Comparative analysis of Artemisia plastomes and insights into the infra-generic phylogenetic relationships of the genus

Comparative analysis of Artemisia plastomes and insights into the infra-generic phylogenetic relationships of the genus | 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 Comparative analysis of Artemisia plastomes and insights into the infra-generic phylogenetic relationships of the genus Xinqiang Guo, Dawei Xue, Yuhuan Wu, Mengjie Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4573083/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 Background The genus Artemisia is a taxonomically difficult group comprising 400–500 species mainly distributed in northern hemisphere. Only a limited number of Artemisia plastomes are currently available. Their structure has not been comparatively analyzed, and the phylogenetic backbone of Artemisia based on plastome-scale data has not been reported with dense taxon sampling. This situation has greatly hindered our understanding on the plastome variation patterns and infra-generic relationships of the genus. With the advancement of next generation sequencing technologies, it is becoming easier to obtain and comparatively analyze the plastome, and use it to construct phylogeny. Results In this study, we newly sequenced 34 Artemisia plastomes representing 30 species and three varieties. Combing with 38 previously published plastomes, a total of 72 complete Artemisia plastomes were comparatively analyzed. The results indicated that the Artemisia plastomes were conserved in terms of structure, GC content, gene number and order. All plastomes have a typical quadripartite structure, comprising 87 protein coding, 37 tRNA, and 8 rRNA genes. The IR regions are similar in length and structure among the compared plastomes, with the generic regions more conserved than intergenic spacer regions. The sequence divergence is higher in LSC and SSC regions than in IR regions. Three protein-coding genes and four non-coding regions, i.e., accD , petG , ycf1 , rpoC1 - rpoC2 , rpoC2 - rps2 , trnG (UCC)- trnfM (CAU), and ndhG - ndhI , were found to be highly diverse, and could be chosen as candidates of DNA barcode. Phylogenetic relationships constructed using protein coding genes of plastomes were divided into several clades that did not match with previous infra-generic divisions of Artemisia , and four subgenera were not monophyletic. Furthermore, they were also inconsistent with those based on nuclear markers. And the phylogenetic position of A. stracheyi is still controversial. Conslusions This study reveals that the Artemisia plastomes are conservative, especially in structure, gene number and order. Phylogenetic relationships constructed using CDS further confirmed the infra-generic divisions of Artemisia were not natural. This study lay a foundation for future evolutionary studies of Artemisia . Artemisia Astearaceae Phylogenomics Plastome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Artemisia is the largest genus of tribe Anthemideae in Asteraceae, comprising 400–500 species [ 1 – 4 ]. Members of this genus are mainly distributed in northern hemisphere, with a few species occurring in Africa, South America and Hawaiian Islands [ 1 – 6 ]. Artemisia is economically important. Members of this genus, i.e., A. argyi H. Lév. & Vaniot, and A. capillaris Thunb., have been widely used as herbal remedies in China, and some species have broad applications as food, or forage. And the most famous one undoubtedly is A . annua L. The discovery of the anti-malaria artemisinin from it was awarded a Nobel Prize in Physiology or Medicine in 2015 [ 7 – 9 ]. Thus Artemisia receives extensive scientific attention, especially in the fields of phytochemistry and pharmacology. Artemisia represents one of the most notoriously difficult groups in plant taxonomy largely due to the complex variation patterns of characters [ 2 , 3 ]. Historically, morphological characters were widely used to divide taxa, and unravel the relationships within the genus. This has resulted in continuously taxonomic re-arrangements [ 1 – 4 , 10 – 16 ]. And infra-generic classifications dividing the genus into subgenera, sections or series were subsequently proposed. Among them, the generally accepted one comprises five subgenera, including subg. Absinthium (Miller) Less., subg. Artemisia , subg. Dracunculus (Besser) Rydb., subg. Seriphidium Besser ex Less., and subg. Tridentatae (Rydb.) McArthur, mainly based on the types of capitula, fertility of disc florets, and hairy of receptacles [ 3 , 4 ]. However, phylogenetic relationships revealed by several studies using a limited number of molecular markers (e.g., ITS, psbA – trnH , rpl32 – trnH , trnL – trnF , trnS – trnC ) were to some degree incongruent with these morphological divisions [ 5 , 17 – 20 ]. Among the subg. Absinthium , subg. Dracunculus , subg. Tridentatae , and subg. Seriphidium , none was monophyletic. Species previously assigned to subg. Artemisia were scattered into several clades. Furthermore, Hobbs & Baldwin found three Hawaiian endemic species ( A. australis , A. kauaiensis and A. mauiensis ) together with A. chinensis clustered into one single clade, and they thus proposed the sixth subgenus, subg. Pacifica , to accommodate these species [ 5 ]. Using nuclear single nucleotide polymorphism (SNP) data obtained by genome-skimming sequencing technology, Jiao et al. reconstructed a phylogeny for Artemisia consisting eight main clades, and accordingly they proposed a revised clade-based infra-generic classification dividing the genus into eight subgenera [ 21 ]. To some extent, these discordances about the infra-generic relationships reflect the complex evolutionary history of Artemisia. Plastid (Chloroplast), commonly found in plants and green algae, are important in plant growth and development [ 22 – 24 ]. Typically, the plastome (plastid genome) is a closed loop with a quadripartite structure containing a large single copy region (LSC), a small single copy region (SSC), and two inverted repeat (IRa and IRb) sequences, is 120–200kb in length. Each genome tends to contain approximately 80 protein-coding genes, 4 rRNAs, and 30 tRNAs [ 25 , 26 ]. Due to its small size, uniparental inheritance, conserved sequence and structure, and high cellular copy number, the plastome has been an advantageous resource for various evolutionary studies [ 27 ]. Previously, some plastid genes ( rbcL , matK ) have been extensively used to estimate phylogenetic relationships at deep and shallow levels [ 26 – 30 ]. Some faster evolving genes (i.e., matK , ndhF , rbcL and rpoC1 ) and spacer regions (i.e., atpF - atpH , psbK - psbI , and trnH - psbA ) have even been developed as DNA barcode markers to identify taxa [ 31 ]. In Artemisia , only a few plastid regions (i.e. psbA – trnH , rpl32 – trnH , trnS – trnC ) concatenated with nuclear regions (ITS and ETS) were used to construct the generic or infra-generic phylogenetic relationships, and to explore the evolutionary history of the genus [ 5 – 7 , 17 – 20 , 32 ]. With the advancement of next generation sequencing (NGS) technologies and the decrease in sequencing cost, it is becoming much easier to obtain complete plastome sequences. The plastome data has exhibited greater potential for resolving challenging phylogenetic relationships in a wide spectrum of plant lineages e.g., Eriocaulon (Eriocaulaceae), Trigonotis (Boraginaceae), Apocynaceae, and Ophioglossaceae [ 26 – 30 , 33 , 34 ], and numerous historically difficult issues in plant phylogenetics have been satisfactorily addressed, indicating the plastomes play an indispensable role in plant phylogenetics. For Artemisia , Kim et al. first conducted a comparative analysis of plastomes of 32 Artemisia species in East Asia [ 35 ]. The results revealed that the coding sequences of accD and ycf1 were under weak positive selection, and highly variable. The plastomes were sufficiently polymorphic to be used as super-barcodes. They further confirmed that subg. Artemisia was not monophyletic. Using a plastome data matrix of 38 species, including 18 species from subg. Seriphidium , Jin et al. found that subg. Seriphidium segregated into two main clades [ 36 ]. Furthermore, their structural analysis indicated that the plastomes are relatively conserved with some variations only in IR borders. As of May 2024, only those of 47 species, two varieties and one form were deposited at the National Center for Biotechnology Information (NCBI). In contrast with the large number of taxa in Artemisia , the percentage of sequenced plastomes did not match well the Artemisia biodiversity. There are still gaps in our knowledge of the general variation pattern of Artemisia plastomes especially their structure, gene order, IR/SC boundary, and IR expansion. A finer-scale phylogenetic relationship constructed using plastid data with more informative characters and denser taxon sampling is still lacking. And comparisons between phylogenies constructed using data from nuclear DNA and plastomes to explore whether there is any cyto-nuclear (i.e., chloroplast–nuclear) discordance are also in need. Considering this situation, we newly sequenced, assembled and annotated 37 Artemisia plastomes of 31 species and three varieties in this study. Combining with 38 previously published Artemisia plastomes from public database, we conducted comparative analyses, and constructed phylogenies in order to: (1) study the plastome variation patterns of this genus; (2) identify variable regions as DNA barcode candidates for future taxon identification; (3) recover the backbone of the Artemisia phylogeny using plastome-scale data set. Overall, this study will improve our knowledge of Artemisia plastomes, provide potential genetic markers for taxa identification, and also advance our understanding on the phylogenetic relationships within the genus. Results Plastome features of Artemisia species A total of 72 Artemisia plastomes were included in this study, representing 63 species, three varieties and one form (Tables 1 , S1, S2). Among them, 34 plastomes of 29 species and three varieties were newly sequenced and assembled (Tables 1 , S1). All plastomes showed a typical quadripartite structure, including a large single copy (LSC), a small single copy (SSC), and two inverted repeated (IRa/b) regions (Fig. 1 ). Their size ranged from 150586 bp ( A. ferganensis ) to 151327 bp ( A. smithii ), with a difference of 741 bp and the mean length of 151108 bp. The length of LSC, SSC and IR regions were 82313–83061 bp, 17735–18883 bp, 24927–24985 bp, respectively. The total GC content ranged from 37.40–37.51% with the mean value of 37.50%. The gene categories were rather conserved. A total of 132 genes, including 87 protein coding genes, 37 tRNA genes, and eight rRNA genes, were comprised in every plastome. The detailed information of these plastomes was provided in Table 1 and Supplementary Tables S1 and S2. Table 1 Summary of the newly sequenced plastomes for 33 Artemisia samples. Taxon Genbank No. Nucleotide length (bp) Number of genes GC content Total LSC SSC IR Protein coding genes rRNA genes tRNA genes (%) A. sichuanensis 151230 82935 18375 24960 87 37 8 37.50% A. adamsii 151231 82955 18337 24969 87 37 8 37.40% A. bhutanica 151269 82949 18398 24961 87 37 8 37.50% A. blepharolepis 150908 82963 18019 24963 87 37 8 37.50% A. comaiensis 151298 82930 18448 24960 87 37 8 37.50% A. fulgens var. meiguensis 151201 82856 18425 24960 87 37 8 37.50% A. gyitangensis 151199 82810 18471 24959 87 37 8 37.50% A. integrifolia 151117 82912 18285 24960 87 37 8 37.40% A. jilongensis 151275 82538 18883 24927 87 37 8 37.50% A. linyoureunensis 151174 82718 18594 24931 87 37 8 37.50% A. mairei 151045 82911 18200 24967 87 37 8 37.50% A. mattfeldii var. etomentosa 151087 82929 18238 24960 87 37 8 37.50% A. minor 151083 82556 18673 24927 87 37 8 37.50% A. mongolica 151193 82795 18478 24960 87 37 8 37.50% A. neosinensis 151172 82832 18422 24959 87 37 8 37.50% A. nortonii 151028 82873 18235 24960 87 37 8 37.50% A. phyllobotrys 151171 82931 18312 24964 87 37 8 37.50% A. selengensis -1 151228 82902 18406 24960 87 37 8 37.50% A. sericea 150870 82852 18094 24962 87 37 8 37.50% A. smithii 151225 83037 18272 24958 87 37 8 37.40% A. stracheyi 151327 82937 18470 24960 87 37 8 37.50% A. stricta 150770 82911 17939 24960 87 37 8 37.50% A. sylvatica 151200 82982 18296 24961 87 37 8 37.40% A. tafellii 151274 82937 18417 24960 87 37 8 37.50% A. tangutica 151191 82833 18452 24953 87 37 8 37.50% A. tournefortiana 151106 82600 18606 24950 87 37 8 37.50% A. tridactyla 150654 82810 17946 24949 87 37 8 37.50% A. viscidissima 151241 82655 18622 24982 87 37 8 37.50% A. waltonii 151038 82909 18209 24960 87 37 8 37.50% A. waltonii var. yushuensis 150636 82981 17735 24960 87 37 8 37.40% A. youngii 151249 82948 18363 24969 87 37 8 37.40% A. yunnanensi -1 151190 82987 18283 24960 87 37 8 37.40% A. yunnanensis -2 151232 82977 18335 24960 87 37 8 37.40% The boundaries between IR and SC regions were compared in 66 Artemisia plastomes representing 62 species, three varieties and one form. All of them have the same type of SC/IR junctions (Figs. 2 , S1). The LSC/IRb junction borders (JLB) were located in the gene rps19 , and four types were recovered. The length of rps19 in LSC was 207–219 bp, and 60–72 bp in IRb region. The dominant type is 219 bp in LSC region and 60 bp in IRb region. The SSC/IRa junction borders (JSA) was located in the gene ycf1 , with 4428–4500 bp in SSC region and 556–565 bp in IRa region. At the IRb/SSC junction borders (JSB), the distances between the gene ndhF and the border range were 42–82 bp. At the LSC/IRa junction borders (JLA), the distances between the gene trnH and the border range were 2–135 bp. The IR regions are highly conserved and similar in length and structure. Additionally, no gene rearrangements, inversions, or losses among these plastomes were found. Plastome sequence divergence The sequence divergence of 23 plastomes was analyzed using mVISTA program, with Artemisia sieversiana (Genbank Accession Number: ON729303) as reference. The Artemisia plastomes are rather conserved (Fig. 3 ). The generic regions are more conserved than intergenic spacer regions, and sequence divergence is higher in LSC and SSC than IR regions. Nucleotide polymorphism (Pi) values show very similar results on sequence divergence (Fig. 4 ). Most of 79 CDSs are rather conserved, with Pi values lower than 0.002, while three ( accD , petG , and ycf1 ) have Pi values between 0.004 and 0.006, and the remaining nine CDSs have Pi values lower than 0.004. Most of the genes with high Pi values (≥ 0.002) are located in the single copy regions (Fig. 4 A, Table S3 ). The non-coding regions exhibit higher nucleotide variability (Fig. 4 B, Table S3 ). The regions ndhG - ndhI , trnG (GCC)- trnfM (CAU), and rpoC2 - rps2 have Pi values higher than 0.06, the Pi value of rpoC1 - rpoC2 is between 0.03 and 0.04, and the others have Pi values lower than 0.03. In IR regions, non-coding regions are highly conserved. Simple sequence repeats (SSRs) in Artemisia plastomes Repeated DNA sequences are important in genome rearrangement [ 39 ]. We investigated simple sequence repeats (SSRs) in the alignment of 72 Artemisia plastomes. In total, 4886 SSRs were detected. The numbers of SSRs varied from 58 to 77 in each plastome. Four plastomes (i.e., A. finita , A. kaschgarica , A. fukudo , and A. tournefortiana ) have more SSRs than others (Fig. 5 , Table S4 ). Mononucleotide repeats are the most abundant (2754, 56.4%), followed by tetra- (987, 20.2%), di- (692, 14.2%), tri- (318, 6.5%), penta- (128, 2.6%), and hexa-nucleotide (7, 0.1%) repeats (Table S4 ). The mono-, di-, tri-, and tetra-nucleotide repeats were found in all plastomes, and penta-nucleotide repeats in 67 plastomes, and hexa-nucleotide repeats only in seven plastomes representing six species and one form, including A. blepharolepis , A. finita , A. freyniana f. discolor , A. fukudo , A. linyoureunensis , A. smithi i, and A. yunnanensis . Most SSRs were located in single copy regions with 3794 in LSC and 673 in SSC regions. Only 418 SSRs were in IR regions. Mono-nucleotide repeats may play an important role in genetic variation than other SSRs types. The A/T repeats account for nearly 97.8% of the mono-nucleotide repeats, and this result is similar with other studies. Di-nucleotide repeats are represented only by AT/TA motif. The detailed information of SSRs in each plastome was provided Supplementary Table S4 . Phylogenetic analysis The topologies of phylogenetic trees constructed from maximum likelihood (ML) and Bayesian inference (BI) methods were basically similar (Figs. 6 , S2, S3). All samples of Artemisia were clustered into one single clade, which was sister to the outgroup Ajania – Chrysanthemum clade. The genus Artemisia was split into two clusters. The basal one (here referred to as Clade 1) further divides into two well supported (ML bootstrap value (BS) = 100%; Bayesian posterior probabilities (PP) = 1) subclades, with one subclade comprising two samples of A . annua and one of A . fukudo , and another subclade comprising 17 of subg. Seriphidium. Another cluster (here referred to as Clade 2) divides into three main subclades, including one subclade comprising only Artemisia stracheyi , one comprising ten samples of subg. Dracunculus and two of A . selengensis of subg. Artemisia , and the remaining one subclade comprising all other samples. However, the Clade 2 was not strongly supported (BS = 81, PP = 0.89). Consistent with previous phylogenetic studies using nuclear markers, our results also confirmed that all the four subgenera, subg. Absinthium , subg. Artemisia , subg. Dracunculus and subg. Seriphidium sampled in this study, were not monophyletic (Figs. 6 , S2, S3). Most of the species of subg. Artemisia were formed a monophyletic group, and the rest species were scattered several clades. The phylogenetic position of A . juncea of subg. Seriphidium was not resolved. The remaining species of subg. Seriphidium formed a monophyletic group. And the subg. Dracunculus was also monophyletic when A . blepharolepis was excluded. Only three species of subg. Absinthium were sampled, including A. sieversiana , A. minor , and A. sericea , and they formed one clade with A. juncea of subg. Seriphidium and A. tournefortiana of subg. Artemisia . Disscussion Characteristics of plastomes and genetic variation of Artemisia To understand the plastome structural variation pattern of Artemisia , a denser sampling within the genus is inevitable. In this study, a total of 72 plastomes newly sequenced or downloaded from public database were comparatively analyzed. Nearly consistent with previous studies, the Artemisia plastomes showed a high degree of similarity in terms of GC content, configuration, gene number and order [ 35 – 38 ]. GC content variation along genomes is a key feature of genomic organization and strongly varies between species. It is usually associated with fundamental elements of genome organization, e.g., recombination [ 39 – 43 ]. In the Artemisia plastomes, GC content is not significantly varied between different species, and ranges from 37.40–37.51%, which is typical in angiosperm plastomes [ 41 ]. In fact, no genome rearrangement has been found in these samples. These also reflect that Artemisia plastomes are rather conservative. In general, the length of Artemisia plastomes also fall within the average length range of eudicots [ 22 ]. Sequence length uniformity was found between different samples of the same species, e.g., A . annua . It is more common that different samples of the same species have different sequence length, e.g., A. argyi , A. lancea , and A. selengensis . Three factors have been proposed to drive the difference in plastome length, including intergenic region variation, difference in gene, and the expansion and contraction of IR regions [ 40 ]. All the plastomes are quadripartite, containing the same number of genes, including 87 protein coding, 37 tRNA, and eight rRNA genes. The 66 plastomes analyzed using CPJSdraw belong to the same IR/SC boundary type. The IR regions only varied 58 bp, LSC regions varied 748 bp, and SSC regions varied 148 bp. Thus the variations in Artemisia plastome length were mainly in LSC regions. Previous analyses of whole plastomes revealed that the plastid regions, accD , ndhF , trnT , ycf1 , rpl32 - trnL , trnE - ropB , trnH - psbA , trnK - rps16 , ndhC - trnV , and ndhG - ndhI are highly variable [ 35 – 38 ]. As pointed out by Shaw et al., the plastid region might not be consistently variable across different groups [ 44 ]. In this study, accD , petG , ycf1 , ndhG - ndhI , trnG (GCC)- trnfM (CAU) and rpoC2 - rps2 have higher variability, and were identified as mutational hotspots for Artemisia plastomes. Several plastid regions including rpl32 – trnH , trnS – trnC have been used to construct the phylogeny of Artemisia [ 19 , 36 ]. However, we found that these regions were not the most informative regions. This may have limited the power of resolving the phylogenetic relationships within the genus. The combination of rbcL and matK was recommended as a core plant barcode by the CBOL Plant Working group [ 31 ]. But the Pi values of rbcL and matK were both lower than 0.002, indicating they have a rather limited discriminative power in Artemisia . Plastid accD and ycf1 are important for plant fitness and leaf development. As observed in other plant groups, the accD and ycf1 have high variable nucleotide sequences in the plastomes analyzed in this study. The genus Artemisia is morphologically complex, and species identification is rather difficult. These hotspot regions could be developed as DNA barcode and used to distinguish taxa. Phylogenetic relationships of Artemisia A well resolved phylogenetic relationship is critical for a better understanding of evolutionary patterns and process of plants at different ranks, especially for the large and morphologically variable group as Artemisia [ 45 ]. In this study, we constructed the phylogeny for Artemisia using the protein coding genes (CDS) of plastomes with a broad taxonomic sampling. Consistent with previous studies, our results further confirmed the conflicts between the morphological infra-generic division of Artemisia and the molecular phylogenetic relationships. All the subgenera sampled here, including subg. Absinthium , subg. Artemisia , subg. Dracunculus , and subg. Seriphidium , were not supported as monophyletic. The subg. Artemisia which is the largest in Artemisia , however, is polyphylotic, with the sampled taxa clustered into several clades. In his treatment of Chinese Artemisia , Ling divided subg. Artemisia into two sections, sect. Artemisia and sect. Abrotanum [ 45 ]. This division was also not supported by our and other phylogenetic studies. As indicated by earlier molecular analyses of Artemisia , the subg. Seriphidium once considered as a morphologically independent genus, Seriphidium (Bess.) Poljak., was strongly supported to include in Artemisia [ 36 ]. The subg. Seriphidium was traditionally considered as monophyletic [ 2 , 46 ]. However, in our phylogenetic reconstruction, this subgenus was divided into two clades: one large monophyletic group and another clade including only one species, A . juncea . The subg. Dracunculus was monophyletic when two samples of A . selengensis belonging to subg. Artemisia were included. Additionally, our analyses also revealed that there exists cyto-nuclear phylogenetic discordance, especially the topology position of subg. Dracunculus and subg. Seriphidium . The phylogenetic topologies of Artemisia recovered by previous studies using nuclear loci, including ETS and ITS, and nuclear single nucleotide polymorphisms (SNPs) used by Jiao et al., are somewhat similar [ 21 ]. The species of subg. Dracunculus together with some species of subg. Artemisia constituted the early divergent clade within Artemisia . The remaining taxa were further clustered into two main clades. Most species of subg. Seriphidium together with some species of subg. Absinthium , and subg. Artemisia formed a clade sister to another clade formed mainly by species of subg. Artemisia and subg. Absinthium . The phylogenetic relationships constructed using CDS of plastomes revealed a somewhat different topology. The earliest diverging clade of Artemisia was constituted by all the species of subg. Seriphidium except A . juncea , together with A . annua and A . fukudo of subg. Artemisia . The other species were clustered into one clade which could be further divided into two main clades. One includes all samples of subg. Dracunculus excluding A . blepharolepis , and two samples of A . selengensis , and the other was constituted by most species of subg. Artemisia , and some species of subg. Absinthium , A. juncea of subg. Seriphidium , and A . blepharolepis of subg. Dracunculus . This topology was also revealed by Jin et al. As reported by previous studies, cytonuclear discordance is commonly observed phenomenon in phylogenetic constructions [ 47 , 48 ]. And in Artemisia , the discordance may reflect the frequent hybridization and introgression. The phylogenetic position of Artemisia stracheyi was still controversial. This species was originally described as new in Artemisia , and recorded to occur in Tibet and adjacent regions [ 49 ]. Ghafoor thought A. stracheyi differs remarkably from the genus Artemisia in several morphological characters, including corolla and ovary densely scaly, stamens included, apical anther appendages triangular-ovate, obtuse, achenes quadrangular-pyramidate [ 50 ]. They thus proposed a new genus, Artemisiella Ghafoor, to accomodate this species, and accordingly published a new combination, i.e., Artemisella stracheyi (C.B. Clarke) Ghafoor. This treatment was not generally accepted by later authors [ 1 , 2 ]. Jiao et al. for the first time sampled this species in their phylogenetic study based on nuclear genome SNPs, and found that Ajania quercifolia and Artemisiella stracheyi consisted a clade sister to Artemisia , they thus accepted the treatment proposed by Ghafoor [ 21 , 50 ]. However, our results based on CDS of plastomes indicated that Artemisiella stracheyi (= Artemisia stracheyi ) nested within Artemisia , and formed an independent clade. Morphologically Artemisiella stracheyi is rather unique in the genus Artemisia by having 2- or 3-pinnatisect leaves with lobules narrowly linear, large (6–10 mm in diam.) involucre, and deciduously pubescent receptacle. This morphological and phylogenetic discordance may reflect the complex evolutionary history of A. stracheyi . So in the near future, phylogenetic analyses using plastome and nuclear data with denser sampling and more molecular data, combing with evidence from morphological, cytological, geographical, and ecological studies, are needed to reveal its evolutionary history of A. stracheyi , and determine its phylogenetic position. As mentioned before, Artemisia is such a large, complex, and economically important taxon. It should remain a priority for taxonomical and evolutionary studies, even though these tasks are rather challenging [72]. In this study, we newly sequenced 34 Artemisia plastomes, but the taxon sampling is still inadequate, especially taxa of subg. Pacifica and subg. Tridentatae which were mainly distributed in Hawaiian Islands and North America, respectively. Using these available plastomes, we constructed a phylogenetic backbone for Artemisia . Based on adding more representative taxon sampling to this phylogenetic backbone, the future studies could more comprehensively reveal the phylogeny of Artemisia . Conclusions In this study, we newly sequenced 34 plastomes representing 29 species and three varieties of Artemisia , and obtained 38 previously published plastomes data representing 34 species and one form. Comparative analyses indicated that the Artemisia plastomes are conservative in structure, gene number and order. The IR regions are similar in length and structure among plastomes compared. Three protein-coding genes and four non-coding regions were found to be highly diverse: accD , petG , ycf1 , rpoC1 - rpoC2 , rpoC2 - rps2 , trnG (UCC)- trnfM (CAU), and ndhG - ndhI . These can be chosen as the candidates of DNA barcoding marker for taxon identification. The phylogenetic relationships constructed using protein coding genes further confirmed the infra-generic divisions of Artemisia were not natural. Previously divided four subgenera were not monophyletic. In the future, phylogenetic relationships constructed using plastomes with denser sampling, and comparisons with those of nuclear markers are still needed. Materials and Methods 5.1 Taxa sampling, DNA extraction and illumina sequencing In this study, we newly sequenced 34 plastomes representing 29 species and three varieties of Artemisia (Table 1 ). Detailed information of taxa, voucher specimen, collection locality was provided in Supplementary Table S1 . The materials were collected during our field trips in China. The voucher specimens were all identified by Xinqiang Guo, the first author of this study, and deposited in Herbarium of South China Botanical Garden, Chinese Academy of Sciences (IBSC). In addition, we downloaded 38 Artemisia plastomes representing 34 species and one form from NCBI Genbank database ( https://www.ncbi.nlm.nih.gov/nuccore/ , as of May 1st, 2024) (Supplementary Table S2 ). A total of 72 Artemisia plastomes were obtained and used in comparative plastome analysis. For phylogenetic analyses, Ajania fruticulosa , Ajania nematoloba , Ajania khartensis , and Chrysanthemum przewalskii were selected as outgroups. Fresh leaves were dried with silica gel and kept in − 80 ℃ refrigerator. High quality total genomic DNA of plant samples was extracted from 10 mg silica gel-dried leaves using a modified CTAB (cetyl trimethyl ammonium bromide) DNA extraction method [ 51 ]. The DNA samples were sent to Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China), and a 150-bp paired-ended library with an average insert size of approximately 400 bp was prepared according to the manufacturer’s manual (Illumina, San Diego, CA, USA), and shotgun sequencing was performed on the Illumina NovaSeq 6000 platform. Approximately 3 Gb of raw reads were generated for each sample. 5.2 Plastome assembly and annotation Trimmomatic v.0.40 [ 52 ] was used to remove adapters and filter low-quality reads. NOVOPlasty v2.5.9 [ 53 ] and GetOrganelle pipeline v1.7.2a [ 54 ] were used to de novo assemble plastomes with suggested default parameters, using the complete plastome DNA sequence of Artemisia annua (Genbank Accession Number: KY085890.1) as a reference. The obtained scaffolds were checked using Bandage v0.8.1 [ 55 ]. Assembled plastomes were annotated by using program GeSeq [ 56 ] and Plastid Genome Annotator (PGA) [ 57 ] with A. annua (GenBank accession no.: KY085890.1) as a reference. To precisely define the start and stop codons, intron boundaries, and tRNA genes, annotations were manually adjusted according the reference plastome in Geneious Primer v2021.0.3 (Biomatters Ltd., Auckland, New Zealand). The 34 newly sequenced plastomes were deposited in GenBank database (Table S1 ). The raw data and plastome sequences downloaded from NCBI were re-assembled and re-annotated following the procedures of the newly sequenced samples. The circular plastid genome maps were visualized using OrganellarGenome DRAW v1.3.1 [ 58 ]. 5.3 Comparative analyses, identification of divergence hotspots and simple sequence repeats We selected 23 plastomes representing 22 species and one variety of Artemisia (Supplementary Table S2 ) to conduct plastome comparisons using the mVISTA program with the Shuffle-LAGAN mode [ 59 ]. The annotation of A. sieversiana (GenBank accession No.: ON729303) was chosen as reference. To identify potential hotspots of nucleotide diversity in 72 Artemisia plastomes, 79 CDS were extracted, and aligned using MUSCLE v. 3.8.31 [ 60 ] with default parameters. Then, Nucleotide diversity (Pi) was estimated using DnaSP v. 6 [ 61 ] with window length set as the whole length of each matrix (Supplementary Table S3 ). To have a comprehensive overview of the IR expansion or contraction in the Artemisia plastomes, we selected 66 plastomes and visualized the borders of IR/SC regions using CPJSdraw v1.0.0 [ 62 ]. Simple sequence repeats (SSRs) of 72 plastomes (Supplementary Table S4 ) were identified using the MISA-web (MicroSAtellite; https://pgrc.ipk-gatersleben.de/misa/ ) [ 63 ] with the threshold repeat numbers of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively. 5.4 Phylogenetic analysis Phylogenetic analyses were based on 75 plastomes, including 72 of Artemisia , three of Ajania , and one of Chrysanthemum . 79 unique CDS of these plastomes were extracted using using PhyloSuit v7.3.1 [ 64 ] and Geneious Primer v2021.0.3. The data sets were aligned using MUSCLE v. 3.8.31 [ 60 ], and manually adjusted using AliView v1.26 [ 65 ]. All the individual CDS matrices were concatenated into a single super-matrix using Geneious Primer v2021.0.3. PartitionFinder 2 [ 66 ] was used to determine the best-fit partitioning scheme and the most suitable substitution model. Bayesian phylogenies were constructed using MrBayes v3.2.7a [ 67 ]. Two parallel analyses each four chains (one cold and three hot chains) were run for 40 million Markov Chain Monte Carlo (MCMC) generations with trees sampled every 1000 generations. The first 25% sampled trees were discarded as burn-in. The remaining trees were used to estimate the posterior probabilities (PP). Tracer v.1.6 [ 68 ] was used to ensure convergence and adequate sampling with the average standard deviation of split frequencies 200. The maximum likelihood (ML) analysis were carried out in RAxML-HPC v8.2.12 [ 69 ], with 1000 bootstrap replicates using a fast bootstrapping algorithm (MLBS), to assess node support. Bootstrap percentage (MLBS and MPBS) values ≥ 70 and PP values ≥ 0.95 were regarded as strong support. The final tree files were visualized in FigTree v1.4.3 ( https://tree.bio.ed.ac.uk/software/figtree/ ) and TreeGraph v2.15.0-887 beta [ 70 ]. Declarations Acknowledgments We thank Long Wang of South China Botanical Garden, Chinese Academy of Sciences for his assistance during the field work, and providing us Artemisia samples. Authors’ contributions MJY and XQG planned the projects, designed the research, analyzed data, and wrote the manuscript. DWX and YHW planned the projected. All authors have read and approved the manuscript. Funding This work was supported by the National Natural Science Foundation of China (32270215), and the Natural Science Foundation of Zhejiang Province of China (LQ24C020002). Availability of data and materials All the plastomes newly sequenced and annotated in this study are deposited in the National Center for Biotechnology and Information (NCBI) under the accessions as summarized in Table 1 and Supplementary Table S1. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China. 2 Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou 311121, China. Data Availability Statement: The data presented in this study are available upon request from the first author and corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References Ling YR, Humphries CJ, Gilbert MG. Artemisia Linnaeus. In: Wu ZY, Raven PH, Hong DY, editors. Flora of China, vol. 20–21. Beijing: Science Press, and St. Louis: Missouri Botanical Garden Press; 2011. pp. 676–737. Ling YR. Artemisia L. In: Ling Y, Ling YR, editors. Flora Reipublicae Popularis Sinicae. Volume 76. 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Supplementary Files FIGURES1.tif FIGURES2.jpg FIGURES3.jpg Supplementarylegends.docx TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4573083","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":324842279,"identity":"69c50b7d-e5b1-4018-9fef-21ebd58681f2","order_by":0,"name":"Xinqiang Guo","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xinqiang","middleName":"","lastName":"Guo","suffix":""},{"id":324842284,"identity":"14c06520-f199-44b2-84ae-b945e4b192d4","order_by":1,"name":"Dawei Xue","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Xue","suffix":""},{"id":324842290,"identity":"177b56a7-33f9-486b-97f3-9b0c3eabe6e3","order_by":2,"name":"Yuhuan Wu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuhuan","middleName":"","lastName":"Wu","suffix":""},{"id":324842294,"identity":"56f95eb0-0935-4b8a-92fa-856a3378bc3c","order_by":3,"name":"Mengjie Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYLACCQMQyXwMyk0gWgtbGglaIIDHjDgtuu29h19YFNgwGNzI+fbw547DDPzsOQYMP3fg1mJ25lyahYRBGlBL7nYDyTOHGSR73hgw9p7Bo+VGjpmBhMFhkJZtEoZtIEaOATNjGx4t99+AtPwHqXwmkQjUYk9Qyw0e4wcSBgdAWtgkDoJskSCk5UyOGTCQk3kkzzwzk2xsS+eROPOs4GAvPi3Hzxh/lvhjJ8d3PPmZ5M82azn+9uSND37i0QIEbNISwEhROADh8YCIA3g1ABPKxw9AUr6BgLJRMApGwSgYuQAA/3hPWSsI8YYAAAAASUVORK5CYII=","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":true,"prefix":"","firstName":"Mengjie","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-06-13 02:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4573083/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4573083/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60371984,"identity":"80949bf4-9277-44a1-b620-97ff3612640e","added_by":"auto","created_at":"2024-07-16 05:19:32","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":752090,"visible":true,"origin":"","legend":"\u003cp\u003eGene map of the \u003cem\u003eArtemisia tafellii\u003c/em\u003e chloroplast genome. Genes shown inside the circle are transcribed clockwise, and those shown outside are transcribed counter-clockwise. Genes belonging to different functional groups are shown in different color. The darker gray color in the inner circle corresponds to the GC content, and the lighter gray color corresponds to the AT content. IR, inverted repeat region; LSC, large single copy; SSC, small single copy.\u003c/p\u003e","description":"","filename":"FIGURE1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/377a09839d63e75d6455c3cd.jpg"},{"id":60371235,"identity":"5eb4887c-f6d4-4b01-8693-03c3924a00bc","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":578529,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the borders of large single-copy (LSC), inverted repeat (IR), and small single-copy (SSC) regions among six \u003cem\u003eArtemisia\u003c/em\u003egenomes. JLB (IRb /LSC), JSB (IRb/SSC), JSA (SSC/IRa) and JLA (IRa/LSC) denote the JSs between each corresponding region in the genome.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/eab473bdf723132d99325c01.png"},{"id":60371236,"identity":"1f164178-2277-458b-b798-78845b46a702","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7348633,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment of 23 \u003cem\u003eArtemisia\u003c/em\u003e plastomes using mVISTA program with \u003cem\u003eA. sieversiana\u003c/em\u003e as reference. The X-axis indicates the sequence length, and the Y-axis indicates the percentage identity, ranging from 50 to 100%. Gray arrows below the genes denote the gene orientation. Bars below the X-axis show the gene position of the plastome region.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/07b61f2f44d520e72ce1f17a.png"},{"id":60371985,"identity":"d0b455d1-a53a-46fc-b01e-4327a7194660","added_by":"auto","created_at":"2024-07-16 05:19:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":605605,"visible":true,"origin":"","legend":"\u003cp\u003eThe nucleotide variability (Pi) values in the 72 \u003cem\u003eArtemisia\u003c/em\u003e plastomes. a. Intergenic regions. b. Protein-coding genes. These regions are arranged according to their location in the plastome.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/bde96d8ba62e0e9d204cf566.png"},{"id":60371244,"identity":"03ebc6f6-f757-4191-8c8c-8150e228994d","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":266505,"visible":true,"origin":"","legend":"\u003cp\u003eThe numbers of the simple sequence repeats (SSRs) in the plastomes of \u003cem\u003eArtemisia\u003c/em\u003e species. mono-, mononucleotides; di-, dinucleotides; tri-, trinucleotides; tetra-, tetranucleotides; penta-, pentanucleotides; hexa-, hexanucleotides.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/28e738d0c80cee15b71c5d16.png"},{"id":60371243,"identity":"d990817c-ea11-4505-9cf3-ca5971060d4d","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":764243,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny of \u003cem\u003eArtemisia\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ebased on 79 protein coding sequences (CDS) of 72 \u003cem\u003eArtemisia\u003c/em\u003e species and four outgroups. a. Consensus phylogenetic tree reconstructed by Bayesian inference (BI) analysis. Numbers near the branches are bootstrap support (BS) percentages obtained from maximum likelihood inference and posterior probabilities (PP) obtained from Bayesian analysis (BS/PP). b. Phylogenetic backbone reconstructed by maximum likelihood (ML) inference.\u003c/p\u003e","description":"","filename":"FIGURE6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/c24726f39eb5363cdda8bd29.jpg"},{"id":101258635,"identity":"9b728326-2999-4f72-9159-91b2e9e5b596","added_by":"auto","created_at":"2026-01-27 19:40:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5998681,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/0eb80637-f197-4383-92b9-9b6c0d768105.pdf"},{"id":60371241,"identity":"2549f859-635d-494c-8a6b-4d127e28c57c","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18422732,"visible":true,"origin":"","legend":"","description":"","filename":"FIGURES1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/de8f92104d581fd927dc9969.tif"},{"id":60371238,"identity":"7c7e179c-6525-4745-968b-7ef31470f4e0","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":115574,"visible":true,"origin":"","legend":"","description":"","filename":"FIGURES2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/a8a09ff0d879c7137a7ebf1b.jpg"},{"id":60371986,"identity":"f2edf6ef-ef8e-429d-9e25-2348db993c27","added_by":"auto","created_at":"2024-07-16 05:19:32","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":115148,"visible":true,"origin":"","legend":"","description":"","filename":"FIGURES3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/22d390fd71aa8af73f33866c.jpg"},{"id":60371239,"identity":"d8c96ed7-b6d8-48b7-ba5c-ea426ea34fc4","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13991,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarylegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/c22e8d46b2515b77d63ca177.docx"},{"id":60372703,"identity":"bf4017cd-e9bf-4502-ba8d-b62a1e97013d","added_by":"auto","created_at":"2024-07-16 05:27:32","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12608,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/6912d9ecc29c04483a31002e.xlsx"},{"id":60371247,"identity":"f3b166eb-8f46-4b42-acce-1cfe2c15618b","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13088,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/a9fd11dc20ac79bfc19324af.xlsx"},{"id":60371988,"identity":"1ddac31d-a726-4775-a218-1f0d3a5bfcce","added_by":"auto","created_at":"2024-07-16 05:19:32","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15287,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/a6bd4ccf5643d2be404fd372.xlsx"},{"id":60371248,"identity":"0666c6b7-3704-486d-aa97-640677efd0ff","added_by":"auto","created_at":"2024-07-16 05:03:32","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":264275,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4573083/v1/1379528c1762c1ff865d5895.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative analysis of Artemisia plastomes and insights into the infra-generic phylogenetic relationships of the genus","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eArtemisia\u003c/em\u003e is the largest genus of tribe Anthemideae in Asteraceae, comprising 400\u0026ndash;500 species [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Members of this genus are mainly distributed in northern hemisphere, with a few species occurring in Africa, South America and Hawaiian Islands [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eArtemisia\u003c/em\u003e is economically important. Members of this genus, i.e., \u003cem\u003eA. argyi\u003c/em\u003e H. L\u0026eacute;v. \u0026amp; Vaniot, and \u003cem\u003eA. capillaris\u003c/em\u003e Thunb., have been widely used as herbal remedies in China, and some species have broad applications as food, or forage. And the most famous one undoubtedly is \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eannua\u003c/em\u003e L. The discovery of the anti-malaria artemisinin from it was awarded a Nobel Prize in Physiology or Medicine in 2015 [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus \u003cem\u003eArtemisia\u003c/em\u003e receives extensive scientific attention, especially in the fields of phytochemistry and pharmacology.\u003c/p\u003e \u003cp\u003e \u003cem\u003eArtemisia\u003c/em\u003e represents one of the most notoriously difficult groups in plant taxonomy largely due to the complex variation patterns of characters [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Historically, morphological characters were widely used to divide taxa, and unravel the relationships within the genus. This has resulted in continuously taxonomic re-arrangements [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. And infra-generic classifications dividing the genus into subgenera, sections or series were subsequently proposed. Among them, the generally accepted one comprises five subgenera, including subg. \u003cem\u003eAbsinthium\u003c/em\u003e (Miller) Less., subg. \u003cem\u003eArtemisia\u003c/em\u003e, subg. \u003cem\u003eDracunculus\u003c/em\u003e (Besser) Rydb., subg. \u003cem\u003eSeriphidium\u003c/em\u003e Besser ex Less., and subg. \u003cem\u003eTridentatae\u003c/em\u003e (Rydb.) McArthur, mainly based on the types of capitula, fertility of disc florets, and hairy of receptacles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, phylogenetic relationships revealed by several studies using a limited number of molecular markers (e.g., ITS, \u003cem\u003epsbA\u003c/em\u003e\u0026ndash;\u003cem\u003etrnH\u003c/em\u003e, \u003cem\u003erpl32\u003c/em\u003e\u0026ndash;\u003cem\u003etrnH\u003c/em\u003e, \u003cem\u003etrnL\u003c/em\u003e\u0026ndash;\u003cem\u003etrnF\u003c/em\u003e, \u003cem\u003etrnS\u003c/em\u003e\u0026ndash;\u003cem\u003etrnC\u003c/em\u003e) were to some degree incongruent with these morphological divisions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Among the subg. \u003cem\u003eAbsinthium\u003c/em\u003e, subg. \u003cem\u003eDracunculus\u003c/em\u003e, subg. \u003cem\u003eTridentatae\u003c/em\u003e, and subg. \u003cem\u003eSeriphidium\u003c/em\u003e, none was monophyletic. Species previously assigned to subg. \u003cem\u003eArtemisia\u003c/em\u003e were scattered into several clades. Furthermore, Hobbs \u0026amp; Baldwin found three Hawaiian endemic species (\u003cem\u003eA. australis\u003c/em\u003e, \u003cem\u003eA. kauaiensis\u003c/em\u003e and \u003cem\u003eA. mauiensis\u003c/em\u003e) together with \u003cem\u003eA. chinensis\u003c/em\u003e clustered into one single clade, and they thus proposed the sixth subgenus, subg. \u003cem\u003ePacifica\u003c/em\u003e, to accommodate these species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Using nuclear single nucleotide polymorphism (SNP) data obtained by genome-skimming sequencing technology, Jiao et al. reconstructed a phylogeny for \u003cem\u003eArtemisia\u003c/em\u003e consisting eight main clades, and accordingly they proposed a revised clade-based infra-generic classification dividing the genus into eight subgenera [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To some extent, these discordances about the infra-generic relationships reflect the complex evolutionary history of \u003cem\u003eArtemisia.\u003c/em\u003e\u003c/p\u003e \u003cp\u003ePlastid (Chloroplast), commonly found in plants and green algae, are important in plant growth and development [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Typically, the plastome (plastid genome) is a closed loop with a quadripartite structure containing a large single copy region (LSC), a small single copy region (SSC), and two inverted repeat (IRa and IRb) sequences, is 120\u0026ndash;200kb in length. Each genome tends to contain approximately 80 protein-coding genes, 4 rRNAs, and 30 tRNAs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Due to its small size, uniparental inheritance, conserved sequence and structure, and high cellular copy number, the plastome has been an advantageous resource for various evolutionary studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Previously, some plastid genes (\u003cem\u003erbcL\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e) have been extensively used to estimate phylogenetic relationships at deep and shallow levels [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Some faster evolving genes (i.e., \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003erbcL\u003c/em\u003e and \u003cem\u003erpoC1\u003c/em\u003e) and spacer regions (i.e., \u003cem\u003eatpF\u003c/em\u003e-\u003cem\u003eatpH\u003c/em\u003e, \u003cem\u003epsbK\u003c/em\u003e-\u003cem\u003epsbI\u003c/em\u003e, and \u003cem\u003etrnH\u003c/em\u003e-\u003cem\u003epsbA\u003c/em\u003e) have even been developed as DNA barcode markers to identify taxa [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In \u003cem\u003eArtemisia\u003c/em\u003e, only a few plastid regions (i.e. \u003cem\u003epsbA\u003c/em\u003e\u0026ndash;\u003cem\u003etrnH\u003c/em\u003e, \u003cem\u003erpl32\u003c/em\u003e\u0026ndash;\u003cem\u003etrnH\u003c/em\u003e, \u003cem\u003etrnS\u003c/em\u003e\u0026ndash;\u003cem\u003etrnC\u003c/em\u003e) concatenated with nuclear regions (ITS and ETS) were used to construct the generic or infra-generic phylogenetic relationships, and to explore the evolutionary history of the genus [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the advancement of next generation sequencing (NGS) technologies and the decrease in sequencing cost, it is becoming much easier to obtain complete plastome sequences. The plastome data has exhibited greater potential for resolving challenging phylogenetic relationships in a wide spectrum of plant lineages e.g., \u003cem\u003eEriocaulon\u003c/em\u003e (Eriocaulaceae), \u003cem\u003eTrigonotis\u003c/em\u003e (Boraginaceae), Apocynaceae, and Ophioglossaceae [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and numerous historically difficult issues in plant phylogenetics have been satisfactorily addressed, indicating the plastomes play an indispensable role in plant phylogenetics. For \u003cem\u003eArtemisia\u003c/em\u003e, Kim et al. first conducted a comparative analysis of plastomes of 32 \u003cem\u003eArtemisia\u003c/em\u003e species in East Asia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The results revealed that the coding sequences of \u003cem\u003eaccD\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e were under weak positive selection, and highly variable. The plastomes were sufficiently polymorphic to be used as super-barcodes. They further confirmed that subg. \u003cem\u003eArtemisia\u003c/em\u003e was not monophyletic. Using a plastome data matrix of 38 species, including 18 species from subg. \u003cem\u003eSeriphidium\u003c/em\u003e, Jin et al. found that subg. \u003cem\u003eSeriphidium\u003c/em\u003e segregated into two main clades [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, their structural analysis indicated that the plastomes are relatively conserved with some variations only in IR borders. As of May 2024, only those of 47 species, two varieties and one form were deposited at the National Center for Biotechnology Information (NCBI). In contrast with the large number of taxa in \u003cem\u003eArtemisia\u003c/em\u003e, the percentage of sequenced plastomes did not match well the \u003cem\u003eArtemisia\u003c/em\u003e biodiversity. There are still gaps in our knowledge of the general variation pattern of \u003cem\u003eArtemisia\u003c/em\u003e plastomes especially their structure, gene order, IR/SC boundary, and IR expansion. A finer-scale phylogenetic relationship constructed using plastid data with more informative characters and denser taxon sampling is still lacking. And comparisons between phylogenies constructed using data from nuclear DNA and plastomes to explore whether there is any cyto-nuclear (i.e., chloroplast\u0026ndash;nuclear) discordance are also in need.\u003c/p\u003e \u003cp\u003eConsidering this situation, we newly sequenced, assembled and annotated 37 \u003cem\u003eArtemisia\u003c/em\u003e plastomes of 31 species and three varieties in this study. Combining with 38 previously published \u003cem\u003eArtemisia\u003c/em\u003e plastomes from public database, we conducted comparative analyses, and constructed phylogenies in order to: (1) study the plastome variation patterns of this genus; (2) identify variable regions as DNA barcode candidates for future taxon identification; (3) recover the backbone of the \u003cem\u003eArtemisia\u003c/em\u003e phylogeny using plastome-scale data set. Overall, this study will improve our knowledge of \u003cem\u003eArtemisia\u003c/em\u003e plastomes, provide potential genetic markers for taxa identification, and also advance our understanding on the phylogenetic relationships within the genus.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePlastome features of\u003c/b\u003e \u003cb\u003eArtemisia\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 72 \u003cem\u003eArtemisia\u003c/em\u003e plastomes were included in this study, representing 63 species, three varieties and one form (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S1, S2). Among them, 34 plastomes of 29 species and three varieties were newly sequenced and assembled (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S1). All plastomes showed a typical quadripartite structure, including a large single copy (LSC), a small single copy (SSC), and two inverted repeated (IRa/b) regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Their size ranged from 150586 bp (\u003cem\u003eA. ferganensis\u003c/em\u003e) to 151327 bp (\u003cem\u003eA. smithii\u003c/em\u003e), with a difference of 741 bp and the mean length of 151108 bp. The length of LSC, SSC and IR regions were 82313\u0026ndash;83061 bp, 17735\u0026ndash;18883 bp, 24927\u0026ndash;24985 bp, respectively. The total GC content ranged from 37.40\u0026ndash;37.51% with the mean value of 37.50%. The gene categories were rather conserved. A total of 132 genes, including 87 protein coding genes, 37 tRNA genes, and eight rRNA genes, were comprised in every plastome. The detailed information of these plastomes was provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Tables S1 and S2.\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\u003eSummary of the newly sequenced plastomes for 33 \u003cem\u003eArtemisia\u003c/em\u003e samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTaxon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenbank No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eNucleotide length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eNumber of genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGC content\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProtein coding genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003erRNA genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003etRNA genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. sichuanensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. adamsii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. bhutanica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. blepharolepis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150908\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. comaiensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. fulgens\u003c/em\u003e var. \u003cem\u003emeiguensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. gyitangensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. integrifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. jilongensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18883\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24927\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. linyoureunensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mairei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mattfeldii\u003c/em\u003e var. \u003cem\u003eetomentosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24927\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. mongolica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. neosinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. nortonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. phyllobotrys\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. selengensis\u003c/em\u003e-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. sericea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. smithii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24958\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. stracheyi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. stricta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17939\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. sylvatica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. tafellii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. tangutica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. tournefortiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. tridactyla\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. viscidissima\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. waltonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. waltonii\u003c/em\u003e var. \u003cem\u003eyushuensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. youngii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. yunnanensi\u003c/em\u003e-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. yunnanensis\u003c/em\u003e-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e37.40%\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\u003eThe boundaries between IR and SC regions were compared in 66 \u003cem\u003eArtemisia\u003c/em\u003e plastomes representing 62 species, three varieties and one form. All of them have the same type of SC/IR junctions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, S1). The LSC/IRb junction borders (JLB) were located in the gene \u003cem\u003erps19\u003c/em\u003e, and four types were recovered. The length of \u003cem\u003erps19\u003c/em\u003e in LSC was 207\u0026ndash;219 bp, and 60\u0026ndash;72 bp in IRb region. The dominant type is 219 bp in LSC region and 60 bp in IRb region. The SSC/IRa junction borders (JSA) was located in the gene \u003cem\u003eycf1\u003c/em\u003e, with 4428\u0026ndash;4500 bp in SSC region and 556\u0026ndash;565 bp in IRa region. At the IRb/SSC junction borders (JSB), the distances between the gene \u003cem\u003endhF\u003c/em\u003e and the border range were 42\u0026ndash;82 bp. At the LSC/IRa junction borders (JLA), the distances between the gene \u003cem\u003etrnH\u003c/em\u003e and the border range were 2\u0026ndash;135 bp. The IR regions are highly conserved and similar in length and structure. Additionally, no gene rearrangements, inversions, or losses among these plastomes were found.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlastome sequence divergence\u003c/h2\u003e \u003cp\u003eThe sequence divergence of 23 plastomes was analyzed using mVISTA program, with \u003cem\u003eArtemisia sieversiana\u003c/em\u003e (Genbank Accession Number: ON729303) as reference. The \u003cem\u003eArtemisia\u003c/em\u003e plastomes are rather conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The generic regions are more conserved than intergenic spacer regions, and sequence divergence is higher in LSC and SSC than IR regions. Nucleotide polymorphism (Pi) values show very similar results on sequence divergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Most of 79 CDSs are rather conserved, with Pi values lower than 0.002, while three (\u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e, and \u003cem\u003eycf1\u003c/em\u003e) have Pi values between 0.004 and 0.006, and the remaining nine CDSs have Pi values lower than 0.004. Most of the genes with high Pi values (\u0026ge;\u0026thinsp;0.002) are located in the single copy regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The non-coding regions exhibit higher nucleotide variability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The regions \u003cem\u003endhG\u003c/em\u003e-\u003cem\u003endhI\u003c/em\u003e, \u003cem\u003etrnG\u003c/em\u003e(GCC)-\u003cem\u003etrnfM\u003c/em\u003e(CAU), and \u003cem\u003erpoC2\u003c/em\u003e-\u003cem\u003erps2\u003c/em\u003e have Pi values higher than 0.06, the Pi value of \u003cem\u003erpoC1\u003c/em\u003e-\u003cem\u003erpoC2\u003c/em\u003e is between 0.03 and 0.04, and the others have Pi values lower than 0.03. In IR regions, non-coding regions are highly conserved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSimple sequence repeats (SSRs) in\u003c/b\u003e \u003cb\u003eArtemisia\u003c/b\u003e \u003cb\u003eplastomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRepeated DNA sequences are important in genome rearrangement [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We investigated simple sequence repeats (SSRs) in the alignment of 72 \u003cem\u003eArtemisia\u003c/em\u003e plastomes. In total, 4886 SSRs were detected. The numbers of SSRs varied from 58 to 77 in each plastome. Four plastomes (i.e., \u003cem\u003eA. finita\u003c/em\u003e, \u003cem\u003eA. kaschgarica\u003c/em\u003e, \u003cem\u003eA. fukudo\u003c/em\u003e, and \u003cem\u003eA. tournefortiana\u003c/em\u003e) have more SSRs than others (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Mononucleotide repeats are the most abundant (2754, 56.4%), followed by tetra- (987, 20.2%), di- (692, 14.2%), tri- (318, 6.5%), penta- (128, 2.6%), and hexa-nucleotide (7, 0.1%) repeats (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The mono-, di-, tri-, and tetra-nucleotide repeats were found in all plastomes, and penta-nucleotide repeats in 67 plastomes, and hexa-nucleotide repeats only in seven plastomes representing six species and one form, including \u003cem\u003eA. blepharolepis\u003c/em\u003e, \u003cem\u003eA. finita\u003c/em\u003e, \u003cem\u003eA. freyniana\u003c/em\u003e f. \u003cem\u003ediscolor\u003c/em\u003e, \u003cem\u003eA. fukudo\u003c/em\u003e, \u003cem\u003eA. linyoureunensis\u003c/em\u003e, \u003cem\u003eA. smithi\u003c/em\u003ei, and \u003cem\u003eA. yunnanensis\u003c/em\u003e. Most SSRs were located in single copy regions with 3794 in LSC and 673 in SSC regions. Only 418 SSRs were in IR regions. Mono-nucleotide repeats may play an important role in genetic variation than other SSRs types. The A/T repeats account for nearly 97.8% of the mono-nucleotide repeats, and this result is similar with other studies. Di-nucleotide repeats are represented only by AT/TA motif. The detailed information of SSRs in each plastome was provided Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe topologies of phylogenetic trees constructed from maximum likelihood (ML) and Bayesian inference (BI) methods were basically similar (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, S2, S3). All samples of \u003cem\u003eArtemisia\u003c/em\u003e were clustered into one single clade, which was sister to the outgroup \u003cem\u003eAjania\u003c/em\u003e\u0026ndash;\u003cem\u003eChrysanthemum\u003c/em\u003e clade. The genus \u003cem\u003eArtemisia\u003c/em\u003e was split into two clusters. The basal one (here referred to as Clade 1) further divides into two well supported (ML bootstrap value (BS)\u0026thinsp;=\u0026thinsp;100%; Bayesian posterior probabilities (PP)\u0026thinsp;=\u0026thinsp;1) subclades, with one subclade comprising two samples of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eannua\u003c/em\u003e and one of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efukudo\u003c/em\u003e, and another subclade comprising 17 of subg. \u003cem\u003eSeriphidium.\u003c/em\u003e Another cluster (here referred to as Clade 2) divides into three main subclades, including one subclade comprising only \u003cem\u003eArtemisia stracheyi\u003c/em\u003e, one comprising ten samples of subg. \u003cem\u003eDracunculus\u003c/em\u003e and two of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eselengensis\u003c/em\u003e of subg. \u003cem\u003eArtemisia\u003c/em\u003e, and the remaining one subclade comprising all other samples. However, the Clade 2 was not strongly supported (BS\u0026thinsp;=\u0026thinsp;81, PP\u0026thinsp;=\u0026thinsp;0.89).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with previous phylogenetic studies using nuclear markers, our results also confirmed that all the four subgenera, subg. \u003cem\u003eAbsinthium\u003c/em\u003e, subg. \u003cem\u003eArtemisia\u003c/em\u003e, subg. \u003cem\u003eDracunculus\u003c/em\u003e and subg. \u003cem\u003eSeriphidium\u003c/em\u003e sampled in this study, were not monophyletic (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, S2, S3). Most of the species of subg. \u003cem\u003eArtemisia\u003c/em\u003e were formed a monophyletic group, and the rest species were scattered several clades. The phylogenetic position of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ejuncea\u003c/em\u003e of subg. \u003cem\u003eSeriphidium\u003c/em\u003e was not resolved. The remaining species of subg. \u003cem\u003eSeriphidium\u003c/em\u003e formed a monophyletic group. And the subg. \u003cem\u003eDracunculus\u003c/em\u003e was also monophyletic when \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eblepharolepis\u003c/em\u003e was excluded. Only three species of subg. \u003cem\u003eAbsinthium\u003c/em\u003e were sampled, including \u003cem\u003eA. sieversiana\u003c/em\u003e, \u003cem\u003eA. minor\u003c/em\u003e, and \u003cem\u003eA. sericea\u003c/em\u003e, and they formed one clade with \u003cem\u003eA. juncea\u003c/em\u003e of subg. \u003cem\u003eSeriphidium\u003c/em\u003e and \u003cem\u003eA. tournefortiana\u003c/em\u003e of subg. \u003cem\u003eArtemisia\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Disscussion","content":"\u003cp\u003e \u003cb\u003eCharacteristics of plastomes and genetic variation of\u003c/b\u003e \u003cb\u003eArtemisia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand the plastome structural variation pattern of \u003cem\u003eArtemisia\u003c/em\u003e, a denser sampling within the genus is inevitable. In this study, a total of 72 plastomes newly sequenced or downloaded from public database were comparatively analyzed. Nearly consistent with previous studies, the \u003cem\u003eArtemisia\u003c/em\u003e plastomes showed a high degree of similarity in terms of GC content, configuration, gene number and order [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. GC content variation along genomes is a key feature of genomic organization and strongly varies between species. It is usually associated with fundamental elements of genome organization, e.g., recombination [\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e–\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In the \u003cem\u003eArtemisia\u003c/em\u003e plastomes, GC content is not significantly varied between different species, and ranges from 37.40–37.51%, which is typical in angiosperm plastomes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In fact, no genome rearrangement has been found in these samples. These also reflect that \u003cem\u003eArtemisia\u003c/em\u003e plastomes are rather conservative. In general, the length of \u003cem\u003eArtemisia\u003c/em\u003e plastomes also fall within the average length range of eudicots [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Sequence length uniformity was found between different samples of the same species, e.g., \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eannua\u003c/em\u003e. It is more common that different samples of the same species have different sequence length, e.g., \u003cem\u003eA. argyi\u003c/em\u003e, \u003cem\u003eA. lancea\u003c/em\u003e, and \u003cem\u003eA. selengensis\u003c/em\u003e. Three factors have been proposed to drive the difference in plastome length, including intergenic region variation, difference in gene, and the expansion and contraction of IR regions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. All the plastomes are quadripartite, containing the same number of genes, including 87 protein coding, 37 tRNA, and eight rRNA genes. The 66 plastomes analyzed using CPJSdraw belong to the same IR/SC boundary type. The IR regions only varied 58 bp, LSC regions varied 748 bp, and SSC regions varied 148 bp. Thus the variations in \u003cem\u003eArtemisia\u003c/em\u003e plastome length were mainly in LSC regions.\u003c/p\u003e \u003cp\u003ePrevious analyses of whole plastomes revealed that the plastid regions, \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003etrnT\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erpl32\u003c/em\u003e-\u003cem\u003etrnL\u003c/em\u003e, \u003cem\u003etrnE\u003c/em\u003e-\u003cem\u003eropB\u003c/em\u003e, \u003cem\u003etrnH\u003c/em\u003e-\u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003etrnK\u003c/em\u003e-\u003cem\u003erps16\u003c/em\u003e, \u003cem\u003endhC\u003c/em\u003e-\u003cem\u003etrnV\u003c/em\u003e, and \u003cem\u003endhG\u003c/em\u003e-\u003cem\u003endhI\u003c/em\u003e are highly variable [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. As pointed out by Shaw et al., the plastid region might not be consistently variable across different groups [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003endhG\u003c/em\u003e-\u003cem\u003endhI\u003c/em\u003e, \u003cem\u003etrnG\u003c/em\u003e(GCC)-\u003cem\u003etrnfM\u003c/em\u003e(CAU) and \u003cem\u003erpoC2\u003c/em\u003e-\u003cem\u003erps2\u003c/em\u003e have higher variability, and were identified as mutational hotspots for \u003cem\u003eArtemisia\u003c/em\u003e plastomes. Several plastid regions including \u003cem\u003erpl32\u003c/em\u003e–\u003cem\u003etrnH\u003c/em\u003e, \u003cem\u003etrnS\u003c/em\u003e–\u003cem\u003etrnC\u003c/em\u003e have been used to construct the phylogeny of \u003cem\u003eArtemisia\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, we found that these regions were not the most informative regions. This may have limited the power of resolving the phylogenetic relationships within the genus. The combination of \u003cem\u003erbcL\u003c/em\u003e and \u003cem\u003ematK\u003c/em\u003e was recommended as a core plant barcode by the CBOL Plant Working group [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. But the Pi values of \u003cem\u003erbcL\u003c/em\u003e and \u003cem\u003ematK\u003c/em\u003e were both lower than 0.002, indicating they have a rather limited discriminative power in \u003cem\u003eArtemisia\u003c/em\u003e. Plastid \u003cem\u003eaccD\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e are important for plant fitness and leaf development. As observed in other plant groups, the \u003cem\u003eaccD\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e have high variable nucleotide sequences in the plastomes analyzed in this study. The genus \u003cem\u003eArtemisia\u003c/em\u003e is morphologically complex, and species identification is rather difficult. These hotspot regions could be developed as DNA barcode and used to distinguish taxa.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic relationships of\u003c/b\u003e \u003cb\u003eArtemisia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA well resolved phylogenetic relationship is critical for a better understanding of evolutionary patterns and process of plants at different ranks, especially for the large and morphologically variable group as \u003cem\u003eArtemisia\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this study, we constructed the phylogeny for \u003cem\u003eArtemisia\u003c/em\u003e using the protein coding genes (CDS) of plastomes with a broad taxonomic sampling. Consistent with previous studies, our results further confirmed the conflicts between the morphological infra-generic division of \u003cem\u003eArtemisia\u003c/em\u003e and the molecular phylogenetic relationships. All the subgenera sampled here, including subg. \u003cem\u003eAbsinthium\u003c/em\u003e, subg. \u003cem\u003eArtemisia\u003c/em\u003e, subg. \u003cem\u003eDracunculus\u003c/em\u003e, and subg. \u003cem\u003eSeriphidium\u003c/em\u003e, were not supported as monophyletic. The subg. \u003cem\u003eArtemisia\u003c/em\u003e which is the largest in \u003cem\u003eArtemisia\u003c/em\u003e, however, is polyphylotic, with the sampled taxa clustered into several clades. In his treatment of Chinese \u003cem\u003eArtemisia\u003c/em\u003e, Ling divided subg. \u003cem\u003eArtemisia\u003c/em\u003e into two sections, sect. \u003cem\u003eArtemisia\u003c/em\u003e and sect. \u003cem\u003eAbrotanum\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This division was also not supported by our and other phylogenetic studies. As indicated by earlier molecular analyses of \u003cem\u003eArtemisia\u003c/em\u003e, the subg. \u003cem\u003eSeriphidium\u003c/em\u003e once considered as a morphologically independent genus, \u003cem\u003eSeriphidium\u003c/em\u003e (Bess.) Poljak., was strongly supported to include in \u003cem\u003eArtemisia\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The subg. \u003cem\u003eSeriphidium\u003c/em\u003e was traditionally considered as monophyletic [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, in our phylogenetic reconstruction, this subgenus was divided into two clades: one large monophyletic group and another clade including only one species, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ejuncea\u003c/em\u003e. The subg. \u003cem\u003eDracunculus\u003c/em\u003e was monophyletic when two samples of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eselengensis\u003c/em\u003e belonging to subg. \u003cem\u003eArtemisia\u003c/em\u003e were included.\u003c/p\u003e \u003cp\u003eAdditionally, our analyses also revealed that there exists cyto-nuclear phylogenetic discordance, especially the topology position of subg. \u003cem\u003eDracunculus\u003c/em\u003e and subg. \u003cem\u003eSeriphidium\u003c/em\u003e. The phylogenetic topologies of \u003cem\u003eArtemisia\u003c/em\u003e recovered by previous studies using nuclear loci, including ETS and ITS, and nuclear single nucleotide polymorphisms (SNPs) used by Jiao et al., are somewhat similar [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The species of subg. \u003cem\u003eDracunculus\u003c/em\u003e together with some species of subg. \u003cem\u003eArtemisia\u003c/em\u003e constituted the early divergent clade within \u003cem\u003eArtemisia\u003c/em\u003e. The remaining taxa were further clustered into two main clades. Most species of subg. \u003cem\u003eSeriphidium\u003c/em\u003e together with some species of subg. \u003cem\u003eAbsinthium\u003c/em\u003e, and subg. \u003cem\u003eArtemisia\u003c/em\u003e formed a clade sister to another clade formed mainly by species of subg. \u003cem\u003eArtemisia\u003c/em\u003e and subg. \u003cem\u003eAbsinthium\u003c/em\u003e. The phylogenetic relationships constructed using CDS of plastomes revealed a somewhat different topology. The earliest diverging clade of \u003cem\u003eArtemisia\u003c/em\u003e was constituted by all the species of subg. \u003cem\u003eSeriphidium\u003c/em\u003e except \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ejuncea\u003c/em\u003e, together with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eannua\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efukudo\u003c/em\u003e of subg. \u003cem\u003eArtemisia\u003c/em\u003e. The other species were clustered into one clade which could be further divided into two main clades. One includes all samples of subg. \u003cem\u003eDracunculus\u003c/em\u003e excluding \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eblepharolepis\u003c/em\u003e, and two samples of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eselengensis\u003c/em\u003e, and the other was constituted by most species of subg. \u003cem\u003eArtemisia\u003c/em\u003e, and some species of subg. \u003cem\u003eAbsinthium\u003c/em\u003e, \u003cem\u003eA. juncea\u003c/em\u003e of subg. \u003cem\u003eSeriphidium\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eblepharolepis\u003c/em\u003e of subg. \u003cem\u003eDracunculus\u003c/em\u003e. This topology was also revealed by Jin et al. As reported by previous studies, cytonuclear discordance is commonly observed phenomenon in phylogenetic constructions [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. And in \u003cem\u003eArtemisia\u003c/em\u003e, the discordance may reflect the frequent hybridization and introgression.\u003c/p\u003e \u003cp\u003eThe phylogenetic position of \u003cem\u003eArtemisia stracheyi\u003c/em\u003e was still controversial. This species was originally described as new in \u003cem\u003eArtemisia\u003c/em\u003e, and recorded to occur in Tibet and adjacent regions [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Ghafoor thought \u003cem\u003eA. stracheyi\u003c/em\u003e differs remarkably from the genus \u003cem\u003eArtemisia\u003c/em\u003e in several morphological characters, including corolla and ovary densely scaly, stamens included, apical anther appendages triangular-ovate, obtuse, achenes quadrangular-pyramidate [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. They thus proposed a new genus, \u003cem\u003eArtemisiella\u003c/em\u003e Ghafoor, to accomodate this species, and accordingly published a new combination, i.e., \u003cem\u003eArtemisella stracheyi\u003c/em\u003e (C.B. Clarke) Ghafoor. This treatment was not generally accepted by later authors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Jiao et al. for the first time sampled this species in their phylogenetic study based on nuclear genome SNPs, and found that \u003cem\u003eAjania quercifolia\u003c/em\u003e and \u003cem\u003eArtemisiella stracheyi\u003c/em\u003e consisted a clade sister to \u003cem\u003eArtemisia\u003c/em\u003e, they thus accepted the treatment proposed by Ghafoor [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, our results based on CDS of plastomes indicated that \u003cem\u003eArtemisiella stracheyi\u003c/em\u003e (= \u003cem\u003eArtemisia stracheyi\u003c/em\u003e) nested within \u003cem\u003eArtemisia\u003c/em\u003e, and formed an independent clade. Morphologically \u003cem\u003eArtemisiella stracheyi\u003c/em\u003e is rather unique in the genus \u003cem\u003eArtemisia\u003c/em\u003e by having 2- or 3-pinnatisect leaves with lobules narrowly linear, large (6–10 mm in diam.) involucre, and deciduously pubescent receptacle. This morphological and phylogenetic discordance may reflect the complex evolutionary history of \u003cem\u003eA. stracheyi\u003c/em\u003e. So in the near future, phylogenetic analyses using plastome and nuclear data with denser sampling and more molecular data, combing with evidence from morphological, cytological, geographical, and ecological studies, are needed to reveal its evolutionary history of \u003cem\u003eA. stracheyi\u003c/em\u003e, and determine its phylogenetic position.\u003c/p\u003e \u003cp\u003eAs mentioned before, \u003cem\u003eArtemisia\u003c/em\u003e is such a large, complex, and economically important taxon. It should remain a priority for taxonomical and evolutionary studies, even though these tasks are rather challenging [72]. In this study, we newly sequenced 34 \u003cem\u003eArtemisia\u003c/em\u003e plastomes, but the taxon sampling is still inadequate, especially taxa of subg. \u003cem\u003ePacifica\u003c/em\u003e and subg. \u003cem\u003eTridentatae\u003c/em\u003e which were mainly distributed in Hawaiian Islands and North America, respectively. Using these available plastomes, we constructed a phylogenetic backbone for \u003cem\u003eArtemisia\u003c/em\u003e. Based on adding more representative taxon sampling to this phylogenetic backbone, the future studies could more comprehensively reveal the phylogeny of \u003cem\u003eArtemisia\u003c/em\u003e.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we newly sequenced 34 plastomes representing 29 species and three varieties of \u003cem\u003eArtemisia\u003c/em\u003e, and obtained 38 previously published plastomes data representing 34 species and one form. Comparative analyses indicated that the \u003cem\u003eArtemisia\u003c/em\u003e plastomes are conservative in structure, gene number and order. The IR regions are similar in length and structure among plastomes compared. Three protein-coding genes and four non-coding regions were found to be highly diverse: \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e-\u003cem\u003erpoC2\u003c/em\u003e, \u003cem\u003erpoC2\u003c/em\u003e-\u003cem\u003erps2\u003c/em\u003e, \u003cem\u003etrnG\u003c/em\u003e(UCC)-\u003cem\u003etrnfM\u003c/em\u003e(CAU), and \u003cem\u003endhG\u003c/em\u003e-\u003cem\u003endhI\u003c/em\u003e. These can be chosen as the candidates of DNA barcoding marker for taxon identification. The phylogenetic relationships constructed using protein coding genes further confirmed the infra-generic divisions of \u003cem\u003eArtemisia\u003c/em\u003e were not natural. Previously divided four subgenera were not monophyletic. In the future, phylogenetic relationships constructed using plastomes with denser sampling, and comparisons with those of nuclear markers are still needed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e5.1 Taxa sampling, DNA extraction and illumina sequencing\u003c/p\u003e \u003cp\u003eIn this study, we newly sequenced 34 plastomes representing 29 species and three varieties of \u003cem\u003eArtemisia\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Detailed information of taxa, voucher specimen, collection locality was provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The materials were collected during our field trips in China. The voucher specimens were all identified by Xinqiang Guo, the first author of this study, and deposited in Herbarium of South China Botanical Garden, Chinese Academy of Sciences (IBSC). In addition, we downloaded 38 \u003cem\u003eArtemisia\u003c/em\u003e plastomes representing 34 species and one form from NCBI Genbank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nuccore/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/nuccore/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, as of May 1st, 2024) (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). A total of 72 \u003cem\u003eArtemisia\u003c/em\u003e plastomes were obtained and used in comparative plastome analysis. For phylogenetic analyses, \u003cem\u003eAjania fruticulosa\u003c/em\u003e, \u003cem\u003eAjania nematoloba\u003c/em\u003e, \u003cem\u003eAjania khartensis\u003c/em\u003e, and \u003cem\u003eChrysanthemum przewalskii\u003c/em\u003e were selected as outgroups.\u003c/p\u003e \u003cp\u003eFresh leaves were dried with silica gel and kept in \u0026minus;\u0026thinsp;80 ℃ refrigerator. High quality total genomic DNA of plant samples was extracted from 10 mg silica gel-dried leaves using a modified CTAB (cetyl trimethyl ammonium bromide) DNA extraction method [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The DNA samples were sent to Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China), and a 150-bp paired-ended library with an average insert size of approximately 400 bp was prepared according to the manufacturer\u0026rsquo;s manual (Illumina, San Diego, CA, USA), and shotgun sequencing was performed on the Illumina NovaSeq 6000 platform. Approximately 3 Gb of raw reads were generated for each sample.\u003c/p\u003e \u003cp\u003e5.2 Plastome assembly and annotation\u003c/p\u003e \u003cp\u003eTrimmomatic v.0.40 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] was used to remove adapters and filter low-quality reads. NOVOPlasty v2.5.9 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and GetOrganelle pipeline v1.7.2a [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] were used to \u003cem\u003ede novo\u003c/em\u003e assemble plastomes with suggested default parameters, using the complete plastome DNA sequence of \u003cem\u003eArtemisia annua\u003c/em\u003e (Genbank Accession Number: KY085890.1) as a reference. The obtained scaffolds were checked using Bandage v0.8.1 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Assembled plastomes were annotated by using program GeSeq [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and Plastid Genome Annotator (PGA) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] with \u003cem\u003eA. annua\u003c/em\u003e (GenBank accession no.: KY085890.1) as a reference. To precisely define the start and stop codons, intron boundaries, and tRNA genes, annotations were manually adjusted according the reference plastome in Geneious Primer v2021.0.3 (Biomatters Ltd., Auckland, New Zealand). The 34 newly sequenced plastomes were deposited in GenBank database (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The raw data and plastome sequences downloaded from NCBI were re-assembled and re-annotated following the procedures of the newly sequenced samples. The circular plastid genome maps were visualized using OrganellarGenome DRAW v1.3.1 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e5.3 Comparative analyses, identification of divergence hotspots and simple sequence repeats\u003c/p\u003e \u003cp\u003eWe selected 23 plastomes representing 22 species and one variety of \u003cem\u003eArtemisia\u003c/em\u003e (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) to conduct plastome comparisons using the mVISTA program with the Shuffle-LAGAN mode [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The annotation of \u003cem\u003eA. sieversiana\u003c/em\u003e (GenBank accession No.: ON729303) was chosen as reference. To identify potential hotspots of nucleotide diversity in 72 \u003cem\u003eArtemisia\u003c/em\u003e plastomes, 79 CDS were extracted, and aligned using MUSCLE v. 3.8.31 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] with default parameters. Then, Nucleotide diversity (Pi) was estimated using DnaSP v. 6 [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] with window length set as the whole length of each matrix (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). To have a comprehensive overview of the IR expansion or contraction in the \u003cem\u003eArtemisia\u003c/em\u003e plastomes, we selected 66 plastomes and visualized the borders of IR/SC regions using CPJSdraw v1.0.0 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Simple sequence repeats (SSRs) of 72 plastomes (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) were identified using the MISA-web (MicroSAtellite; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pgrc.ipk-gatersleben.de/misa/\u003c/span\u003e\u003cspan address=\"https://pgrc.ipk-gatersleben.de/misa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] with the threshold repeat numbers of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively.\u003c/p\u003e \u003cp\u003e5.4 Phylogenetic analysis\u003c/p\u003e \u003cp\u003ePhylogenetic analyses were based on 75 plastomes, including 72 of \u003cem\u003eArtemisia\u003c/em\u003e, three of \u003cem\u003eAjania\u003c/em\u003e, and one of \u003cem\u003eChrysanthemum\u003c/em\u003e. 79 unique CDS of these plastomes were extracted using using PhyloSuit v7.3.1 [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and Geneious Primer v2021.0.3. The data sets were aligned using MUSCLE v. 3.8.31 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and manually adjusted using AliView v1.26 [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. All the individual CDS matrices were concatenated into a single super-matrix using Geneious Primer v2021.0.3.\u003c/p\u003e \u003cp\u003ePartitionFinder 2 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] was used to determine the best-fit partitioning scheme and the most suitable substitution model. Bayesian phylogenies were constructed using MrBayes v3.2.7a [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Two parallel analyses each four chains (one cold and three hot chains) were run for 40\u0026nbsp;million Markov Chain Monte Carlo (MCMC) generations with trees sampled every 1000 generations. The first 25% sampled trees were discarded as burn-in. The remaining trees were used to estimate the posterior probabilities (PP). Tracer v.1.6 [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] was used to ensure convergence and adequate sampling with the average standard deviation of split frequencies\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and effective sample sizes (ESS) of all parameters\u0026thinsp;\u0026gt;\u0026thinsp;200. The maximum likelihood (ML) analysis were carried out in RAxML-HPC v8.2.12 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], with 1000 bootstrap replicates using a fast bootstrapping algorithm (MLBS), to assess node support. Bootstrap percentage (MLBS and MPBS) values\u0026thinsp;\u0026ge;\u0026thinsp;70 and PP values\u0026thinsp;\u0026ge;\u0026thinsp;0.95 were regarded as strong support. The final tree files were visualized in FigTree v1.4.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"https://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TreeGraph v2.15.0-887 beta [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Long Wang of South China Botanical Garden, Chinese Academy of Sciences for his assistance during the field work, and providing us \u003cem\u003eArtemisia\u003c/em\u003e samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMJY and XQG planned the projects, designed the research, analyzed data, and wrote the manuscript. DWX and YHW planned the projected. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32270215), and the Natural Science Foundation of Zhejiang Province of China (LQ24C020002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the plastomes newly sequenced and annotated in this study are deposited in the National Center for Biotechnology and Information (NCBI) under the accessions as summarized in Table 1 and Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\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\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\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China. \u003csup\u003e2\u003c/sup\u003eZhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou 311121, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data presented in this study are available upon request from the first author and corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLing YR, Humphries CJ, Gilbert MG. \u003cem\u003eArtemisia\u003c/em\u003e Linnaeus. 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BMC Bioinformatics 2010;11:7.\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":"Artemisia, Astearaceae, Phylogenomics, Plastome","lastPublishedDoi":"10.21203/rs.3.rs-4573083/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4573083/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\u003eArtemisia\u003c/em\u003e is a taxonomically difficult group comprising 400\u0026ndash;500 species mainly distributed in northern hemisphere. Only a limited number of \u003cem\u003eArtemisia\u003c/em\u003e plastomes are currently available. Their structure has not been comparatively analyzed, and the phylogenetic backbone of \u003cem\u003eArtemisia\u003c/em\u003e based on plastome-scale data has not been reported with dense taxon sampling. This situation has greatly hindered our understanding on the plastome variation patterns and infra-generic relationships of the genus. With the advancement of next generation sequencing technologies, it is becoming easier to obtain and comparatively analyze the plastome, and use it to construct phylogeny.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we newly sequenced 34 \u003cem\u003eArtemisia\u003c/em\u003e plastomes representing 30 species and three varieties. Combing with 38 previously published plastomes, a total of 72 complete \u003cem\u003eArtemisia\u003c/em\u003e plastomes were comparatively analyzed. The results indicated that the \u003cem\u003eArtemisia\u003c/em\u003e plastomes were conserved in terms of structure, GC content, gene number and order. All plastomes have a typical quadripartite structure, comprising 87 protein coding, 37 tRNA, and 8 rRNA genes. The IR regions are similar in length and structure among the compared plastomes, with the generic regions more conserved than intergenic spacer regions. The sequence divergence is higher in LSC and SSC regions than in IR regions. Three protein-coding genes and four non-coding regions, i.e., \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e-\u003cem\u003erpoC2\u003c/em\u003e, \u003cem\u003erpoC2\u003c/em\u003e-\u003cem\u003erps2\u003c/em\u003e, \u003cem\u003etrnG\u003c/em\u003e(UCC)-\u003cem\u003etrnfM\u003c/em\u003e(CAU), and \u003cem\u003endhG\u003c/em\u003e-\u003cem\u003endhI\u003c/em\u003e, were found to be highly diverse, and could be chosen as candidates of DNA barcode. Phylogenetic relationships constructed using protein coding genes of plastomes were divided into several clades that did not match with previous infra-generic divisions of \u003cem\u003eArtemisia\u003c/em\u003e, and four subgenera were not monophyletic. Furthermore, they were also inconsistent with those based on nuclear markers. And the phylogenetic position of \u003cem\u003eA. stracheyi\u003c/em\u003e is still controversial.\u003c/p\u003e\u003ch2\u003eConslusions\u003c/h2\u003e \u003cp\u003eThis study reveals that the \u003cem\u003eArtemisia\u003c/em\u003e plastomes are conservative, especially in structure, gene number and order. Phylogenetic relationships constructed using CDS further confirmed the infra-generic divisions of \u003cem\u003eArtemisia\u003c/em\u003e were not natural. This study lay a foundation for future evolutionary studies of \u003cem\u003eArtemisia\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Comparative analysis of Artemisia plastomes and insights into the infra-generic phylogenetic relationships of the genus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-16 05:03:27","doi":"10.21203/rs.3.rs-4573083/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":"e1bd6029-eadd-4d20-a32e-74ae074b2c9c","owner":[],"postedDate":"July 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-27T19:39:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-16 05:03:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4573083","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4573083","identity":"rs-4573083","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}