Phylogenetic analysis and divergence time estimation of Lycium species in China based on the chloroplast genomes

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Despite the economic and ecological importance of Lycium , its phylogeny, interspecific relationships, and evolutionary history remain relatively unknown. In this study, we constructed a phylogeny and estimated divergence time based on the chloroplast genomes (CPGs) of 15 species, including subspecies, of the genus Lycium from China. Results: We sequenced and annotated 15 CPGs in this study. Comparative analysis of these genomes from these Lycium species revealed a typical quadripartite structure, with a total sequence length ranging from 154,890 to 155,677 base pairs (bp). The CPGs was highly conserved and moderately differentiated. Through annotation, we identified a total of 128–132 genes. Analysis of the boundaries of inverted repeat (IR) regions showed consistent positioning: the junctions of the IRb/LSC region were located in rps 19 in all Lycium species, IRb/SSC between the ycf 1 and ndh F genes, and SSC/IRa within the ycf 1 gene. Sequence variation in the SSC region exceeded that in the IR region. We did not detect major expansions or contractions in the IR region or rearrangements or insertions in the CPGs of the 15 Lycium species. Comparative analyses revealed five hotspot regions in the CPG: trn R(UCU), atp F- atp H, ycf 3- trn S(GGA), trn S(GGA), and trn L-UAG, which could potentially serve as molecular markers. In addition, phylogenetic tree construction based on the CPG indicated that the 15 Lycium species formed a monophyletic group and were divided into two typical subbranches and three minor branches. Molecular dating suggested that Lycium diverged from its sister genus approximately 17.7 million years ago (Mya) and species diversification within the Lycium species of China primarily occurred during the recent Pliocene epoch. Conclusion: The divergence time estimation presented in this study will facilitate future research on Lycium , aid in species differentiation, and facilitate diverse investigations into this economically and ecologically important genus. Lycium Plastome structure Comparative analysis Phylogenetic relationship Divergence time Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Lycium is a crucial shrub genus in the Solanaceae family, consisting of approximately 70 species found globally across Southern Africa, Europe, Asia, America, and Australia [ 1 – 2 ]. However, China hosts 15 species, including subspecies, primarily growing in the arid and semi-arid regions of Ningxia, Xinjiang, Inner Mongolia, and other areas [ 3 – 7 ]. These plants have been utilized as food and herbal medicine in China for millennia [ 8 – 9 ], with various parts like fruits, leaves, root bark, and young shoots used as local foods and/or medicines [ 10 ]. These plants impart potential pharmacological effects such as anti-aging properties, reduction of blood glucose and serum lipids, and immune regulation [ 11 ]. Several studies have demonstrated that extracts of these plants can prevent and treat diseases such as night sweats, diabetes, cough, vomiting, hypertension, and ulcers [ 10 , 12 – 13 ]. Overall, previous studies have focused on the growth, development, medicinal value, and breeding of Lycium [ 14 – 17 ]. Despite its economic and ecological significance, much remains unknown about Lycium phylogeny, interspecific relationships, and evolutionary history. While numerous studies have identified and analyzed phylogenetic relationships within the Lycium genus using DNA barcode fragments [ 18 – 22 ], research on CPG phylogeny and lineage diversification is lacking. Chloroplasts are essential organelles involved in photosynthesis and carbon fixation in plant cells. The advancement in sequencing technologies has made whole CP sequencing more accessible [ 23 – 25 ]. Compared with DNA fragments, CPGs contain significantly more informative sites for analyzing nucleotide diversity and reconstructing phylogenies among closely related species [ 26 – 28 ]. CPGs typically range from 75 to 250 kb in length, with numerous copies in a given cell, are maternally inherited in most plants, and have conserved gene content and order [ 29 , 30 ]. The plastome is characterized by two large inverted repeat regions (IRa and IRb) separated by two single-copy regions, referred to as the large single-copy region (LSC) and the small single-copy region (SSC) [ 31 ]. Recently, CPGs have been used for comparative and phylogenetic analyses, proving useful in species identification, genetic diversity assessment, nucleotide diversity assessment, resolving phylogenetic relationships, and evolutionary history [ 32 – 36 ]. Despite the use of CPGs, few molecular phylogenetic studies have attempted to resolve infrafamilial relationships within Lycium using broad taxon sampling. Most of these studies were based on one or a few molecular loci or had sampling limitations, leaving our understanding of the phylogenetic relationships and divergence timescales of the genus unclear [ 18 , 37 – 39 ]. In this study, we sequenced and aligned the CPGs of 15 species, including subspecies, of the genus Lycium from China. Our main objectives were to (a) construct a phylogeny based on the CPGs of 36 species from the Solanaceae family; (b) date the divergence of the Lycium clade; and (c) examine structural changes in the CPGs of the sampled Lycium species. Results Characteristics of Lycium chloroplast genomes After quality control and pre-processing, a minimum of 6 Gb of whole-genome sequencing data were obtained for each of the 16 species included in this study (Table S1). These clean reads were used to assemble complete CPs using a reference-guided approach. All the newly assembled CPGs displayed a typical quadripartite structure, with two IR regions separating the LSC and SSC regions (Fig. 1). For each of the 15 Lycium species, the CPGs size ranged from 154,890 bp ( L. changjicum ) to 155,677 bp ( L. amarum ) (Table S1). All the CPGs had a typical quadripartite circular structure (Fig. 1) consisting of an LSC and an SSC region separated by a pair of IR regions (Fig. 1 and Table S1). The LSC region’s length varied from 85,892 bp ( L. changjicum ) to 86,635 bp ( L. amarum ), and the lengths of the SSC and IR regions ranged from 18,190 bp ( L. cylindricum ) to 18,215 bp ( L. barbarum ) and 25,394 bp ( L. ruthenicum ) to 25,469 bp ( L. cylindricum ), respectively (Table S1). The GC content of the entire plasmid sequence and the LSC, SSC, and IR regions was similar across all Lycium CPGs. Specifically, the GC content of the entire plasmid sequence was 37.8–37.9%; while the GC content of the IR regions was 43.1–43.2%, which was higher than that of the LSC and SSC regions (35.8–35.9% and 32.3–32.4%, respectively; Table S1). Additionally, the number of annotated genes in each CPGs ranged from 128 ( L. amarum ) to 132 ( L. ningxiaens ), and included 35–37 tRNA and 8 rRNA genes. Figue 1 Gene map of the CPGs of fifteen Lycium species. Genes belonging to different functional groups are shown in different colors. The darker gray area in the inner circle indicates the GC content and the lighter gray indicates the AT content of the genome. The thick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into the small single-copy (SSC) and large single-copy (LSC) regions. Comparative genomics and divergence hotspots Using L. chinense as a reference, the CPGs of the 15 Lycium species were visually compared with those obtained using the mVISTA online database. The results showed that the CPGs of the 15 species were conserved, especially the coding regions (Fig. 2). Considering the entire plastome, the SSC and LSC regions displayed marked divergence compared to the IR regions. The proportion of non-coding regions was greater than that of protein-coding regions, and divergence hotspots were largely located in the intergenic spacer regions (Fig. 2). These divergence hotspots usually consist of highly variable sequences that can be used as potential DNA barcodes for phylogenetic analyses and to determine relationships between species. Therefore, to further understand DNA polymorphisms (Pi), mutation hotspot regions in the CPGs of the15 Lycium plants were screened using DnaSP software (Fig. 3). Pi analysis revealed that Pi values ranged from 0 to 0.00851, and the CPGs were relatively structurally conserved, small, and highly variable among the species. We identified five mutation hotspot regions (Pi > 0.0006) that could be used as potential molecular markers. Of these, trn R(UCU), atp F- atp H, ycf 3- trn S(GGA), and trn S(GGA) are located in the LSC region, while trn L-UAG is located in the SSC region (Fig. 3). None of the mutation hotspots are located in the IR region. Figue 2 Sequence alignment of the CPGs of Lycium species. The alignment was performed using the mVISTA program and the L. chinense CP was used as a reference. The Y-axis: the degree of identity ranging from 50 to 100%. Coding and non-coding regions were marked in blue and red, respectively. Black arrows indicated the position and direction of each gene. CNS: conserved non-coding sequences. Figue 3 Sliding window test of nucleotide diversity (Pi) in the multiple alignments of 15 Lycium species species (window length: 600 bp; step size: 200 bp). X-axis: the position of the midpoint of the window; Y-axis: the nucleotide diversity of each window. Figue 4 Comparisons of the borders of the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions among the CPGs of fifteen Lycium species. Boundaries between IR and SC regions The boundaries of the LSC, SSC, and IR regions were highly consistent within Lycium and no obvious expansion or contraction of the IR region was detected in the 15 CPGs (Fig. 4). Here, trn H was shown to be the first gene in the LSC region at the junction between IRa and LSC (i.e., IRa/LSC). At the other end of the LSC region, the IRb/LSC junctions were located in the rps 19 gene sequence in all Lycium species, with the length of the rps 19 gene located in the IRb region, varying from 46 to 50 bp. At both ends of the SSC region, IRb/SSC junctions were located between the ycf 1 and ndh F genes. However, SSC/IRa was found in the ycf 1 gene, with the length of the ycf 1 gene located in the IRa region varying from 995 to 1004 bp. Phylogenetic analyses of Solanaceae To infer the phylogenetic relationships of the 36 Solanaceae species, we included two Convolvulaceae species whose CPGs are publicly available in the GenBank database. These species ( Calystegia hederacea and Convolvulus arvensis ) were used as the outgroup for phylogenetic analyses. The final concatenated dataset included 55 plastid genes and 43,044 sites, after trimming poorly aligned regions and gaps with missing genes. In the phylogenetic trees, all nodes in the ML and BI analyses of each dataset generated almost congruent topologies with high bootstrap support (BS) and posterior probability (PP) values (Fig. 5 and S1, S2, S3, S4). Thirty-six plant species belonging to the Solanaceae family were divided among three typical branches. Branches of Physochlaina physaloides , Przewalskia tangutica , Scopolia carniolica , Atropanthe sinensis , Solanum betaceum , Anisodus acutangulus , Hyoscyamus niger , and Atropa belladonna were resolved as sisters of Lycium . Further, in the CDS phylogeny, Lycium species were clustered on one large branch, confirming that the independence of this genus is highly supported (BS; PP = 100%, 1) (Fig. S1 and S2). The 15 species of plants belonging to Lycium were divided into two typical sub-branches and three minor branches: clade I-1, comprising L. barbarum var. auranticarpum , L. cylindricum , L. yunnanense , L. barbarum , L. dasystemum var. rubricaulium , and L. dasystemum on one sub-branch (BS; PP = 100%, 1); clade I-2 consisting of L. chinense var. potaninii , L. truncatum , L. amarum , and L. chinense on another sub-branch (BS; PP = 100%, 1), and clade II comprising L. barbarum var. implicatum , L. qingshuiheense , L.ningxiaense , L. changjicum , and L. ruthenicum on a different branch (BS; PP = 100%, 1). Figue 5 Phylogeny and clade divergence of Solanaceae and outgroups based on 55 plastome protein-coding genes. Stars indicate fossil calibrations used in this analysis. Geological periods are marked with background colors. Mya: million years ago; Pal: Paleocene; Pli: Pliocene; Ple: Pleistocene Table 1 Estimated ages for Lycium subclades Subclade name * Mean age (Mya) 95% highest posterior density interval (HPD) Subclade 1 17.7 14.0-21.3 Subclade 2 4.95 1.09–3.86 Subclade 3 1.7 0.64–2.76 *Subclades are labeled in Fig. 5 Divergence time estimation We estimated the divergence timescales of the major clades within Lycium according to the calibration of the gene tree constructed based on 55 plastid genes. The split between Lycium and its sister group was estimated to have occurred 17.7 Mya. The crown ages of all subclades in the genus Lycium were dated mainly within the Pleistocene, suggesting that numerous species of this genus originally diversified in the recent past (6 Mya) (Fig. 5 and Table 1). Discussion Previous studies have reported that most angiosperms have CPGs ranging in size from 120 to 170 kb, with the IR region typically spanning 20–30 kb [ 16 , 27 – 28 ]. In the present study, a comparative analysis of the CPGs indicated that those of 15 Lycium species from China were at the larger end of the spectrum for angiosperm organelle genomes, ranging from 154,890 bp ( L. changjicum ) to 155,677 bp ( L. amarum ). All Lycium species exhibited a typical quadripartite structure that is similar to other higher plants, comprising the LSC region (85,892–86,635 bp), SSC region (18,190–18,215 bp), and two identical IR regions (25,394–25,469 bp). The GC content of the entire plastid sequence and the LSC, SSC, and IR regions was similar across all Lycium CPGs. In agreement with numerous studies on angiosperms, the IR regions exhibited the highest GC content [ 27 ]. The conversion between sequences and higher GC content may contribute to the greater conservation of IR regions [ 28 , 29 ]. In angiosperms, the IR region is relatively conserved in sequence and structure. The narrowing and widening of its edges are not only important factors for length variation, but also the main cause of the emergence of pseudogenes [ 34 – 35 ]. While cp genes have evolved slowly and are relatively conserved in terms of sequence and structure, boundary contraction and expansion in the IR regions are common phenomena. In our study, we analyzed 15 CPGs within the highly conserved Lycium species and noticed that no major expansions or contractions occurred in the IR regions. Highly variable regions offer valuable phylogenetic information. For example, variable regions aid in species kinship identification and gene pool construction [ 38 – 41 ]. A good DNA barcode must be a short, representative DNA fragment with high variability and amenable to amplification [ 42 ]. In this study, both the sequence and structure of Lycium CPGs were highly conserved. mVISTA analysis revealed that most of the variation in nucleotide sequences occurred in noncoding regions, consistent with previous reports, suggesting this variation as a common feature of angiosperms [ 43 – 45 ]. In the Lycium , several highly variable regions, such as mat k, rps 16– trn K and trn H– psb A, are recognized as potential DNA barcoding sites [ 39 , 46 ]. In addition, our nucleotide diversity (Pi) analysis led to the identification of five highly variable regions with Pi values greater than 0.006, including three non-coding tRNA regions ( trn S, trn L, and trn R) and two intergenic regions ( atp F- atp H and ycf 3- trn S(GGA). In conclusion, these mutation hotspot regions play an important role in the identification and characterization of Lycium plant species. Lycium is a genus of shrubs with significant economic and ecological importance. Therefore, considerable efforts have been dedicated to cultivated practices, growth and development, medicinal application, and breeding [ 10 – 17 ]. However, to date, few studies have explored its phylogenetic relationships and divergent timescales [ 11 ]. The CPGs are central to molecular biology research and have become a prominent focus. In particular, species identification, phylogenetic relationships, and the reconstruction of evolutionary history via whole-genome sequencing have become important tools because of improvements in sequencing technology and low costs. Phylogenetic trees constructed based on a single or few gene sequences often yield inconsistent or even conflicting topologies due to variations in evolutionary rates and horizontal shifts between genes. This complication affects the determination of accurate evolutionary relationships among species [ 47 – 48 ]. In this study, we constructed a phylogenetic tree using the BI and ML methods. The CPGs of 15 Lycium species converged into branches with high support (Fig. S3 and S4). Notably, L. cylindricum , L. yunnanense , L. dasystemum , and L. dasystemum were similar to L. barbarum and closely related, while L. chinense was classified in the same branch as L. truncatum and L. amarum (BS; PP = 100%, 1.00, respectively), indicating that the three species are similar. Based on the phylogenetic tree results, we speculate that L. qingshuiheense , L. ningxiaense , and L. changjicum may have originated from L. ruthenicum . Although L. barbarum var. implicatum and L. qingshuiheense formed a small, distinct branch, the support rate was low, indicating that they may be the same species. This study demonstrates that high-resolution CPG sequences provide valuable resources for broad research on genetic information and species identification of Lycium spp. The highly conserved and stable alignment of the 55 plastid genes allowed us to calibrate the divergence and origin of Lycium (Fig. 5). We used three tentative calibrations to estimate the diversification. While the estimated ages should be used with caution, our findings indicate that the Lycium diverged from the sister genus around 17.7 Mya, and the two successive clades within the Lycium diverged 4.95 and 1.7 Mya, suggesting relatively late clade diversifications. Specifically, most species diversification within the subclades of Lycium , as estimated from these plastid genes, appeared to have occurred in the recent past, mostly after 5 Mya., despite the fact that numerous species are currently acknowledged in genera. This observation may partly account for the widespread hybridization observed among these young species [ 46 ], likely resulting from incomplete reproductive isolation. The divergence timescales estimated here for the major subclades will serve as a basic timescale for diverse studies on this economically and ecologically important genus. Conclusions We analyzed the complete CPGs of 15 Lycium species and found that all exhibited a quadripartite structure, typical of most angiosperms. The CPG arrangement was highly conserved, with great sequence variation observed in the SSC region compared with that in the IR region. We did not detect major expansions or contractions in the IR region, nor did we find any rearrangements or insertions in the CPGs of the 15 Lycium species. We identified highly variable regions within Lycium that are likely to be useful for species delimitation. Phylogenetic tree construction based on the CPGs showed that all 15 Lycium species formed a monophyletic group, divided into two typical subbranches and three minor branches. Molecular dating suggested that Lycium diverged from its sister genus around 17.7 Mya, with species diversification of the Lycium species of China occurring mainly within the recent Pliocene epoch. Overall, our findings and the estimated divergence times will facilitate future studies on Lycium , assist in species differentiation, and facilitate diverse studies of this economically and ecologically significant genus. Materials and methods Taxon sampling, DNA extraction, and plastome sequencing A total of 38 CPGs representing Solanaceae and related families were included in this study (Table S2 ). 36 CPGs from Solanaceae were selected, including all Lycium species found in China. Further, two additional CPGs from related families in Convolvulaceae were chosen as outgroups for phylogenetic analysis. Among these 38 CPGs, 16 complete CPGs were newly sequenced, and the others were obtained from GenBank (Table S2 ). The leaves used in this study were collected from natural populations in China. The plant materials were identified by Dr. Lei Zhang and the voucher specimens (Table S2 ) were deposited in the Herbarium of North Minzu University (NMU; Yinchuan, China). For each species, we extracted total DNA from dried leaves preserved them in silica gel using the CTAB protocol [ 49 ]. Paired-end libraries with an insert size of 500 base pairs (bp) were constructed by Illumina (Qingdao, Shandong, China) following sequencing with a HiSeq × Ten System (Jizhi, Qingdao, Shandong, China). Chloroplast genome and annotation At least two gigabases (Gb) of 2 × 150 bp short read data were generated for each sample. Reads with quality scores of less than 7 and with more than 10% ambiguous nucleotides were filtered. The remaining reads were assembled using NOVOPlasty version 2.7.2 [ 50 ] software. The contigs were aligned into sequence in Geneious version 9.1.8 [ 51 ] software using the L. chinense CPG as a reference. The CPGs were annotated using Plann version 1.1 [ 52 ]. Protein-coding genes were extracted using customized Python scripts. Alignment of chloroplast genes across all species was performed using PRANK version 130410 [ 53 ] software. Poorly aligned regions were trimmed using Gblocks version 0.91b [ 54 ] with the option “−t = c,” selected to set the type of sequence to codons. Genes that were absent in at least one species were excluded, and the aligned sequences were combined into a super matrix. Additionally, circular maps of the CPGs were created using OGDRAW version 1.2 [ 55 ], and all annotated CPGs were submitted to GenBank [ 56 ]. Comparative genomics and structural analyses The structural variation and identification of arrangement events across Lycium was conducted for the 15 CPGs of Lycium . The results of the comparative analysis of the CPGs were visualized with the mVISTA program [ 57 ] and the annotated CPG of L. chinense was used as the reference in the LAGAN mode [ 58 ]. The junction sites of the four structural regions (IRA, LSC, SSC, and IRB) and adjacent genes in 15 Lycium CPGs were visualized and compared using IRscope [ 59 ] software to obtain a macroscopic view of the CPG structure. Following sequence alignment, nucleotide diversity (Pi) analysis of the CPG was performed using DnaSP version 6.0 [ 60 ]. Phylogenetic inference and divergence time estimation We generated two datasets for phylogenetic analysis: a protein-coding region (CDS) set and a whole plastome (WP) set. Protein-coding genes (PCGs) were extracted from the GenBank formatted file containing 38 CPGs using customized Perl scripts that removed the start and end codons. After excluding possible pseudogenes, 55 PCGs were retained in all species. Each PCG was aligned using PRANK version130410 based on the translated amino acid sequences. Genes that had been lost in at least one species were discarded and then the remaining aligned sequences were concatenated into a super matrix. Independent phylogenetic analyses were performed for each dataset (CDS and WP) using the maximum likelihood (ML) and Bayesian inference (BI) methodologies. We used RAxML version 8.1.24 [ 61 ] to conduct ML analyses with a general time reversible model with a gamma distribution (GTR + Γ). The best-scoring ML tree was obtained using the rapid hill-climbing algorithm (i.e., the option “-f d”) with 1,000 bootstrap replicates. The optimal model (GTR + I + G) was identified using jModeltest software, and BI analysis was conducted using MrBayes version 3.2.6 [ 62 ]. Additionally, FigTree version 1.4.2 [ 63 ] was used to visualize phylogeny. We estimated divergence times from the plastome dataset using an approximate likelihood method, as implemented in MCMCtree in PAML version 4 [ 64 ] software, with independent relaxed-clock and birth–death sampling [ 65 ] strategies. Fossil dates were used as calibration points to reduce bias for more accurate age estimates [ 66 ]. However, the fossil records of Solanaceae are very limited. Three fossils were used to constrain the internal nodes: (1) According to the ancient seed fossil of Solanum nigrum [ 67 ], 5.3–11.6 Mya was the age range assigned to the split between Solanum nigrum and its sister species Tubocapsicum anomalum . (2) The split between Solanum and Lycium was assigned an age range of 19.0-23.3 Mya as previously estimated [ 68 ]. (3) The root of the phylogeny was restricted to an age range of 46.2–53.7 Mya based on the secondary age constraints described by Särkinen et al. [ 68 ]. The best-fit GTR + Γ model was selected and the prior of the substitution rate (rgene) was modeled by a Γ distribution as Γ (2, 200, 1). We set parameters for the birth–death process with species sampling and σ 2 values of (1, 1, 0.1) and G (1, 10, 1), respectively. We executed the MCMC runs for 2,000 generations as burn-in and then sampled every 750 generations until 20,000 samples were obtained. We compared two MCMC runs for convergence using random seeds and obtained similar results. Declarations Ethics approval and consent to participate Plant samples were collected conforming to the national / international legislation and institutional guidelines. Availability of data and materials The 16 newly assembled and annotated CPGs have been submitted to NCBI (https://www. ncbi. nlm. nih. gov), with accession numbers listed in table S1. Conflicts of Interest The authors declare no conflict of interest. Funding This study was supported by the Ningxia Natural Science Foundation (2022AAC05063, 2021AAC02005) and the National Natural Science Foundation of China (32260049). The anonymous reviewers and editors are sincerely acknowledged. Authors’ contributions Lei Zhang and Guoqi Zheng developed the concept of the study. Lei Zhang and Erdong Zhang conducted the experiment and data analysis. Lei Zhang, Yuqing Wei and Guoqi Zheng drafted the manuscript. Yuqing Wei and Guoqi Zheng supervised the study. All authors revised the manuscript. References Zhang JX, Guan SH, Feng RH, Wang Y, Wu ZY, Zhang YB, Chen XH, Bi KS, Guo DA. Neolignanamides, lignanamides, and other phenolic compounds from the root bark of Lycium chinense . Journal of Natural Products. 2013;76:51–58. Turchetto C, Fagundes NJ, Segatto AL, Kuhlemeier C, Solis Neffa VG, Speranza PR, Bonatto SL, Freitas LB. 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Nucleic Acids Research. 2013;41.W575–W581. Sayers EW, Cavanaugh M, Clark K, Ostell J, Pruitt KD, Karsch-Mizrachi I. GenBank. Nucleic Acids Research. 2020;48:D84–D86. Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic acids research. 2004;32:W273–W279. Brudno M, Malde S, Poliakov A, Do CB, Couronne O, Dubchak I, Batzoglou S. Glocal alignment: finding rearrangements during alignment. Bioinformatics. 2003;19:i54– i62. Amiryousefi A, Hyvönen J, Poczai P. IRscope: an online program to visualize the junction sites of chloroplast genomes. Bioinformatics. 2018;34:3030–3031. Rozas J, Ferrer-Mata A, Sánchez-Delbarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology & Evolution. 2017;34:3299–3302. Stamatakis A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget Bret, Liu L, Suchard MA, Huelsenbeck JP, Notes A. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology. 2012;61(3):539–542. Rambaut A. FigTree v1. 4. University of Edinburgh, Edinburgh, UK. 2012. Available at: http://tree.bio.ed.ac.uk/software/figtree. Yang ZH. PAML 4: Phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution. 2007; 24:1586–1591. Rannala B. Yang Z. Inferring speciation times under an episodic molecular clock. Systematic Biology. 2007;56:453–466. Crepet WL. Nixon KC. Gandolfo MA. Fossil evidenceand phylogeny: the age of major angiosperm clades based on mesofossil and macrofossil evidence from cretaceous deposits. American Journal of Botany. 2004;91:1666–1682. Vander BJ. Miocene floras in the Lower Rhenish basin and their ecological interpretation. Review of Palaeobotany and Playnology. 1987;52:299–366. Särkinen T, Bohs L, Olmstead RG, Knapp S. A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Ecology Evolution. 2013;13:214. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigues.docx SupplementaryTables.xls Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 04 May, 2024 Reviewers agreed at journal 02 Apr, 2024 Reviews received at journal 22 Mar, 2024 Reviewers agreed at journal 19 Mar, 2024 Reviewers invited by journal 08 Mar, 2024 Editor assigned by journal 08 Mar, 2024 Editor invited by journal 04 Mar, 2024 Submission checks completed at journal 04 Mar, 2024 First submitted to journal 01 Mar, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4002205","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276424035,"identity":"20ceca7b-9f8f-44c5-ace1-926c3911f824","order_by":0,"name":"Lei Zhang","email":"","orcid":"","institution":"National Ethnic Affairs Commission of the People’s Republic of China, North Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":276424036,"identity":"4eacabde-7ca5-4a3e-b8a4-f44e638e9175","order_by":1,"name":"Erdong Zhang","email":"","orcid":"","institution":"National Ethnic Affairs Commission of the People’s Republic of China, North Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Erdong","middleName":"","lastName":"Zhang","suffix":""},{"id":276424037,"identity":"def72b73-e768-4e55-ba5e-585cdc23af38","order_by":2,"name":"Yuqing Wei","email":"","orcid":"","institution":"National Ethnic Affairs Commission of the People’s Republic of China, North Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Wei","suffix":""},{"id":276424038,"identity":"8bd59616-4153-4eb4-a7d2-dff66f6524db","order_by":3,"name":"Guoqi Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACCRiDmbHx8d9/Njz8/A1Ea2FuNuBhS5ORnHGAWC0M7G0SPGyHbQwaEvDrkJ/dfOzhl1+H5fiOM7ZJSPCc5zFgOMD44WMObi2Mc46lG8v2HTaWPMzYbGEgcZvHnLmBWXLmNtxamCVyzKQlew4nbjjM2HgjweA2j2XDATZmXjxa2CTyv8G0NEgcSDjHY3AgAb8WHokcNskPP8BamiQbDhwgrEVCIs1MmrEhHewXY8aGZB7JGQeb8fpFfkbyM8kff6zl+M4ff/iYscHOnp+/+eCHj3i0gIOAtw1IHoDzGRvwqwcp+fEHRcsoGAWjYBSMAlQAAMqqU9yZpagQAAAAAElFTkSuQmCC","orcid":"","institution":"Ningxia University","correspondingAuthor":true,"prefix":"","firstName":"Guoqi","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-03-01 05:47:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4002205/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4002205/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52121883,"identity":"864e2e83-6eb1-4841-9244-ca4acf1b7cda","added_by":"auto","created_at":"2024-03-07 04:51:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":513493,"visible":true,"origin":"","legend":"\u003cp\u003eGene map of the CPGs of fifteen \u003cem\u003eLycium\u003c/em\u003e species. Genes belonging to different functional groups are shown in different colors. The darker gray area in the inner circle indicates the GC content and the lighter gray indicates the AT content of the genome. The thick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into the small single-copy (SSC) and large single-copy (LSC) regions.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/19f0be83b5679eeff434cc8d.jpg"},{"id":52121885,"identity":"4cecfd93-00ff-4797-acf6-cd85149c877c","added_by":"auto","created_at":"2024-03-07 04:51:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9505910,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment of the CPGs of \u003cem\u003eLycium\u003c/em\u003e species. The alignment was performed using the mVISTA program and the \u003cem\u003eL. chinense\u003c/em\u003e CP was used as a reference. The Y-axis: the degree of identity ranging from 50 to 100%. Coding and non-coding regions were marked in blue and red, respectively. Black arrows indicated the position and direction of each gene. CNS: conserved non-coding sequences.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/53288661ac6aea060b5c2898.jpg"},{"id":52121882,"identity":"ea5592fb-e8d0-4add-9b2f-8dc961754178","added_by":"auto","created_at":"2024-03-07 04:51:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1202641,"visible":true,"origin":"","legend":"\u003cp\u003eSliding window test of nucleotide diversity (Pi) in the multiple alignments of 15 \u003cem\u003eLycium\u003c/em\u003especies species (window length: 600 bp; step size: 200 bp). X-axis: the position of the midpoint of the window; Y-axis: the nucleotide diversity of each window.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/a97ec374328e9c891528c4b0.jpg"},{"id":52121884,"identity":"2cad3ac8-fd59-4af9-a0aa-dc66f9ba956f","added_by":"auto","created_at":"2024-03-07 04:51:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5348522,"visible":true,"origin":"","legend":"\u003cp\u003eComparisons of the borders of the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions among the CPGs of fifteen \u003cem\u003eLycium\u003c/em\u003e species.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/b3b360d427e92e56e156d73d.jpg"},{"id":52121886,"identity":"8d2b5853-617c-46ab-a8f6-7cdc3991d6b1","added_by":"auto","created_at":"2024-03-07 04:51:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1961102,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny and clade divergence of Solanaceae and outgroups based on 55 plastome protein-coding genes. Stars indicate fossil calibrations used in this analysis. Geological periods are marked with background colors. Mya: million years ago; Pal: Paleocene; Pli: Pliocene; Ple: Pleistocene\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/5727856cd6e895839cbb018f.jpg"},{"id":52121892,"identity":"3d19279c-29b9-4cae-844e-c165c775055e","added_by":"auto","created_at":"2024-03-07 04:51:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1124366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/a0d468ff-803c-4124-8b91-f7b7f6a4c38f.pdf"},{"id":52121887,"identity":"3a30007e-ce6f-437b-8875-42b45b9dece6","added_by":"auto","created_at":"2024-03-07 04:51:11","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":688202,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigues.docx","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/ef6bad95a253d62f50da2795.docx"},{"id":52121888,"identity":"158f824a-57ed-4d7c-aa57-39a495990665","added_by":"auto","created_at":"2024-03-07 04:51:11","extension":"xls","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":29696,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xls","url":"https://assets-eu.researchsquare.com/files/rs-4002205/v1/9778ea599e38c07e41270891.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phylogenetic analysis and divergence time estimation of Lycium species in China based on the chloroplast genomes","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eLycium\u003c/em\u003e is a crucial shrub genus in the Solanaceae family, consisting of approximately 70 species found globally across Southern Africa, Europe, Asia, America, and Australia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, China hosts 15 species, including subspecies, primarily growing in the arid and semi-arid regions of Ningxia, Xinjiang, Inner Mongolia, and other areas [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These plants have been utilized as food and herbal medicine in China for millennia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], with various parts like fruits, leaves, root bark, and young shoots used as local foods and/or medicines [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These plants impart potential pharmacological effects such as anti-aging properties, reduction of blood glucose and serum lipids, and immune regulation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several studies have demonstrated that extracts of these plants can prevent and treat diseases such as night sweats, diabetes, cough, vomiting, hypertension, and ulcers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Overall, previous studies have focused on the growth, development, medicinal value, and breeding of \u003cem\u003eLycium\u003c/em\u003e [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite its economic and ecological significance, much remains unknown about \u003cem\u003eLycium\u003c/em\u003e phylogeny, interspecific relationships, and evolutionary history. While numerous studies have identified and analyzed phylogenetic relationships within the \u003cem\u003eLycium\u003c/em\u003e genus using DNA barcode fragments [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], research on CPG phylogeny and lineage diversification is lacking.\u003c/p\u003e \u003cp\u003eChloroplasts are essential organelles involved in photosynthesis and carbon fixation in plant cells. The advancement in sequencing technologies has made whole CP\u003c/p\u003e \u003cp\u003esequencing more accessible [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Compared with DNA fragments, CPGs contain significantly more informative sites for analyzing nucleotide diversity and reconstructing phylogenies among closely related species [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. CPGs typically range from 75 to 250 kb in length, with numerous copies in a given cell, are maternally inherited in most plants, and have conserved gene content and order [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The plastome is characterized by two large inverted repeat regions (IRa and IRb) separated by two single-copy regions, referred to as the large single-copy region (LSC) and the small single-copy region (SSC) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Recently, CPGs have been used for comparative and phylogenetic analyses, proving useful in species identification, genetic diversity assessment, nucleotide diversity assessment, resolving phylogenetic relationships, and evolutionary history [\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the use of CPGs, few molecular phylogenetic studies have attempted to resolve infrafamilial relationships within \u003cem\u003eLycium\u003c/em\u003e using broad taxon sampling. Most of these studies were based on one or a few molecular loci or had sampling limitations, leaving our understanding of the phylogenetic relationships and divergence timescales of the genus unclear [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, we sequenced and aligned the CPGs of 15 species, including subspecies, of the genus \u003cem\u003eLycium\u003c/em\u003e from China. Our main objectives were to (a) construct a phylogeny based on the CPGs of 36 species from the Solanaceae family; (b) date the divergence of the \u003cem\u003eLycium\u003c/em\u003e clade; and (c) examine structural changes in the CPGs of the sampled \u003cem\u003eLycium\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacteristics of\u003c/strong\u003e \u003cstrong\u003eLycium\u003c/strong\u003e chloroplast genomes\u003c/p\u003e\n\u003cp\u003eAfter quality control and pre-processing, a minimum of 6 Gb of whole-genome sequencing data were obtained for each of the 16 species included in this study (Table S1). These clean reads were used to assemble complete CPs using a reference-guided approach. All the newly assembled CPGs displayed a typical quadripartite structure, with two IR regions separating the LSC and SSC regions (Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eFor each of the 15 \u003cem\u003eLycium\u003c/em\u003e species, the CPGs size ranged from 154,890 bp (\u003cem\u003eL. changjicum\u003c/em\u003e) to 155,677 bp (\u003cem\u003eL. amarum\u003c/em\u003e) (Table S1). All the CPGs had a typical quadripartite circular structure (Fig.\u0026nbsp;1) consisting of an LSC and an SSC region separated by a pair of IR regions (Fig.\u0026nbsp;1 and Table S1). The LSC region\u0026rsquo;s length varied from 85,892 bp (\u003cem\u003eL. changjicum\u003c/em\u003e) to 86,635 bp (\u003cem\u003eL. amarum\u003c/em\u003e), and the lengths of the SSC and IR regions ranged from 18,190 bp (\u003cem\u003eL. cylindricum\u003c/em\u003e) to 18,215 bp (\u003cem\u003eL. barbarum\u003c/em\u003e) and 25,394 bp (\u003cem\u003eL. ruthenicum\u003c/em\u003e) to 25,469 bp (\u003cem\u003eL. cylindricum\u003c/em\u003e), respectively (Table S1). The GC content of the entire plasmid sequence and the LSC, SSC, and IR regions was similar across all \u003cem\u003eLycium\u003c/em\u003e CPGs. Specifically, the GC content of the entire plasmid sequence was 37.8\u0026ndash;37.9%; while the GC content of the IR regions was 43.1\u0026ndash;43.2%, which was higher than that of the LSC and SSC regions (35.8\u0026ndash;35.9% and 32.3\u0026ndash;32.4%, respectively; Table S1). Additionally, the number of annotated genes in each CPGs ranged from 128 (\u003cem\u003eL. amarum\u003c/em\u003e) to 132 (\u003cem\u003eL. ningxiaens\u003c/em\u003e), and included 35\u0026ndash;37 tRNA and 8 rRNA genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigue 1\u003c/strong\u003e Gene map of the CPGs of fifteen \u003cem\u003eLycium\u003c/em\u003e species. Genes belonging to different functional groups are shown in different colors. The darker gray area in the inner circle indicates the GC content and the lighter gray indicates the AT content of the genome. The thick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into the small single-copy (SSC) and large single-copy (LSC) regions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative genomics and divergence hotspots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing \u003cem\u003eL. chinense\u003c/em\u003e as a reference, the CPGs of the 15 \u003cem\u003eLycium\u003c/em\u003e species were visually compared with those obtained using the mVISTA online database. The results showed that the CPGs of the 15 species were conserved, especially the coding regions (Fig.\u0026nbsp;2). Considering the entire plastome, the SSC and LSC regions displayed marked divergence compared to the IR regions. The proportion of non-coding regions was greater than that of protein-coding regions, and divergence hotspots were largely located in the intergenic spacer regions (Fig.\u0026nbsp;2). These divergence hotspots usually consist of highly variable sequences that can be used as potential DNA barcodes for phylogenetic analyses and to determine relationships between species. Therefore, to further understand DNA polymorphisms (Pi), mutation hotspot regions in the CPGs of the15 \u003cem\u003eLycium\u003c/em\u003e plants were screened using DnaSP software (Fig.\u0026nbsp;3). Pi analysis revealed that Pi values ranged from 0 to 0.00851, and the CPGs were relatively structurally conserved, small, and highly variable among the species. We identified five mutation hotspot regions (Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.0006) that could be used as potential molecular markers. Of these, \u003cem\u003etrn\u003c/em\u003eR(UCU), \u003cem\u003eatp\u003c/em\u003eF-\u003cem\u003eatp\u003c/em\u003eH, \u003cem\u003eycf\u003c/em\u003e3-\u003cem\u003etrn\u003c/em\u003eS(GGA), and \u003cem\u003etrn\u003c/em\u003eS(GGA) are located in the LSC region, while \u003cem\u003etrn\u003c/em\u003eL-UAG is located in the SSC region (Fig.\u0026nbsp;3). None of the mutation hotspots are located in the IR region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigue 2\u003c/strong\u003e Sequence alignment of the CPGs of \u003cem\u003eLycium\u003c/em\u003e species. The alignment was performed using the mVISTA program and the \u003cem\u003eL. chinense\u003c/em\u003e CP was used as a reference. The Y-axis: the degree of identity ranging from 50 to 100%. Coding and non-coding regions were marked in blue and red, respectively. Black arrows indicated the position and direction of each gene. CNS: conserved non-coding sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigue 3\u003c/strong\u003e Sliding window test of nucleotide diversity (Pi) in the multiple alignments of 15 \u003cem\u003eLycium\u003c/em\u003e species species (window length: 600 bp; step size: 200 bp). X-axis: the position of the midpoint of the window; Y-axis: the nucleotide diversity of each window.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigue 4\u003c/strong\u003e Comparisons of the borders of the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions among the CPGs of fifteen \u003cem\u003eLycium\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBoundaries between IR and SC regions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe boundaries of the LSC, SSC, and IR regions were highly consistent within \u003cem\u003eLycium\u003c/em\u003e and no obvious expansion or contraction of the IR region was detected in the 15 CPGs (Fig.\u0026nbsp;4). Here, \u003cem\u003etrn\u003c/em\u003eH was shown to be the first gene in the LSC region at the junction between IRa and LSC (i.e., IRa/LSC). At the other end of the LSC region, the IRb/LSC junctions were located in the \u003cem\u003erps\u003c/em\u003e19 gene sequence in all \u003cem\u003eLycium\u003c/em\u003e species, with the length of the \u003cem\u003erps\u003c/em\u003e19 gene located in the IRb region, varying from 46 to 50 bp. At both ends of the SSC region, IRb/SSC junctions were located between the \u003cem\u003eycf\u003c/em\u003e1 and \u003cem\u003endh\u003c/em\u003eF genes. However, SSC/IRa was found in the \u003cem\u003eycf\u003c/em\u003e1 gene, with the length of the \u003cem\u003eycf\u003c/em\u003e1 gene located in the IRa region varying from 995 to 1004 bp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analyses of Solanaceae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo infer the phylogenetic relationships of the 36 Solanaceae species, we included two Convolvulaceae species whose CPGs are publicly available in the GenBank database. These species (\u003cem\u003eCalystegia hederacea\u003c/em\u003e and \u003cem\u003eConvolvulus arvensis\u003c/em\u003e) were used as the outgroup for phylogenetic analyses. The final concatenated dataset included 55 plastid genes and 43,044 sites, after trimming poorly aligned regions and gaps with missing genes. In the phylogenetic trees, all nodes in the ML and BI analyses of each dataset generated almost congruent topologies with high bootstrap support (BS) and posterior probability (PP) values (Fig.\u0026nbsp;5 and S1, S2, S3, S4). Thirty-six plant species belonging to the Solanaceae family were divided among three typical branches. Branches of \u003cem\u003ePhysochlaina physaloides\u003c/em\u003e, \u003cem\u003ePrzewalskia tangutica\u003c/em\u003e, \u003cem\u003eScopolia carniolica\u003c/em\u003e, \u003cem\u003eAtropanthe sinensis\u003c/em\u003e, \u003cem\u003eSolanum betaceum\u003c/em\u003e, \u003cem\u003eAnisodus acutangulus\u003c/em\u003e, \u003cem\u003eHyoscyamus niger\u003c/em\u003e, and \u003cem\u003eAtropa belladonna\u003c/em\u003e were resolved as sisters of \u003cem\u003eLycium\u003c/em\u003e. Further, in the CDS phylogeny, \u003cem\u003eLycium\u003c/em\u003e species were clustered on one large branch, confirming that the independence of this genus is highly supported (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1) (Fig. S1 and S2). The 15 species of plants belonging to \u003cem\u003eLycium\u003c/em\u003e were divided into two typical sub-branches and three minor branches: clade I-1, comprising \u003cem\u003eL. barbarum\u003c/em\u003e var. \u003cem\u003eauranticarpum\u003c/em\u003e, \u003cem\u003eL. cylindricum\u003c/em\u003e, \u003cem\u003eL. yunnanense\u003c/em\u003e, \u003cem\u003eL. barbarum\u003c/em\u003e, \u003cem\u003eL. dasystemum\u003c/em\u003e var. \u003cem\u003erubricaulium\u003c/em\u003e, and \u003cem\u003eL. dasystemum\u003c/em\u003e on one sub-branch (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1); clade I-2 consisting of \u003cem\u003eL. chinense\u003c/em\u003e var. \u003cem\u003epotaninii\u003c/em\u003e, \u003cem\u003eL. truncatum\u003c/em\u003e, \u003cem\u003eL. amarum\u003c/em\u003e, and \u003cem\u003eL. chinense\u003c/em\u003e on another sub-branch (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1), and clade II comprising \u003cem\u003eL. barbarum\u003c/em\u003e var. \u003cem\u003eimplicatum\u003c/em\u003e, \u003cem\u003eL. qingshuiheense\u003c/em\u003e, \u003cem\u003eL.ningxiaense\u003c/em\u003e, \u003cem\u003eL. changjicum\u003c/em\u003e, and \u003cem\u003eL. ruthenicum\u003c/em\u003e on a different branch (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigue 5\u003c/strong\u003e Phylogeny and clade divergence of Solanaceae and outgroups based on 55 plastome protein-coding genes. Stars indicate fossil calibrations used in this analysis. Geological periods are marked with background colors. Mya: million years ago; Pal: Paleocene; Pli: Pliocene; Ple: Pleistocene\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEstimated ages for \u003cem\u003eLycium\u003c/em\u003e subclades\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSubclade name\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean age (Mya)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% highest posterior density interval (HPD)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSubclade 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.0-21.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSubclade 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.09\u0026ndash;3.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSubclade 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.64\u0026ndash;2.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e*Subclades are labeled in Fig. 5\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDivergence time estimation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe estimated the divergence timescales of the major clades within \u003cem\u003eLycium\u003c/em\u003e according to the calibration of the gene tree constructed based on 55 plastid genes. The split between \u003cem\u003eLycium\u003c/em\u003e and its sister group was estimated to have occurred 17.7 Mya. The crown ages of all subclades in the genus \u003cem\u003eLycium\u003c/em\u003e were dated mainly within the Pleistocene, suggesting that numerous species of this genus originally diversified in the recent past (6 Mya) (Fig.\u0026nbsp;5 and Table\u0026nbsp;1).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have reported that most angiosperms have CPGs ranging in size from 120 to 170 kb, with the IR region typically spanning 20\u0026ndash;30 kb [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, a comparative analysis of the CPGs indicated that those of 15 \u003cem\u003eLycium\u003c/em\u003e species from China were at the larger end of the spectrum for angiosperm organelle genomes, ranging from 154,890 bp (\u003cem\u003eL. changjicum\u003c/em\u003e) to 155,677 bp (\u003cem\u003eL. amarum\u003c/em\u003e). All \u003cem\u003eLycium\u003c/em\u003e species exhibited a typical quadripartite structure that is similar to other higher plants, comprising the LSC region (85,892\u0026ndash;86,635 bp), SSC region (18,190\u0026ndash;18,215 bp), and two identical IR regions (25,394\u0026ndash;25,469 bp). The GC content of the entire plastid sequence and the LSC, SSC, and IR regions was similar across all \u003cem\u003eLycium\u003c/em\u003e CPGs. In agreement with numerous studies on angiosperms, the IR regions exhibited the highest GC content [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The conversion between sequences and higher GC content may contribute to the greater conservation of IR regions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn angiosperms, the IR region is relatively conserved in sequence and structure. The narrowing and widening of its edges are not only important factors for length variation, but also the main cause of the emergence of pseudogenes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. While cp genes have evolved slowly and are relatively conserved in terms of sequence and structure, boundary contraction and expansion in the IR regions are common phenomena. In our study, we analyzed 15 CPGs within the highly conserved \u003cem\u003eLycium\u003c/em\u003e species and noticed that no major expansions or contractions occurred in the IR regions.\u003c/p\u003e \u003cp\u003eHighly variable regions offer valuable phylogenetic information. For example, variable regions aid in species kinship identification and gene pool construction [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A good DNA barcode must be a short, representative DNA fragment with high variability and amenable to amplification [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, both the sequence and structure of \u003cem\u003eLycium\u003c/em\u003e CPGs were highly conserved. mVISTA analysis revealed that most of the variation in nucleotide sequences occurred in noncoding regions, consistent with previous reports, suggesting this variation as a common feature of angiosperms [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the \u003cem\u003eLycium\u003c/em\u003e, several highly variable regions, such as \u003cem\u003emat\u003c/em\u003ek, \u003cem\u003erps\u003c/em\u003e16\u0026ndash;\u003cem\u003etrn\u003c/em\u003eK and \u003cem\u003etrn\u003c/em\u003eH\u0026ndash;\u003cem\u003epsb\u003c/em\u003eA, are recognized as potential DNA barcoding sites [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In addition, our nucleotide diversity (Pi) analysis led to the identification of five highly variable regions with Pi values greater than 0.006, including three non-coding tRNA regions (\u003cem\u003etrn\u003c/em\u003eS, \u003cem\u003etrn\u003c/em\u003eL, and \u003cem\u003etrn\u003c/em\u003eR) and two intergenic regions (\u003cem\u003eatp\u003c/em\u003eF-\u003cem\u003eatp\u003c/em\u003eH and \u003cem\u003eycf\u003c/em\u003e3-\u003cem\u003etrn\u003c/em\u003eS(GGA). In conclusion, these mutation hotspot regions play an important role in the identification and characterization of \u003cem\u003eLycium\u003c/em\u003e plant species.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLycium\u003c/em\u003e is a genus of shrubs with significant economic and ecological importance. Therefore, considerable efforts have been dedicated to cultivated practices, growth and development, medicinal application, and breeding [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, to date, few studies have explored its phylogenetic relationships and divergent timescales [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The CPGs are central to molecular biology research and have become a prominent focus. In particular, species identification, phylogenetic relationships, and the reconstruction of evolutionary history via whole-genome sequencing have become important tools because of improvements in sequencing technology and low costs. Phylogenetic trees constructed based on a single or few gene sequences often yield inconsistent or even conflicting topologies due to variations in evolutionary rates and horizontal shifts between genes. This complication affects the determination of accurate evolutionary relationships among species [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, we constructed a phylogenetic tree using the BI and ML methods. The CPGs of 15 \u003cem\u003eLycium\u003c/em\u003e species converged into branches with high support (Fig. S3 and S4). Notably, \u003cem\u003eL. cylindricum\u003c/em\u003e, \u003cem\u003eL. yunnanense\u003c/em\u003e, \u003cem\u003eL. dasystemum\u003c/em\u003e, and \u003cem\u003eL. dasystemum\u003c/em\u003e were similar to \u003cem\u003eL. barbarum\u003c/em\u003e and closely related, while \u003cem\u003eL. chinense\u003c/em\u003e was classified in the same branch as \u003cem\u003eL. truncatum\u003c/em\u003e and \u003cem\u003eL. amarum\u003c/em\u003e (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1.00, respectively), indicating that the three species are similar. Based on the phylogenetic tree results, we speculate that \u003cem\u003eL. qingshuiheense\u003c/em\u003e, \u003cem\u003eL. ningxiaense\u003c/em\u003e, and \u003cem\u003eL. changjicum\u003c/em\u003e may have originated from \u003cem\u003eL. ruthenicum\u003c/em\u003e. Although \u003cem\u003eL. barbarum\u003c/em\u003e var. \u003cem\u003eimplicatum\u003c/em\u003e and \u003cem\u003eL. qingshuiheense\u003c/em\u003e formed a small, distinct branch, the support rate was low, indicating that they may be the same species. This study demonstrates that high-resolution CPG sequences provide valuable resources for broad research on genetic information and species identification of \u003cem\u003eLycium\u003c/em\u003e spp.\u003c/p\u003e \u003cp\u003eThe highly conserved and stable alignment of the 55 plastid genes allowed us to calibrate the divergence and origin of \u003cem\u003eLycium\u003c/em\u003e (Fig.\u0026nbsp;5). We used three tentative calibrations to estimate the diversification. While the estimated ages should be used with caution, our findings indicate that the \u003cem\u003eLycium\u003c/em\u003e diverged from the sister genus around 17.7 Mya, and the two successive clades within the \u003cem\u003eLycium\u003c/em\u003e diverged 4.95 and 1.7 Mya, suggesting relatively late clade diversifications. Specifically, most species diversification within the subclades of \u003cem\u003eLycium\u003c/em\u003e, as estimated from these plastid genes, appeared to have occurred in the recent past, mostly after 5 Mya., despite the fact that numerous species are currently acknowledged in genera. This observation may partly account for the widespread hybridization observed among these young species [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], likely resulting from incomplete reproductive isolation. The divergence timescales estimated here for the major subclades will serve as a basic timescale for diverse studies on this economically and ecologically important genus.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe analyzed the complete CPGs of 15 \u003cem\u003eLycium\u003c/em\u003e species and found that all exhibited a quadripartite structure, typical of most angiosperms. The CPG arrangement was highly conserved, with great sequence variation observed in the SSC region compared with that in the IR region. We did not detect major expansions or contractions in the IR region, nor did we find any rearrangements or insertions in the CPGs of the 15 \u003cem\u003eLycium\u003c/em\u003e species. We identified highly variable regions within \u003cem\u003eLycium\u003c/em\u003e that are likely to be useful for species delimitation. Phylogenetic tree construction based on the CPGs showed that all 15 \u003cem\u003eLycium\u003c/em\u003e species formed a monophyletic group, divided into two typical subbranches and three minor branches. Molecular dating suggested that \u003cem\u003eLycium\u003c/em\u003e diverged from its sister genus around 17.7 Mya, with species diversification of the \u003cem\u003eLycium\u003c/em\u003e species of China occurring mainly within the recent Pliocene epoch. Overall, our findings and the estimated divergence times will facilitate future studies on \u003cem\u003eLycium\u003c/em\u003e, assist in species differentiation, and facilitate diverse studies of this economically and ecologically significant genus.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eTaxon sampling, DNA extraction, and plastome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 38 CPGs representing Solanaceae and related families were included in this study (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). 36 CPGs from Solanaceae were selected, including all \u003cem\u003eLycium\u003c/em\u003e species found in China. Further, two additional CPGs from related families in Convolvulaceae were chosen as outgroups for phylogenetic analysis. Among these 38 CPGs, 16 complete CPGs were newly sequenced, and the others were obtained from GenBank (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). The leaves used in this study were collected from natural populations in China. The plant materials were identified by Dr. Lei Zhang and the voucher specimens (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e) were deposited in the Herbarium of North Minzu University (NMU; Yinchuan, China). For each species, we extracted total DNA from dried leaves preserved them in silica gel using the CTAB protocol [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. Paired-end libraries with an insert size of 500 base pairs (bp) were constructed by Illumina (Qingdao, Shandong, China) following sequencing with a HiSeq \u0026times; Ten System (Jizhi, Qingdao, Shandong, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChloroplast genome and annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt least two gigabases (Gb) of 2 \u0026times; 150 bp short read data were generated for each sample. Reads with quality scores of less than 7 and with more than 10% ambiguous nucleotides were filtered. The remaining reads were assembled using NOVOPlasty version 2.7.2 [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e] software. The contigs were aligned into sequence in Geneious version 9.1.8 [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e] software using the \u003cem\u003eL. chinense\u003c/em\u003e CPG as a reference. The CPGs were annotated using Plann version 1.1 [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. Protein-coding genes were extracted using customized Python scripts. Alignment of chloroplast genes across all species was performed using PRANK version 130410 [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e] software. Poorly aligned regions were trimmed using Gblocks version 0.91b [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e] with the option \u0026ldquo;\u0026minus;t\u0026thinsp;=\u0026thinsp;c,\u0026rdquo; selected to set the type of sequence to codons. Genes that were absent in at least one species were excluded, and the aligned sequences were combined into a super matrix. Additionally, circular maps of the CPGs were created using OGDRAW version 1.2 [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e], and all annotated CPGs were submitted to GenBank [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative genomics and structural analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structural variation and identification of arrangement events across \u003cem\u003eLycium\u003c/em\u003e was conducted for the 15 CPGs of \u003cem\u003eLycium\u003c/em\u003e. The results of the comparative analysis of the CPGs were visualized with the mVISTA program [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e] and the annotated CPG of \u003cem\u003eL. chinense\u003c/em\u003e was used as the reference in the LAGAN mode [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. The junction sites of the four structural regions (IRA, LSC, SSC, and IRB) and adjacent genes in 15 \u003cem\u003eLycium\u003c/em\u003e CPGs were visualized and compared using IRscope [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e] software to obtain a macroscopic view of the CPG structure. Following sequence alignment, nucleotide diversity (Pi) analysis of the CPG was performed using DnaSP version 6.0 [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic inference and divergence time estimation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe generated two datasets for phylogenetic analysis: a protein-coding region (CDS) set and a whole plastome (WP) set. Protein-coding genes (PCGs) were extracted from the GenBank formatted file containing 38 CPGs using customized Perl scripts that removed the start and end codons. After excluding possible pseudogenes, 55 PCGs were retained in all species. Each PCG was aligned using PRANK version130410 based on the translated amino acid sequences. Genes that had been lost in at least one species were discarded and then the remaining aligned sequences were concatenated into a super matrix. Independent phylogenetic analyses were performed for each dataset (CDS and WP) using the maximum likelihood (ML) and Bayesian inference (BI) methodologies. We used RAxML version 8.1.24 [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e] to conduct ML analyses with a general time reversible model with a gamma distribution (GTR\u0026thinsp;+\u0026thinsp;\u0026Gamma;). The best-scoring ML tree was obtained using the rapid hill-climbing algorithm (i.e., the option \u0026ldquo;-f d\u0026rdquo;) with 1,000 bootstrap replicates. The optimal model (GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G) was identified using jModeltest software, and BI analysis was conducted using MrBayes version 3.2.6 [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]. Additionally, FigTree version 1.4.2 [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e] was used to visualize phylogeny.\u003c/p\u003e\n\u003cp\u003eWe estimated divergence times from the plastome dataset using an approximate likelihood method, as implemented in MCMCtree in PAML version 4 [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e] software, with independent relaxed-clock and birth\u0026ndash;death sampling [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e] strategies. Fossil dates were used as calibration points to reduce bias for more accurate age estimates [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]. However, the fossil records of Solanaceae are very limited. Three fossils were used to constrain the internal nodes: (1) According to the ancient seed fossil of Solanum nigrum [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e], 5.3\u0026ndash;11.6 Mya was the age range assigned to the split between Solanum nigrum and its sister species \u003cem\u003eTubocapsicum anomalum\u003c/em\u003e. (2) The split between Solanum and \u003cem\u003eLycium\u003c/em\u003e was assigned an age range of 19.0-23.3 Mya as previously estimated [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e]. (3) The root of the phylogeny was restricted to an age range of 46.2\u0026ndash;53.7 Mya based on the secondary age constraints described by S\u0026auml;rkinen et al. [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e]. The best-fit GTR\u0026thinsp;+\u0026thinsp;\u0026Gamma; model was selected and the prior of the substitution rate (rgene) was modeled by a \u0026Gamma; distribution as \u0026Gamma; (2, 200, 1). We set parameters for the birth\u0026ndash;death process with species sampling and \u0026sigma;\u003csup\u003e2\u003c/sup\u003e values of (1, 1, 0.1) and G (1, 10, 1), respectively. We executed the MCMC runs for 2,000 generations as burn-in and then sampled every 750 generations until 20,000 samples were obtained. We compared two MCMC runs for convergence using random seeds and obtained similar results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant samples were collected conforming to the national / international legislation and institutional guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16 newly assembled and annotated CPGs have been submitted to NCBI (https://www. ncbi. nlm. nih. gov), with accession numbers listed in table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Ningxia Natural Science Foundation (2022AAC05063, 2021AAC02005) and the National Natural Science Foundation of China (32260049). The anonymous reviewers and editors are sincerely acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLei Zhang and Guoqi Zheng developed the concept of the study. Lei Zhang and Erdong Zhang conducted the experiment and data analysis. Lei Zhang, Yuqing Wei and Guoqi Zheng drafted the manuscript. Yuqing Wei and Guoqi Zheng supervised the study. All authors revised the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang JX, Guan SH, Feng RH, Wang Y, Wu ZY, Zhang YB, Chen XH, Bi KS, Guo DA. Neolignanamides, lignanamides, and other phenolic compounds from the root bark of \u003cem\u003eLycium chinense\u003c/em\u003e. Journal of Natural Products. 2013;76:51\u0026ndash;58.\u003c/li\u003e\n\u003cli\u003eTurchetto C, Fagundes NJ, Segatto AL, Kuhlemeier C, Solis Neffa VG, Speranza PR, Bonatto SL, Freitas LB. 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A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Ecology Evolution. 2013;13:214.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lycium, Plastome structure, Comparative analysis, Phylogenetic relationship, Divergence time","lastPublishedDoi":"10.21203/rs.3.rs-4002205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4002205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eLycium\u003c/em\u003e is an economically and ecologically important genus of shrubs, consisting of approximately 70 species distributed worldwide, 15 of which are located in China. Despite the economic and ecological importance of \u003cem\u003eLycium\u003c/em\u003e, its phylogeny, interspecific relationships, and evolutionary history remain relatively unknown. In this study, we constructed a phylogeny and estimated divergence time based on the chloroplast genomes (CPGs) of 15 species, including subspecies, of the genus \u003cem\u003eLycium\u003c/em\u003efrom China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe sequenced and annotated 15 CPGs in this study. Comparative analysis of these genomes from these \u003cem\u003eLycium\u003c/em\u003e species revealed a typical quadripartite structure, with a total sequence length ranging from 154,890 to 155,677 base pairs (bp). The CPGs was highly conserved and moderately differentiated. Through annotation, we identified a total of 128–132 genes. Analysis of the boundaries of inverted repeat (IR) regions showed consistent positioning: the junctions of the IRb/LSC region were located in \u003cem\u003erps\u003c/em\u003e19 in all \u003cem\u003eLycium\u003c/em\u003e species, IRb/SSC between the \u003cem\u003eycf\u003c/em\u003e1 and \u003cem\u003endh\u003c/em\u003eF genes, and SSC/IRa within the \u003cem\u003eycf\u003c/em\u003e1 gene. Sequence variation in the SSC region exceeded that in the IR region. We did not detect major expansions or contractions in the IR region or rearrangements or insertions in the CPGs of the 15 \u003cem\u003eLycium\u003c/em\u003e species. Comparative analyses revealed five hotspot regions in the CPG: \u003cem\u003etrn\u003c/em\u003eR(UCU), \u003cem\u003eatp\u003c/em\u003eF-\u003cem\u003eatp\u003c/em\u003eH, \u003cem\u003eycf\u003c/em\u003e3-\u003cem\u003etrn\u003c/em\u003eS(GGA), \u003cem\u003etrn\u003c/em\u003eS(GGA), and \u003cem\u003etrn\u003c/em\u003eL-UAG, which could potentially serve as molecular markers. In addition, phylogenetic tree construction based on the CPG indicated that the 15 \u003cem\u003eLycium\u003c/em\u003e species formed a monophyletic group and were divided into two typical subbranches and three minor branches. Molecular dating suggested that \u003cem\u003eLycium\u003c/em\u003e diverged from its sister genus approximately 17.7 million years ago (Mya) and species diversification within the \u003cem\u003eLycium\u003c/em\u003e species of China primarily occurred during the recent Pliocene epoch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe divergence time estimation presented in this study will facilitate future research on \u003cem\u003eLycium\u003c/em\u003e, aid in species differentiation, and facilitate diverse investigations into this economically and ecologically important genus.\u003c/p\u003e","manuscriptTitle":"Phylogenetic analysis and divergence time estimation of Lycium species in China based on the chloroplast genomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 04:51:04","doi":"10.21203/rs.3.rs-4002205/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-05-04T13:29:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"dbfc7ea1-fa0e-4b8e-8418-eca7b0ee0d59","date":"2024-04-02T16:33:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-22T16:41:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"fd75ca86-1456-44d1-a2eb-922e6a39218d","date":"2024-03-19T17:38:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-08T12:33:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-08T11:53:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-04T17:41:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-04T15:35:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-03-01T05:40:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e144701-aecb-42f5-9676-bedfb195be39","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-31T09:33:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 04:51:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4002205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4002205","identity":"rs-4002205","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}