and high quality services for the global research community. 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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-8552740","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":573774287,"identity":"de05e4d6-f406-410c-85cf-1970a92ed0eb","order_by":0,"name":"Xinya Ma","email":"","orcid":"","institution":"Dali Universit","correspondingAuthor":false,"prefix":"","firstName":"Xinya","middleName":"","lastName":"Ma","suffix":""},{"id":573774288,"identity":"70975607-e01a-4541-b692-5478f3996879","order_by":1,"name":"Xinghui Xu","email":"","orcid":"","institution":"Dali Universit","correspondingAuthor":false,"prefix":"","firstName":"Xinghui","middleName":"","lastName":"Xu","suffix":""},{"id":573774290,"identity":"a5831152-77cc-4fe0-a261-a9e152a54106","order_by":2,"name":"Chaoyang Luo","email":"","orcid":"","institution":"Dali Universit","correspondingAuthor":false,"prefix":"","firstName":"Chaoyang","middleName":"","lastName":"Luo","suffix":""},{"id":573774292,"identity":"e1cf84b0-73f7-40e0-8543-eec86da2206e","order_by":3,"name":"Zhenyu Xiong","email":"","orcid":"","institution":"Dali Universit","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Xiong","suffix":""},{"id":573774293,"identity":"5c9dd102-a940-43ef-8cef-34f51297a75b","order_by":4,"name":"Juan Zhang","email":"","orcid":"","institution":"Yunnan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Zhang","suffix":""},{"id":573774302,"identity":"5ddd669a-4a67-48b4-9388-9be46c1e4f30","order_by":5,"name":"Yuan Mu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYHACxgcwFjODAQMDHxFamIHqGCTgWtiI0MImgdDCQIQW3Rk5ZhUfaurq+KXPHmAuKNgmz8Z+xvADQ80dnFrMbuSY3Zxx7LCEZF9eAvMMg9uGbTw5xhIMx57h1XKbh+2AhMEZHvPfPAa3GdsYcgwkGBsO49VS/OdfnYT9GR4DZqAW+zb+N8Y/CGlhZmxjljDggWhJbJPIMcNvy5lnxZK9fYclZ0BtSW6TeFZmkXAMj5bjyRs//PhWx8/fA9Ly57ZtP3/y5hsfanBrYWDgMMAikoBHAwMD+wPCIqNgFIyCUTCyAQD1+U7PPNI+RgAAAABJRU5ErkJggg==","orcid":"","institution":"Dali Universit","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Mu","suffix":""}],"badges":[],"createdAt":"2026-01-08 14:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8552740/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8552740/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101349550,"identity":"526e9d40-b71a-4abd-8b15-3685d8d70e04","added_by":"auto","created_at":"2026-01-28 18:11:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165804,"visible":true,"origin":"","legend":"\u003cp\u003eCircular map of the chloroplast genome of \u003cem\u003eAgave \u003c/em\u003esp.\u003cem\u003e \u003c/em\u003eGenes are color coded according to their functions. Genes located on the outer edge transcribe in an anticlockwise direction, whereas those on the inner side transcribe clockwise.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552740/v1/c7a12e4d27532e50fd3d4fbd.jpg"},{"id":101349495,"identity":"d49dbc5f-19d6-48f0-a1ef-c2b1d54ccdb1","added_by":"auto","created_at":"2026-01-28 18:10:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100887,"visible":true,"origin":"","legend":"\u003cp\u003eThe Bar chart of codon usage for amino acids of 79 protein-coding genes in the chloroplast genome of \u003cem\u003eAgave \u003c/em\u003esp.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552740/v1/9df23d09ce7634142102d0d4.jpg"},{"id":101349468,"identity":"25bb59e3-2863-40b5-b258-142d0d93ef23","added_by":"auto","created_at":"2026-01-28 18:10:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115364,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed using Maximum Likelihood (ML) method based on the complete chloroplast genome sequences of 42 Asparagaceae species.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552740/v1/118b0946c09ee29aa0911feb.jpg"},{"id":101881516,"identity":"a33bfa13-0a1f-47fc-939b-ef9147e7a92b","added_by":"auto","created_at":"2026-02-04 15:12:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1063824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8552740/v1/10d11957-d981-4b16-8169-0ed08ace35f1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of the complete chloroplast genome of Agave sp. (Asparagales: Asparagaceae: Agavoideae) and its phylogenetic analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAgave\u003c/em\u003e originates from the arid and semi-arid regions of the Americas and has diversified under different environmental conditions. The \u003cem\u003eAgave\u003c/em\u003e genus has about 210 species, of which roughly 160 are found in Mexico, (Eguiarte et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gonz\u0026aacute;lez Trillo et al., 2024). \u003cem\u003eAgave\u003c/em\u003e is widely cultivated around the world and extensively used for producing food, beverages, fibers, and medicines. In addition, due to its strong drought resistance and resource-use efficiency, it has become a potential group for addressing global climate change (Eguiarte et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gonz\u0026aacute;lez Trillo et al., 2024). Generally, \u003cem\u003eAgave\u003c/em\u003e shows potential to compete with or complement traditional bioenergy crops, providing a direction for the development of semi-arid lands (Torres et al., 2015; Gonz\u0026aacute;lez Trillo et al., 2024).\u003c/p\u003e \u003cp\u003eThe classification and identification of \u003cem\u003eAgave\u003c/em\u003e have long faced challenges. Widespread morphological variation caused by arid and semi-arid environments, natural hybridization, and convergent evolution together make it difficult to define species. Genetic and morphological differences within populations directly affect the quality fluctuations of \u003cem\u003eAgave\u003c/em\u003e products (Torres et al., 2015). In previous studies, the core chloroplast fragments \u003cem\u003erbcL\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e were used to explore the phylogeny of the \u003cem\u003eAgave genus\u003c/em\u003e, suggesting that \u003cem\u003eAgave sensu stricto\u003c/em\u003e is not a monophyletic group. \u003cem\u003eManfreda\u003c/em\u003e, \u003cem\u003ePolianthes\u003c/em\u003e, and \u003cem\u003eProchnyanthes\u003c/em\u003e are parallel lineages to \u003cem\u003eAgave sensu stricto\u003c/em\u003e, but due to the limited variable sites of the molecular markers, the precise relationships among the groups could not be clearly determined (Bogler et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Good-Avila et al (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) integrated nuclear gene \u003cem\u003eITS\u003c/em\u003e and chloroplast fragments for a combined analysis, proposing the hypothesis of two divergence events in \u003cem\u003eAgave sensu lato\u003c/em\u003e (clade \u003cem\u003eAgave sensu stricto\u003c/em\u003e, \u003cem\u003ePolianthes\u003c/em\u003e, \u003cem\u003eManfreda\u003c/em\u003e, and \u003cem\u003eProchnyanthes\u003c/em\u003e, which together represent 30% of the genus). However, due to insufficient resolution of the chloroplast data, the branch support was relatively low. Later, by further expanding the sampling of \u003cem\u003ematk\u003c/em\u003e and nuclear genes, they not only confirmed the paraphyly of \u003cem\u003eAgave sensu stricto\u003c/em\u003e but also found that this classification characteristic was not directly associated with the evolution of pollination syndromes (Flores-Abreu et al,.2019). By using the highly variable nuclear gene \u003cem\u003eITS\u003c/em\u003e sequences to construct a phylogenetic tree, the broadly defined \u003cem\u003eAgave\u003c/em\u003e genus was successfully divided into five well-supported clades, basically clarifying the paraphyletic nature of the narrowly defined groups, while also pointing out the obvious limitations of traditional chloroplast fragment analysis (Jim\u0026eacute;nez-Barron O et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, relying solely on studies of nuclear \u003cem\u003eITS\u003c/em\u003e and traditional chloroplast fragments still makes it difficult to verify whether there is a conflict in the co-evolution of the nuclear and plastid genomes. Single-gene markers cannot completely avoid issues such as multi-copy incongruence and interference from random mutations, so the phylogenetic relationships of this group still need to be further investigated.\u003c/p\u003e \u003cp\u003eThe chloroplast genome is characterized by structural conservation, high copy number, and a slow evolutionary rate, and it contains regions with different evolutionary rates, such as coding and non-coding regions (Hang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It can provide abundant molecular trait information, compensating for the limitations of a single marker or fragment. The complete sequence of the chloroplast genome has been widely used in plant phylogenetic studies (Wu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ma et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previous studies of the complete chloroplast genomes have widely used in genus \u003cem\u003eAgave\u003c/em\u003e, which supported the monophyly of \u003cem\u003eAgave sensu stricto\u003c/em\u003e from a maternal inheritance perspective (Gonz\u0026aacute;lez-Trillo et al., 2024; Yang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, nearly 1,278 chloroplast genomes of Asparagaceae are stored in NCBI (National Center for Biotechnology Information), but only 12 of them belong to \u003cem\u003eAgave sensu lato\u003c/em\u003e. This severely restricts the in-depth study of this group. Therefore, this study aims to obtain the complete chloroplast genome sequence of \u003cem\u003eAgave\u003c/em\u003e sp. discuss the phylogenetic analysis. This study will provide fundamental data support for future research on chloroplast-related evolution, ecology, and resource utilization.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection of plant material\u003c/h2\u003e \u003cp\u003eThe fresh leaves were collected in Dali University, Dali, Yunnan Province, China (25\u0026deg;40\u0026prime;25\u0026Prime;N 100\u0026deg;09\u0026prime;22\u0026Prime;E) in March 2025. (specimen number: IEHBR-2025001)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA extraction and chloroplast genome sequencing and assembly\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from leaf samples using the kit according to the instructions provided by Tiangen. The quality and quantity of DNA were evaluated by agarose gel electrophoresis, NanoDrop 2000, and Qubit 3.0 fluorometer. DNA was prepared for sequencing using NEB kits, and PCR fragment amplification was performed. The resulting libraries were purified and quality-assessed using the Agilent 5400 system, and their concentrations were determined. High-throughput sequencing (paired-end 150 bp) was conducted on the Illumina HiSeq X system at BGI Genomics (Shaanxi, China) (Illumina, San Diego, CA). Gene annotation was performed using Geneious software, with the \u003cem\u003eAgave durangensis\u003c/em\u003e genome as a reference. Annotations were manually reviewed to ensure accuracy, including the start and stop codons of protein-coding genes and the boundaries of introns and exons. A circular map of the Seravschanicus chloroplast genome was obtained using OGDRAW. Codon usage characteristics were analyzed with CodonW software, systematically examining the structure and features of the \u003cem\u003eAgave\u003c/em\u003e sp. chloroplast genome, and comprehensively assessing structural characteristics, codon usage, and relative synonymous codon usage (RSCU) (Sharp PM et al., 1987). Finally, the chloroplast genome was submitted to GenBank with the accession number.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eTo further explore the position of \u003cem\u003eAgave\u003c/em\u003e sp. and phylogenetic relationships family Asparagaceae, this study constructed a phylogenetic tree using the complete chloroplast genome. The analysis covered 42 species of the Asparagaceae family, \u003cem\u003eAsparagus cochinchinensis\u003c/em\u003e and \u003cem\u003eZephyranthes mesochloa\u003c/em\u003e used as outgroups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sequence acquisition were performed using PhyloSuite v1.2.2 (Zhang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Preliminary alignments were conducted with MAFFT v7.505 (Katoh and Standley, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and the alignment results were optimized using MACSE v2.07 (Ranwez et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ModelFinder (Kalyaanamoorthy et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was used to determine the optimal model based on the Bayesian Information Criterion (BIC), which was found to be TVM. Maximum likelihood (ML) phylogenetic analysis was carried out in IQ-TREE v1.6.12 (Nguyen et al., 2015) with the branch support tested using 1000 ultrafast bootstrap replicates. Visualization and annotation of the phylogenetic tree were performed with the online tool iTOL v6 (Letunic and Bork, 2021), and images were optimized using Adobe Photoshop 2023 to meet publication requirements.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic characteristics of chloroplast genomes in 42 species for phylogenetic analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003especies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank NO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003especies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenBank NO\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave attenuata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032696.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_035999.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave amaniensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_058311.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta plantaginea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053555.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave americana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032053.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta sieboldiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_060662.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave angustifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_059876.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta tsushimensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_060664.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave cantula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_070107.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta tsushimensis var.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_060663.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave durangensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_072313.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta ventricosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_032706.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave fourcroydes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_059874.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta venusta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_046895.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave hybrid cultivar\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_045534.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHosta yingeri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMZ919315.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAgave sisalana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_059875.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eManfreda virginica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_032707.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBeschorneria septentrionalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032699.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePolianthes\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKX931464.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCamassia scilloides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032700.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSchoenolirion croceum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_032710.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eChlorogalum pomeridianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032701.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca brevifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW281833.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHesperaloe campanulata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032702.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca brevifolia x Yucca jaegeriana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW281856.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHesperaloe parviflora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032703.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca filamentosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_032712.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHesperocallis undulata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032704.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca gloriosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_086555.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHesperoyucca whipplei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_032705.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca jaegeriana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW281819.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHosta capitata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMZ919305.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca queretaroensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_032713.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHosta clausa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_046896.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca schidigera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_032714.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHosta clausa var.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOL628765.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eYucca treculeana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_061319.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHosta jonesii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMZ919311.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eZephyranthes mesochloa\u003c/em\u003e(\u003cem\u003eoutgroup\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_057554.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHosta longipes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_070392.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAsparagus cochinchinensis\u003c/em\u003e(\u003cem\u003eoutgroup\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_060472.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFunctional classification of genes in the chloroplast genome of \u003cem\u003eAgave\u003c/em\u003e sp.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategoryfor gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup of genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eName of genes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eSelf-replication\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of ribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl20\u003c/em\u003e, \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erpl32\u003c/em\u003e, \u003cem\u003erpl23\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003erpl14\u003c/em\u003e, \u003cem\u003erpl33\u003c/em\u003e, \u003cem\u003erpl16\u003c/em\u003e, \u003cem\u003erpl36\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall subunit of ribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps11\u003c/em\u003e, \u003cem\u003erps12\u003c/em\u003e(\u003cem\u003eX3\u003c/em\u003e), \u003cem\u003erps14\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003erps18\u003c/em\u003e, \u003cem\u003erps4\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003erps7\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003erps8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA dependent RNA polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpoA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e, \u003cem\u003erpoC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erRNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn5\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003errn4.5(\u003c/em\u003eX2), \u003cem\u003errn16\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003errn23\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etRNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrnR-UCU\u003c/em\u003e, \u003cem\u003etrnE-UUC\u003c/em\u003e, \u003cem\u003etrnT-GGU\u003c/em\u003e, \u003cem\u003etrnS-GGA\u003c/em\u003e, \u003cem\u003etrnI-CAU\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnV-GAC\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnR-ACG\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnL-UAA\u003c/em\u003e, \u003cem\u003etrnG-GCC\u003c/em\u003e, \u003cem\u003etrnD-GUC\u003c/em\u003e, \u003cem\u003etrnY-GUA\u003c/em\u003e, \u003cem\u003etrnP-UGG\u003c/em\u003e, \u003cem\u003etrnM-CAU\u003c/em\u003e, \u003cem\u003etrnL-CAA(X2)\u003c/em\u003e, \u003cem\u003etrnS-GCU\u003c/em\u003e, \u003cem\u003etrnW-CCA\u003c/em\u003e, \u003cem\u003etrnF-GAA\u003c/em\u003e, \u003cem\u003etrnT-UGU\u003c/em\u003e, \u003cem\u003etrnS-UGA\u003c/em\u003e, \u003cem\u003etrnV-UAC\u003c/em\u003e, \u003cem\u003etrnG-UCC\u003c/em\u003e, \u003cem\u003etrnL-UAG\u003c/em\u003e, \u003cem\u003etrnI-GAU\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnH-GUG\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnfM-CAU\u003c/em\u003e, \u003cem\u003etrnQ-UUG\u003c/em\u003e, \u003cem\u003etrnN-GUU\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnK-UUU\u003c/em\u003e, \u003cem\u003etrnA-UGC\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003etrnC-GCA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eGene for photosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsaA\u003c/em\u003e, \u003cem\u003epsaB\u003c/em\u003e, \u003cem\u003epsaC\u003c/em\u003e, \u003cem\u003epsaI\u003c/em\u003e, \u003cem\u003epsaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsbL\u003c/em\u003e, \u003cem\u003epsbZ\u003c/em\u003e, \u003cem\u003epsbM\u003c/em\u003e, \u003cem\u003epsbN\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003epsbB\u003c/em\u003e, \u003cem\u003epsbC\u003c/em\u003e, \u003cem\u003epsbD\u003c/em\u003e, \u003cem\u003epsbE\u003c/em\u003e, \u003cem\u003epsbF\u003c/em\u003e, \u003cem\u003epsbT\u003c/em\u003e, \u003cem\u003epsbH\u003c/em\u003e, \u003cem\u003epsbI\u003c/em\u003e, \u003cem\u003epsbJ\u003c/em\u003e, \u003cem\u003epsbK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of NADH-dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003endhG\u003c/em\u003e, \u003cem\u003endhH\u003c/em\u003e, \u003cem\u003endhI\u003c/em\u003e, \u003cem\u003endhJ\u003c/em\u003e, \u003cem\u003endhK\u003c/em\u003e, \u003cem\u003endhA\u003c/em\u003e, \u003cem\u003endhB\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003endhC\u003c/em\u003e, \u003cem\u003endhD\u003c/em\u003e, \u003cem\u003endhE\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of cytochrome b/f complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epetL\u003c/em\u003e, \u003cem\u003epetN\u003c/em\u003e, \u003cem\u003epetA\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e, \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunit for ATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatpI\u003c/em\u003e, \u003cem\u003eatpA\u003c/em\u003e, \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, \u003cem\u003eatpF\u003c/em\u003e, \u003cem\u003eatpH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of rubisco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003eOther genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTranslational initiation factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003einfA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclpP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvelope membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunit of Acetyl-carboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-type cytochrome synthesis gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOpen reading frames(ORF,\u003cem\u003eycf\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003eycf2\u003c/em\u003e(\u003cem\u003eX2\u003c/em\u003e), \u003cem\u003eycf3\u003c/em\u003e, \u003cem\u003eycf4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eChloroplast genome features\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eAgave\u003c/em\u003e sp. cp genome is 157,489 bp in size and contains two IR regions (IRa and IRb; each 26,612 bp), separating the LSC (86,018 bp) and SSC (18,247 bp) regions, forming the quadripartite structure characteristic of angiosperms. It contains 113 unique genes, including 4 ribosomal rRNAs, 79 CDS, and 30 tRNA genes. We found 17 duplicated genes in the IR regions, including 4 rRNAs, 5 CDS, and 8 tRNA genes. The overall guanine-cytosine (GC) content of the chloroplast genome is 38.57%, with the GC content of the LSC, IR, and SSC regions being 35.9%, 43.1%, and 31.8%, respectively. Analysis of GC content by different gene types showed that rRNAs have a GC content of 55.4%, tRNAs 53.1%, and coding sequences 38.2%. The total genome size, the size of each region, and gene content are consistent with those reported for \u003cem\u003eAgave durangensis\u003c/em\u003e, \u003cem\u003eAgave sisalana\u003c/em\u003e, and \u003cem\u003eAgave amaniensis\u003c/em\u003e (Gonz\u0026aacute;lez-Trillo et al., 2024; Yang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRelative synonymous codon usage\u003c/h2\u003e \u003cp\u003eThe codon preference of the chloroplast genome is directly related to the genome's AT/GC content and can reflect the AT-rich characteristics of the \u003cem\u003eAgave\u003c/em\u003e chloroplast genome. The chloroplast genome of \u003cem\u003eAgave\u003c/em\u003e sp. contains 79 protein-coding genes (PCGs). Relative synonymous codon usage (RSCU) was calculated, and the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) are shown. These genes are encoded by 19,582 codons and exhibit highly conserved codon usage bias (CUB). Leucine (Leu) is the most frequently used amino acid, appearing 1,963 times, while cysteine (Cys) is the least common, occurring 215 times. RSCU analysis shows that AGU has the highest RSCU value at 3.43, while UCG has the lowest at 0.10. Among these codons, 29 have RSCU values greater than 1, and 27 have values less than 1. Additionally, the codon for methionine (Met), AUG, and the codon for tryptophan (Trp), UGG, both have an RSCU value of 1 (Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic reconstruction\u003c/h3\u003e\n\u003cp\u003eThe analysis of the complete chloroplast genome sequence constructed a maximum likelihood phylogenetic tree with strong support. Phylogenetic analysis indicates that this tree has four major clades, each with a bootstrap value of 100%. The genera \u003cem\u003eYucca\u003c/em\u003e and \u003cem\u003eHosta\u003c/em\u003e cluster together on a separate branch, representing a relatively independent evolutionary line within the Asparagaceae, forming a typical monophyletic group, which was consistent with previous morphological and molecular studies (Bogler et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eCamassia scilloides\u003c/em\u003e and \u003cem\u003eChlorogalum pomeridianum\u003c/em\u003e are sister species, forming a paraphyletic relationship with \u003cem\u003eHesperocallis undulata\u003c/em\u003e, \u003cem\u003eHesperoyucca whipplei\u003c/em\u003e, \u003cem\u003eSchoenolirion croceum\u003c/em\u003e, and the \u003cem\u003eHesperaloe genus\u003c/em\u003e, which aligns with previous research findings (Yang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eAgave\u003c/em\u003e sp. is closely related to \u003cem\u003eAgave attenuata\u003c/em\u003e, forming a sister group. The traditionally defined \u003cem\u003eAgave sensu stricto\u003c/em\u003e is not monophyletic and diverged over a short period. \u003cem\u003eAgave sensu lato\u003c/em\u003e is paraphyletic, including \u003cem\u003ePolianthes\u003c/em\u003e and \u003cem\u003eManfreda virginica\u003c/em\u003e. This is consistent with phylogeny from nuclear \u003cem\u003eITS\u003c/em\u003e gene (Jim\u0026eacute;nez-Barron et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The phylogenetic tree clearly illustrates the evolutionary branching relationships of the genera \u003cem\u003eAgave\u003c/em\u003e, \u003cem\u003eYucca\u003c/em\u003e, \u003cem\u003eHosta\u003c/em\u003e, and other closely related groups within the Asparagaceae.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents the complete chloroplast genome sequence of \u003cem\u003eAgave\u003c/em\u003e sp., with a cp genome size of 157,489 bp, featuring a typical quadripartite structure and 113 unique genes, similar to other agave species. Our chloroplast phylogenetic analysis of 42 species supports the paraphyly within the broad genus \u003cem\u003eAgave\u003c/em\u003e while \u003cem\u003eYucca\u003c/em\u003e, \u003cem\u003eAgave sensu lato\u003c/em\u003e, and \u003cem\u003eHosta\u003c/em\u003e are well-supported monophyletic groups. Our results provide a valuable insight into chloroplast evolution within the family Asparagaceae.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXinya Ma: Conceptualization, Methodology, Investigation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.Xionghui Xu: Methodology, Investigation.Chaoyang Luo: Conceptualization.Zhenyu Xiong: Methodology, Visualization.Juan Zhang: Visualization.Yuan Mu: Conceptualization, Methodology, Investigation, Project administration, Supervision, Funding acquisition, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe dataset generated and analyzed in this study is available in the NCBI-Nucleotide database. For the biological samples included in this study (with the study-generated identifier: PX665995), the relevant accessions are: NC_032696.1, NC_058311.1, NC_032053.1, NC_059876.1, NC_070107.1, NC_072313.1, NC_059874.1, NC_045534.1, NC_059875.1, NC_032699.1, NC_032700.1, NC_032701.1, NC_032702.1, NC_032703.1, NC_032704.1, NC_032705.1, MZ919305.1, NC_046896.1, OL628765.1, MZ919311.1, NC_070392.1, NC_035999.1, NC_053555.1, NC_060662.1, NC_060664.1, NC_060663.1, NC_032706.1, NC_046895.1, MZ919315.1, NC_032707.1, KX931464.1, NC_032710.1, MW281833.1, MW281856.1, NC_032712.1, NC_086555.1, MW281819.1, NC_032713.1, NC_032714.1, NC_061319.1, NC_057554.1, NC_060472.1\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEguiarte LE, Jim\u0026eacute;nez Barr\u0026oacute;n OA, Aguirre-Planter E et al (2021) Evolutionary ecology of Agave: distribution patterns, phylogeny, and coevolution (an homage to Howard S. Gentry). 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Mol Phylogenet Evol 133:176\u0026ndash;188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez-Barron O, Garc\u0026iacute;a-Sandoval R, Magall\u0026oacute;n S et al (2020) Phylogeny, Diversification Rate, and Divergence Time of \u003cem\u003eAgave sensu lato\u003c/em\u003e (Asparagaceae), a Group of Recent Origin in the Process of Diversification. Front Plant Sci 11:536135\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Huang X, Tan S et al (2021) The complete chloroplast genome of \u003cem\u003eAgave sisalana\u003c/em\u003e. Mitochondrial DNA Part B 6(7):1855\u0026ndash;1856\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, Tan S, Qin X et al (2022) The complete chloroplast genome of \u003cem\u003eAgave amaniensis\u003c/em\u003e (Asparagales: Asparagaceae: Agavoideae). 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Nat Methods (6): 587\u0026ndash;589\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen LT, Schmidt HA, von Haeseler A et al 20145. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol (1): 268\u0026ndash;274\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Bork P (2024) Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 52(W1):W78\u0026ndash;W82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Jiang D, Guo K et al (2023) Comparative analysis of codon usage patterns in chloroplast genomes of ten \u003cem\u003eEpimedium\u003c/em\u003e species. BMC Genom Data 24(1):3\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":"Asparagaceae, Agave sp., Chloroplast genome, phylogenetic analysis, Agave sensu lato","lastPublishedDoi":"10.21203/rs.3.rs-8552740/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8552740/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenus \u003cem\u003eAgave\u003c/em\u003e is one of the most diverse and complex group of the family Asparagaceae, originates from the Americas (with Mexico as its native range) and holds significant economic value as a widely distributed crop. However, the systematic phylogenetic relationship of this group is still controversial. In this study, we successfully sequenced, assembled, and annotated the chloroplast genome of \u003cem\u003eAgave\u003c/em\u003e sp. The result showed: 1) The chloroplast genome of \u003cem\u003eAgave\u003c/em\u003e sp. was 157,489bp in length with a GC content of 38.57%. It consisted of a large single-copy region (LSC, 86,018 bp), a pair of inverted repeat regions (IR, 26,612bp), and a small single-copy region (SSC, 18,247bp). In addition, the chloroplast genome of \u003cem\u003eAgave\u003c/em\u003e sp. contains a total of 113 unique genes, including 79 protein-coding genes (PCGs), 30 tRNAs, and 4 rRNAs. 2) phylogenetic analysis of Asparagaceae indicate that the genera \u003cem\u003eYucca\u003c/em\u003e and \u003cem\u003eHosta\u003c/em\u003e each form a distinct monophyletic clade, clustering independently on separate branches. \u003cem\u003eAgave\u003c/em\u003e sp. shows a close evolutionary relationship with \u003cem\u003eAgave attenuata\u003c/em\u003e, forming a sister group. Furthermore, our phylogenetic tree provides support for the taxonomic classification of \u003cem\u003eAgave sensu lato\u003c/em\u003e. This study enriches the genomic data of Agave and offers crucial reference information for subsequent research on the evolutionary history, taxonomic classification, and resource utilization of the Asparagaceae family.\u003c/p\u003e","manuscriptTitle":"Characterization of the complete chloroplast genome of Agave sp. (Asparagales: Asparagaceae: Agavoideae) and its phylogenetic analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 18:10:33","doi":"10.21203/rs.3.rs-8552740/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":"5eb5344d-0f20-4f45-9d67-946323443b47","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T12:41:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 18:10:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8552740","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8552740","identity":"rs-8552740","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}