Hybridization of Lycium qingshuiheense and L. ningxiaense revealed using morphological, SSR markers, and chloroplast genomic data

Hybridization of Lycium qingshuiheense and L. ningxiaense revealed using morphological, SSR markers, and chloroplast genomic data | 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 Hybridization of Lycium qingshuiheense and L. ningxiaense revealed using morphological, SSR markers, and chloroplast genomic data Lei Zhang, Chaopan Zhang, Erdong Zhang, Yuqing Wei, Guoqi Zheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5874276/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in BMC Genomics → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Interspecific hybridization is a common natural phenomenon that plays a crucial role in the formation and maintenance of species. In recent years, the origins of hybrids have been extensively investigated across various plant groups. Lycium has received relatively little attention regarding the occurrence of natural hybrids, despite its economic and ecological significance. In this study, hybridization of L. qingshuiheense and L. ningxiaense was identified based on morphological traits, nuclear simple sequence repeat (SSR) markers, and chloroplast genomic data. Results: We selected 90 specimens of four species across their distributional ranges to examine 12 morphological traits and characterize clusters with distinct phenological traits. Similarly, we genotyped 67 individuals from four species across 10 populations using 10 SSR markers. The morphological analyses revealed a clear division between individuals of L. barbarum and L. ruthenicum while most individuals of L. qingshuiheense and L. ningxiaense were distributed between L. barbarum and L. ruthenicum . The genetic clusters were consistent with the morphological clusters. However, most of the L. ningxiaense samples comprised F1 hybrids, while L. qingshuiheense showed a distinct genetic pattern compared to that of L. ningxiaense due to backcross or interspecific genetic infiltration. Among the four Lycium species, the composition and structureal features of chloroplast genomes from L. qingshuiheense and L. ningxiaense were identical and clustered on a branch with L. ruthenicum in the phylogenetic tree. Conclusion: Combining the morphological, SSR markers, and cp genomes analysis, we affirmed that L. qingshuiheense and L. ningxiaense are hybrids originating from crossing L. ruthenicum (♀) and L. barbarum (♂). This study elucidated the origin of two Lycium species, which contributes to our understanding of the evolutionary mechanisms underlying interspecific hybridization in Lycium . Furthermore, it provides a scientific foundation for the development, utilization, and improvement of Lycium varieties. Lycium morphological analyses simple sequence repeat (SSR) chloroplast genome hybridization identification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Lycium is a genus of shrubs belonging to the Solanaceae family. Lycium species show robust adaptability to different surroundings, making them pioneer plants in preventing wind and sand erosion and resisting land salinization [ 1 – 2 ]. This genus consists of more than 80 species, primarily distributed across South America, southwest North America, and Africa, with a limited number of species found in the temperate regions of Eurasia [ 3 – 4 ]. In China, the recorded number of Lycium species ranges from 8 to 15, including subspecies, primarily growing in the arid and semi-arid regions of Ningxia, Xinjiang, Inner Mongolia, and other provinces [ 5 – 6 ]. For centuries, certain species within the Lycium genus, including L. barbarum and L. ruthenicum , have been used in China both as traditional herbal remedies and dietary staples [ 7 – 8 ]. Various parts of Lycium species, like fruits and root bark, are used to treat a variety of ailments and are also to create tonics, because they provide benefits such as immune regulation and reduction of blood glucose and serum lipids [ 9 – 10 ]. Lycium species exhibits significant genetic diversity. However, species delimitation of Lycium has remained problematic due to extensive morphological variation and frequent interspecific hybridization [ 11 – 12 ]. Several previously described species, including some from China, may actually be hybrids or intraspecific variations of other species, leading to taxonomic uncertainty. However, these ambiguous species have not been comprehensively investigated [ 13 ]. With ongoing advancements in molecular biology, the use of molecular techniques in species delimitation research has increased [ 14 – 15 ]. Simple sequence repeats (SSRs or microsatellites) possess the advantages of codominant inheritance and distinct marker characteristics, making them an ideal tool for studying genetic diversity and population genetics [ 16 – 17 ]. Recent studies using nuclear SSR genotyping have explored the interspecific relationships and hybridization of Lycium [ 18 – 19 ], demonstrating significant genetic variation among species and differentiating between Lycium spp. and hybrids [ 20 – 25 ]. Additionally, chloroplasts (cp) possess their own genome, making them a useful tool for identifying the maternal parent of hybrid offspring and thus clarifying phylogenetic relationships [ 26 – 27 ]. Several recent studies have used cp genomes for species delimitation and phylogenetic analysis to resolve phylogenetic relationships [ 28 – 31 ]. In this study, we used four Lycium species: L. ningxiaense R. J. Wang and Q. Liao [ 5 , 32 ], L. qingshuiheense X. L. Jiang and J. N. Li [ 6 ], L. barbarum and L. ruthenicum . These species were chosen due to their close morphological similarities and geographical overlap, which can lead to potential confusion in species identification. L. qingshuiheense and L. ningxiaense were found in central Ningxia, China, and share morphological traits with L. barbarum and L. ruthenicum , which are widespread in northwest China. Despite these similarities, L. barbarum and L. ruthenicum can be distinguished based on their distribution ranges and morphological features. L. ruthenicum has linear or subcylindric leaves and purple-black, globose fruit, while L. barbarum has lanceolate or long-elliptic leaves and red or orange-yellow, oblong or ovoid fruit [ 4 ]. However, field observations indicate that L. qingshuiheense and L. ningxiaense display intermediate morphological traits, including leaf shape, flower characteristics, and fruit color, suggesting the possibility of hybridization between L. barbarum and L. ruthenicum . Thus, it is speculated that the L. qingshuiheense and L. ningxiaense may be the result of hybridization. To clarify the relationship among these species, this study integrated molecular data, which has proven effective in resolving species boundaries where morphological traits alone are insufficient. As recently recommended for integrated species delimitation [ 33 ], this study used 10 nuclear SSRs (or microsatellite loci), cp genomes, and morphological data to assess the genetic composition and structure of L. qingshuiheense and L. ningxiaense . The current study aimed to address the following questions: (1) Are L. qingshuiheense and L. ningxiaense the product of hybridization? (2) What is the genetic composition and structure resulting from this hybridization? (3) Do the parent species involved in hybridization differ between L. qingshuiheense and L. ningxiaense ? Results Morphological clustering We conducted principal coordinate analysis (PCoA) and hierarchical clustering on 12 observed and measured traits of 90 Lycium specimens. These traits each accounted for > 5% of the variance in the data and included PC1 to PC4 (PC1 = 51.22%; PC2 = 16.63%; PC3 = 10.24%; PC4 = 7.95%) and combined they represented 84.99% of variance. All other PCs individually accounted for < 5% of the variance in the data. The greatest amount of shape variance was observed across PC1 and PC2 (Fig. 1 A). The arrangement of the scatterplot was based on (PCo1) and (PCo2), individuals of L. barbarum and L. ruthenicum formed two clear groups, while most individuals of L. qingshuiheense and L. ningxiaense were distributed between L. barbarum and L. ruthenicum , with only a few samples distributed in the L. ruthenicum group. All other PCs showed similar relationships. Separate analysis of variance (ANOVA) tests on the 12 quantitative traits across 90 specimens representing four distinct species revealed several findings: L. barbarum demonstrated significantly larger measurements compared to other species in seven traits: petiole length, leaf length, leaf width, leaf area, corolla radius, corolla lobes, and fruit color (R) (Fig. 1 B; S1). Contrastingly, L. ruthenicum displayed longer floral tubes relative to other species (Fig. 1 C). In addition, the fruit color(G) of L. qingshuiheense and L. ningxiaense was notably similar (Fig. 1 D), with the variation between these two species being more pronounced than that observed between L. barbarum and L. ruthenicum (Fig. 1 D). For the remaining quantitative traits, no significant differences were detected among the four species (Fig. S1 ). Clustering analyses of nuclear SSR (nSSR) microsatellite data Structural analysis of the 10 nSSR loci for 67 individuals from the four Lycium species showed that the data comprised two genetic clusters (K = 2), based on both the Pritchard and Evanno methods (Fig. 2 A). When K = 2, The first cluster was composed primarily of L. barbarum . In contrast, the second cluster included all L. ruthenicum individuals (Fig. 2 B). The genetic composition of L. qingshuiheense and L. ningxiaense represented a genetic mix of the two clusters. The PCoA of the nSSR data showed that L. barbarum and L. ruthenicum formed distinct clusters, with all the sampled individuals of both L. qingshuiheense and L. ningxiaense dispersed between them, indicating mixed origins. However, compared with L. ningxiaense , L. qingshuiheense had more genetic components from L. ruthenicum (Fig. 2 C). Characteristics of the cp genomes of the four Lycium species The length of the 27 cp genomes from the four Lycium species ranged from 154,830 bp in L. ruthenicum to 155,148 bp in L. barbarum (Fig. 3 ; Table S1 ). All the cp genomes exhibited characteristic quadripartite circular structures, consisting of a large single copy (LSC) region and a small single copy (SSC) region, which were separated by two inverted repeat (IR) regions (Fig. 3 ). The lengths of the LSC regions varied from 85,831 to 85,991 bp, while the lengths of the SSC and IR regions ranged from 18,190 to 18,209 bp and from 25,395 to 25,458 bp, respectively (Table S1 ). The GC content of the cp genomes was found to be 37.9%, whereas the GC content within the IR regions ranged between 43.14% and 43.19%. This is notably higher than that observed in the LSC and SSC regions, which were measured at 35.92%-35.98% and 32.31%-32.35%, respectively (Table S1 ). Notably, the total length, LSC length, SSC length, IR length, and the GC content of the entire genome and that of individual regions of L. qingshuiheense and L. ningxiaense were similar (closer to L. ruthenicum than to L. barbarum ). Additionally, all cp genomes contained 132 annotated genes, including 87 protein-coding genes (PCGs), 8 rRNA genes, and 37 tRNA genes (Table 1 ). Table 1 Chloroplast genome genes annotation of four Lycium species Classification Gene family Gene name Protein-coding genes (87) Photosystem I psa A, B, C, I, J Photosystem II psb A, B, C, D, E, F, H, I, J, K, L, M, N, T, Z Cytochrome b/f complex pet A, B*, D*, G, L, N ATP synthase atp A, B, E, F*, H, I NADH plastoquinone oxidoreductase ndh A**, B**(x2), C, D, E, F, G, H, I, J, K ribulose−1,5-bisphosphate carboxylase rbc L Small subunit ofribosome rps 11, 12**(x2), 14, 15, 16*, 18, 19, 2, 3, 4, 7(x2), 8 Large subunit ofribosome rpl 14, 16*, 2*(x2), 20, 22, 23(x2), 32, 33, 36 DNA dependent RNA polymerase rpo A, B, C1*, C2 Maturase mat K Acetyl-CoAcarboxylase acc D Cytochrome C heme attachment protein ccs A Chloroplast envelop membrane protein cem A Clp protease clp P ** Hypothetical chloroplast conserved open reading frame ycf 1(x2), 2(x2), 3**, 4, 15(x2) rRNA genes (8) rRNA genes rrn 16(x2), 23(x2), 4.5(x2), 5(x2) tRNA genes (37) tRNA genes trn A(UGC)*(x2), C(GCA), D(GUC), E(UUC), F(GAA), fM(CAU), G(GCC), G(UCC)*, H(GUG, I(CAU)(x2), I(GAU)*(x2), K(UUU)*, L(CAA)(x2), L(UAA)*, L(UAG), M(CAU), N(GUU)(x2), P(UGG), Q(UUG), (ACG)(x2), (UCU), S(GCU), S(GGA), S(UGA), T(GGU), T(UGU), V(GAC)(x2), V(UAC)*, W(CCA), Y(GUA) * represents a gene with one intron, ** represents a gene with two introns, (x2) repeat genes Phylogenetic analysis of the four Lycium species based on their cp genomes We utilized L. chinese as an outgroup to elucidate the phylogenetic relationships among the four species based on their cp genomes. The final concatenated dataset comprised 79 plastid genes and 67,422 sites (Table S2 ). In the phylogenetic trees, most nodes generated from both the maximum likelihood (ML) and Bayesian inference (BI) analyses exhibited nearly congruent topologies (Fig. 4 ; Figs. S2, S3, S4). A total of 27 individuals from four Lycium species were divided into two typical branches. Clade 1, comprised L. barbarum (BS; PP = 100%, 1); and Clade 2, consisted of L. qingshuiheense , L. ningxiaense , and L. ruthenicumon (BS; PP = 100%, 1) (Fig. 4 ; Figs. S2, S3, S4). Discussion This study investigated the hybrid origins of L. qingshuiheense and L. ningxiaense , focusing on species delimitation and their genetic and morphological characteristics in relation to L. barbarum and L. ruthenicum . Our results provide evidence for their hybrid origin, as both L. qingshuiheense and L. ningxiaense exhibited intermediate morphological traits and genetic profiles, which is consistent with the admixture of the two parental species. Previous research indicates that the morphological analysis is highly effective for distinguishing between closely related species [ 16 , 34 ]. Specifically, it is used to differentiate between overall changes in gross morphology, even if the individual morphology of different species overlaps [ 35 – 36 ]. Morphologically, L. qingshuiheense and L. ningxiaense displayed leaf size and shape, corolla size, and fruit color that fall between those of L. barbarum and L. ruthenicum (Fig. 1 ; S1). The intermediate traits observed in our study support the hypothesis of hybridization, which is in line with previous research showing that hybrid species often exhibit characteristics blending those of their parent species [ 15 , 37 – 39 ]. Genetic analyses using SSR markers and cp genome sequencing further supported this conclusion, as both species showed genetic profiles indicative of hybridization, with alleles closely resembling those of the parental species (Fig. 2 ). Interspecific hybridization is a common phenomenon in nature [ 40 – 41 ], and hybrid swarms or hybrid zones provide valuable insights into interspecific gene flow and hybrid fitness [ 42 – 44 ]. Although the origins of hybrids have been explored in numerous plant groups, such as Populus [ 45 ], Rhododendron [ 46 ] and Mimulus [ 47 ], little attention has been paid to natural hybrids occurring in the Lycium genus. The current study fills this knowledge gap by providing evidence of hybridizations between the distantly related species L. barbarum and L. ruthenicum in the origin of L. qingshuiheense and L. ningxiaense . Our reasons for this conclusion are as follows. First, SSR marker data showed clear genetic admixture as alleles for both putative parental species were present in each individual of L. qingshuiheense and L. ningxiaense , which strongly suggested their hybrid origin. Moreover, most populations of the L. ningxiaense comprised F1 hybrids, with some individuals showing evidence of backcrossing with each of the two parent species (Fig. 2 ). This predominance of F1 hybrids is rare, as hybrid swarms typically contain a mix of F1s, F2s, and backcrosses [ 48 – 51 ]. This phenomenon of F1s appearing in L. ningxiaense is possibly due to strong reproductive isolation between the parent species, limiting gene flow and preventing the generation of hybrids beyond the F1 generation. Field observations further supported our hypothesis, as populations of L. qingshuiheense and L. ningxiaense were relatively small and produced few seeds. Moreover, it remains unknown whether these seeds germinate. We also failed to find young seedlings from the habitat of L. qingshuiheense and L. ningxiaense , which seems to support the conclusion that the populations of L. ningxiaense mainly comprise F1s. The presence of these hybrid populations consisting mainly of F1s suggests that the new and recent hybridizations between two parental species may have continuously produced more F1s to repopulate the L. qingshuiheense and L. ningxiaense hybrid zones and have maintained the unique F1 hybrid zones. Although they are hybrids, L. qingshuiheense show a different genetic pattern than that of L. ningxiaense due to backcross or interspecific genetic infiltration. Further, comparative analysis of the cp genomes in the current study further supported the hybrid hypothesis. The four Lycium species contained a total of 132 annotated genes, which comprised 87 protein-coding genes (PCGs), 37 tRNA genes, and 8 RNA genes. The size of the chloroplast genomes varied from 154,830 to 155,148 bp (Table 1 ). This cp genome result was consistent with Zhang et al.[ 52 ], suggesting that the cp genomes of Lycium were conserved in structure. Among the four species, cp genome composition and structure, including basic characteristics, GC content, and gene number of L. qingshuiheense and L. ningxiaense were identical (Table S1 ). In addition, all individuals of L. qingshuiheense and L. ningxiaense clustered on a branch with L. ruthenicum (Fig. 4 ), suggesting that L. ruthenicum should be the maternal parent of the hybrid. The geographical overlap between L. barbarum and L. ruthenicum also supports the hybrid origin of L. qingshuiheense and L. ningxiaense , as these species coexist in the same region, providing the opportunity for interspecific hybridization. Thus, the genetic and morphological evidence obtained in this study strongly suggests that L. qingshuiheense and L. ningxiaense are natural crosses of L. ruthenicum and L. barbarum . Conclusions This study examined four Lycium species using a combination of morphological and molecular approaches and cp genome sequence data. These findings suggest that individuals of L. qingshuiheense and L. ningxiaense are, hybrids of L. barbarum and L. ruthenicum . However, further research investigating chromosomal and genomic data is necessary to clarify species formation and highlight local adaptation processes. This includes examining factors such as chromosome number, population history, and gene flow to accurately ascertain the true identity of the hybrid group. Only through rigorous analyses can appropriate taxonomic revisions be conducted for L. qingshuiheense and L. ningxiaense . Materials and methods Field survey and sample collection Before the field investigations, we scrutinized all the specimens of the four species in the primary herbaria: PE (Herbarium, Institute of Botany, CAS, Beijing, China), KUN (Herbarium, Kunming Institute of Botany, CAS, Kunming, China), WUK (Herbarium, College of Life Sciences, Northwest A&F University, Yangling, China), and IBSC (South China Botanical Garden Herbarium, Guangzhou, China), along with the digital images of the specimens from the Chinese Virtual Herbarium database ( http://www.cvh.ac.cn/ ), including that of L. qingshuiheense and L. ningxiaense held at PE. We explored two populations in the localities from where L. qingshuiheense and L. ningxiaense were previously collected and obtained specimens from 10 individual plants. As L. qingshuiheense and L. ningxiaense are exclusively found in central Ningxia, we collected specimens from 57 individuals from eight populations of L. barbarum and L. ruthnicum distributed in Ningxia (Fig. 5 ; Table 2 ). To avoid misidentification, we conducted a thorough verification of the typical morphological characteristics of leaves, flowers, and fruits during the specimen collection process. For all accessible populations of these four species in the field, healthy leaves were collected from mature plants that were situated at least 50 m apart. We obtained healthy fresh leaves from each tree and promptly dried them in silica gel for DNA extraction. In addition, for each population, branchlets with flowers or fruits were collected from each mature plant for the preparation of voucher specimens. Subsequently, these specimens were identified by Dr. Lei Zhang ( [email protected] ) and stored in the Herbarium of North Minzu University, Yinchuan, China. We recorded the geographic coordinates, including latitude and longitude, as well as the elevation for each sampled population (Table 2 ; Fig. 1 ). In total, 67 individuals and 35 specimens from 10 representative populations of four species was available for population genetic and morphological analyses. Table 2 Detailed information for the four Lycium species used for data analysis in this study Species Latitude Longitude Elevation(m) Collection site Collection number Herbarium deposit* Lycium barbarum 38.3385 106.4062 1088 Lingwu, NingXia zlnmu2022012 NMU 39.1822 106.7945 1037 Shizuishan, NingXia zlnmu2022044 NMU 37.4717 105.9586 1204 Zhongwei, NingXia zlnmu2022282 NMU 37.2757 105.3236 1178 Zhongwei, NingXia zlnmu2022308 NMU 36.8431 106.1341 1551 Guyuan, NingXia zlnmu2022310 NMU 37.5575 106.2893 1193 Wuzhong, NingXia zlnmu2023119 NMU Lycium ruthenicum 35.2318 103.2303 1072 Yinchuan, NingXia zlnmu2022214 NMU 37.2757 105.3236 1178 Zhongwei, NingXia zlnmu2022235 NMU Lycium ningxiaense 37.2756 105.3240 1189 Zhongwei, NingXia zlnmu2022234 NMU Lycium qingshuiheense 37.2757 105.3236 1178 Zhongwei, NingXia zlnmu2022307 NMU *Abbreviations: NMU, Herbarium of North MinZu University. Morphological analysis To obtain comprehensive information on the four species, 90 specimens from different individuals, including 31 specimens collected during the study and 59 specimens from herbaria collections were analyzed. The sampling process included selecting specimens and addressed the distributional ranges of the four species found in northern China (Table S3). Ten morphological traits related to leaves, flowers, and fruits were measured, focusing on traits that have been using to distinguish species in previous taxonomic treatments [ 4 – 5 , 32 ]. Two or three mature leaves, flowers, and fruits from each of the 98 specimens were selected for measurement using ImageJ version v1.53t [ 53 ]. Final morphometric analyses were conducted on four leaf traits (petiole length and leaf length, width, and area), five flower traits (flower length, corolla radius, tube length, corolla lobe length, and angle between the corolla and tube), and fruit color. Subsequently, PCoA was conducted using the R package ggrepel version 0.9.5 ( https://ggrepel.slowkow.com ) and visualized using ggplot2 [ 54 ]. The variability in each quantitative trait were shown in boxplots generated by the R program [ 55 ]. ANOVA analysis was performed for each quantitative trait using SPSS version 19 [ 56 ]. Genetic analysis Total genomic DNA was extracted from the dried leaves using the hexadecyltrimethylammonium bromide method [ 57 ]. We used 10 nSSR primers (Table S4) to genotype our samples [ 58 – 59 ]. Polymerase chain reactions (PCRs) were performed using a 25 µL reaction mix comprising 2.5 µL of 10× Taq buffer, 0.5 mM of each dNTP, 50–100 ng of diluted genomic DNA, 0.5 µL of each primer, and 0.5 units of Taq polymerase (Vazyme Biotech, Nanjing, China). The cycling conditions were as follows: a single cycle at 95°C for 5 min, followed by 36 cycles of 95°C for 45 s, 55°C for 40 s, and 72°C for 80 s, with a final extension cycle at 72°C for 10 min. The amplified products were analyzed on an ABI 3830xl DNA analyzer (Applied Biosystems, Inc., Foster City, CA, USA) at Nuohe Biological Technology (Tianjin, China). We analyzed the genetic composition and structure of the four Lycium species populations (Table S5) using STRUCTURE v2.3.4 [ 60 ] with Bayesian hybrid mixture computation. Ten replicates were performed for each genetic cluster, with K values spanning from 1 to 10 [ 61 ]. Each run comprised 1,000,000 Markov chain Monte Carlo repetitions after a burn-in period of 500,000 iterations. We determined the most likely number of clusters using a combination of the Evanno [ 62 ] and Pritchard [ 63 ] methods performed using Structure Harvester software [ 64 ]. For the SSR data, we conducted a PCoA using the ape package in R [ 65 ]. cp genome assembly and annotation At least two gigabases of 2× 150 bp short read data were generated for each sample. After filtering out reads with quality scores 10% ambiguous nucleotides, clean reads were generated to ensure high-quality data for subsequent analysis. All of the remaining reads were assembled using NOVOPlasty version 2.7.2 [ 66 ] software with k-mer = 39, read length = 150, and insert size = 350. The contigs were aligned into sequences in Geneious version 9.1.8 software [ 67 ]. Finally, the CPGs were annotated using Plann version 1.1 [ 68 ] software based on the L. chinense cp genome as a reference. Additionally, The circular gene map images of the cp genome were created by OGDRAW version 1.2 [ 69 ], and all cp genomes annotated for the first time were submitted to GenBank [ 70 ]. Phylogenetic analysis In our study, two datasets of 27 cp genomes were used to reconstruct phylogenetic relationships: the whole chloroplast genome sequences (WPs) and the aligned protein-coding sequences (CDSs). Using Perl scripts to extract PCGs from GenBank formatted files and exclude possible pseudogenes, 79 PCGs were retained across all species. Each PCG was aligned using MAFFT v.7 [ 71 ], and the aligned sequences were assembled into a supermatrix. Independent phylogenetic analyses were conducted for each dataset (CDSs and WPs) utilizing both maximum likelihood (ML) and Bayesian inference (BI) methodologies. RAxML version 8.1.24 [ 72 ] was employed to perform ML analyses was obtained using the rapid hill-climbing algorithm (i.e., the option “-f d”) with 1,000 bootstrap replicates under the gamma distribution (GTR + Γ) model. The optimal model (GTR + I + G) was identified using jModeltest software, and BI analysis was performed out using MrBayes version 3.2.6 [ 73 ]. Furthermore, we used FigTree version 1.4.2 [ 74 ] software to visualize all phylogeny topology results. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All sequence data has been uploaded to the NCBI Genbank database https://www.ncbi.nlm.nih.gov. All specimens have been deposited in public herbaria. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the Ningxia Natural Science Foundation (2022AAC05063, 2021AAC02005), the National Natural Science Foundation of China (32260049, 32260409) and the graduate innovation project of North Minzu University (YCX24409). The reviewers and editors are sincerely acknowledged. Author contributions ZL and ZGQ conceived and designed the experiments, and manuscript preparation. ZCP and ZED assisted with laboratory and herbarium work. ZED assisted with laboratory work and experimental design. WYQ undertook the experiments and analyses. WYQ and ZGQ assisted with manuscript preparation. 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(2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 14: 2611–20. Pritchard J, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–59. Earl DA, vonHoldt BM. (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv. Genet. Resour. 2012;4: 359–61. Paradis E, Claude J, Strimmer K. APE: analyses of phylogenetics and evolution in R language. Bioinformatics. 2004;20:289–90. Dierckxsens N, Mardulyn P, Smits G. NOVOPlasty: de novo assembly of organ-elle genomes from whole genome data. Nucleic Acids Res. 2017;45(4):e18. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S. Geneious basic: an integrated and extendable desktop software platform for the orga-nization and analysis of sequence data. Bioinformatics. 2012;28:1647–9. Huang DI, Cronk QC, Plann. 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigues.docx SupplementaryTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 09 Apr, 2025 Reviews received at journal 07 Apr, 2025 Reviews received at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers invited by journal 26 Mar, 2025 Submission checks completed at journal 24 Mar, 2025 First submitted to journal 21 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5874276","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434123712,"identity":"ccb34a41-276b-4bfd-84c1-43e49e11c6e0","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":434123713,"identity":"5099dbe0-8b1e-42ee-9239-2ba32241a416","order_by":1,"name":"Chaopan Zhang","email":"","orcid":"","institution":"National Ethnic Affairs Commission of the People’s Republic of China, North Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Chaopan","middleName":"","lastName":"Zhang","suffix":""},{"id":434123714,"identity":"3dd2a7e3-19be-4a89-a7b3-c1f56891121e","order_by":2,"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":434123715,"identity":"a78b6934-286e-448a-8247-46342aa2ca21","order_by":3,"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":434123716,"identity":"4963a5ab-02ed-4aff-8d89-cff8cda53777","order_by":4,"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":"2025-01-21 14:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5874276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5874276/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-12150-3","type":"published","date":"2025-10-28T15:58:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79353025,"identity":"797a91b7-f2d6-4c8d-abb8-bf9f74be46fc","added_by":"auto","created_at":"2025-03-27 10:46:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1223072,"visible":true,"origin":"","legend":"\u003cp\u003eTraits analysis for four \u003cem\u003eLycium\u003c/em\u003e species. A. PCoA result of total trait variation for 90 specimens; B-D. Results of ANOVA analysis for single trait variation of 90 specimens (B. leaf width; C. corolla tube width; D. fruit color). Letters above each of box plots indicate signifcant diferences..\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/063ecfb2964f7731817e3b3b.jpg"},{"id":79353027,"identity":"43380d55-2954-40ef-8b6b-17ab6e3702c9","added_by":"auto","created_at":"2025-03-27 10:46:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4519877,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of the 10 microsatellite SSR data from 10 populations of the four \u003cem\u003eLycium\u003c/em\u003e species studied. (A) The optimal K value was estimated using (B) the distribution of delta K (K=2) and Bayesian clustering plots. (B) Structure analysis results. (C) Principal Coordinates Analysis (PCA) results. Colours in B and C correspond to the species.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/bdeb2cb63d06be40fbed24c4.jpg"},{"id":79353030,"identity":"1a425544-b82c-48fd-83ea-278b93a2817d","added_by":"auto","created_at":"2025-03-27 10:46:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1092354,"visible":true,"origin":"","legend":"\u003cp\u003eGene map of chloroplast genomes of the four \u003cem\u003eLycium\u003c/em\u003e species.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/c5f799dcb88ff4391af729cc.jpg"},{"id":79353893,"identity":"e1878937-716d-4aa8-9c11-cdda0d85ce66","added_by":"auto","created_at":"2025-03-27 10:54:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3118177,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree obtained using the maximum likelihood (ML) methods for the four \u003cem\u003eLycium\u003c/em\u003e species based on 79 PCGs. Colours correspond to the species.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/afa7c5e1629ae8990a76d69e.jpg"},{"id":79354213,"identity":"8afb9db8-340d-4608-8173-79364ec190dd","added_by":"auto","created_at":"2025-03-27 11:02:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3823185,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of 10 populations of the four \u003cem\u003eLycium\u003c/em\u003e species studied here from Ningxia, China. Colours correspond to the species.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/dad867c89e229a2aa4b0a907.jpg"},{"id":95040666,"identity":"401613c2-fbb8-43d5-bbdc-c2e18184eaa1","added_by":"auto","created_at":"2025-11-03 16:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14697801,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/42f55a9f-78df-492d-afb6-e455abbcb059.pdf"},{"id":79353898,"identity":"2d8b4d92-4245-47e6-9d70-4dd361631a84","added_by":"auto","created_at":"2025-03-27 10:54:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":200781,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigues.docx","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/80c5f1ff0b15b874f70ec99f.docx"},{"id":79353033,"identity":"279acc6f-f1eb-4de7-99a1-3fe43ed2dbf3","added_by":"auto","created_at":"2025-03-27 10:46:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5874276/v1/44294cba0ee418bcd15adf00.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hybridization of Lycium qingshuiheense and L. ningxiaense revealed using morphological, SSR markers, and chloroplast genomic data","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eLycium\u003c/em\u003e is a genus of shrubs belonging to the Solanaceae family. \u003cem\u003eLycium\u003c/em\u003e species show robust adaptability to different surroundings, making them pioneer plants in preventing wind and sand erosion and resisting land salinization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This genus consists of more than 80 species, primarily distributed across South America, southwest North America, and Africa, with a limited number of species found in the temperate regions of Eurasia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In China, the recorded number of \u003cem\u003eLycium\u003c/em\u003e species ranges from 8 to 15, including subspecies, primarily growing in the arid and semi-arid regions of Ningxia, Xinjiang, Inner Mongolia, and other provinces [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For centuries, certain species within the \u003cem\u003eLycium\u003c/em\u003e genus, including \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e, have been used in China both as traditional herbal remedies and dietary staples [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Various parts of \u003cem\u003eLycium\u003c/em\u003e species, like fruits and root bark, are used to treat a variety of ailments and are also to create tonics, because they provide benefits such as immune regulation and reduction of blood glucose and serum lipids [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eLycium\u003c/em\u003e species exhibits significant genetic diversity. However, species delimitation of \u003cem\u003eLycium\u003c/em\u003e has remained problematic due to extensive morphological variation and frequent interspecific hybridization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Several previously described species, including some from China, may actually be hybrids or intraspecific variations of other species, leading to taxonomic uncertainty. However, these ambiguous species have not been comprehensively investigated [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. With ongoing advancements in molecular biology, the use of molecular techniques in species delimitation research has increased [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Simple sequence repeats (SSRs or microsatellites) possess the advantages of codominant inheritance and distinct marker characteristics, making them an ideal tool for studying genetic diversity and population genetics [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent studies using nuclear SSR genotyping have explored the interspecific relationships and hybridization of \u003cem\u003eLycium\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], demonstrating significant genetic variation among species and differentiating between \u003cem\u003eLycium spp.\u003c/em\u003e and hybrids [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, chloroplasts (cp) possess their own genome, making them a useful tool for identifying the maternal parent of hybrid offspring and thus clarifying phylogenetic relationships [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Several recent studies have used cp genomes for species delimitation and phylogenetic analysis to resolve phylogenetic relationships [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we used four \u003cem\u003eLycium\u003c/em\u003e species: \u003cem\u003eL. ningxiaense\u003c/em\u003e R. J. Wang and Q. Liao [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eL. qingshuiheense\u003c/em\u003e X. L. Jiang and J. N. Li [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e. These species were chosen due to their close morphological similarities and geographical overlap, which can lead to potential confusion in species identification. \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were found in central Ningxia, China, and share morphological traits with \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e, which are widespread in northwest China. Despite these similarities, \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e can be distinguished based on their distribution ranges and morphological features. \u003cem\u003eL. ruthenicum\u003c/em\u003e has linear or subcylindric leaves and purple-black, globose fruit, while \u003cem\u003eL. barbarum\u003c/em\u003e has lanceolate or long-elliptic leaves and red or orange-yellow, oblong or ovoid fruit [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, field observations indicate that \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e display intermediate morphological traits, including leaf shape, flower characteristics, and fruit color, suggesting the possibility of hybridization between \u003cem\u003eL. barbarum and L. ruthenicum\u003c/em\u003e. Thus, it is speculated that the \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e may be the result of hybridization.\u003c/p\u003e \u003cp\u003eTo clarify the relationship among these species, this study integrated molecular data, which has proven effective in resolving species boundaries where morphological traits alone are insufficient. As recently recommended for integrated species delimitation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], this study used 10 nuclear SSRs (or microsatellite loci), cp genomes, and morphological data to assess the genetic composition and structure of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e. The current study aimed to address the following questions: (1) Are \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e the product of hybridization? (2) What is the genetic composition and structure resulting from this hybridization? (3) Do the parent species involved in hybridization differ between \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e?\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMorphological clustering\u003c/h2\u003e \u003cp\u003eWe conducted principal coordinate analysis (PCoA) and hierarchical clustering on 12 observed and measured traits of 90 \u003cem\u003eLycium\u003c/em\u003e specimens. These traits each accounted for \u0026gt;\u0026thinsp;5% of the variance in the data and included PC1 to PC4 (PC1\u0026thinsp;=\u0026thinsp;51.22%; PC2\u0026thinsp;=\u0026thinsp;16.63%; PC3\u0026thinsp;=\u0026thinsp;10.24%; PC4\u0026thinsp;=\u0026thinsp;7.95%) and combined they represented 84.99% of variance. All other PCs individually accounted for \u0026lt;\u0026thinsp;5% of the variance in the data. The greatest amount of shape variance was observed across PC1 and PC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The arrangement of the scatterplot was based on (PCo1) and (PCo2), individuals of \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e formed two clear groups, while most individuals of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were distributed between \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e, with only a few samples distributed in the \u003cem\u003eL. ruthenicum\u003c/em\u003e group. All other PCs showed similar relationships.\u003c/p\u003e \u003cp\u003eSeparate analysis of variance (ANOVA) tests on the 12 quantitative traits across 90 specimens representing four distinct species revealed several findings: \u003cem\u003eL. barbarum\u003c/em\u003e demonstrated significantly larger measurements compared to other species in seven traits: petiole length, leaf length, leaf width, leaf area, corolla radius, corolla lobes, and fruit color (R) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; S1). Contrastingly, \u003cem\u003eL. ruthenicum\u003c/em\u003e displayed longer floral tubes relative to other species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, the fruit color(G) of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e was notably similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), with the variation between these two species being more pronounced than that observed between \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). For the remaining quantitative traits, no significant differences were detected among the four species (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClustering analyses of nuclear SSR (nSSR) microsatellite data\u003c/h3\u003e\n\u003cp\u003eStructural analysis of the 10 nSSR loci for 67 individuals from the four \u003cem\u003eLycium\u003c/em\u003e species showed that the data comprised two genetic clusters (K\u0026thinsp;=\u0026thinsp;2), based on both the Pritchard and Evanno methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). When K\u0026thinsp;=\u0026thinsp;2, The first cluster was composed primarily of \u003cem\u003eL. barbarum\u003c/em\u003e. In contrast, the second cluster included all \u003cem\u003eL. ruthenicum\u003c/em\u003e individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The genetic composition of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e represented a genetic mix of the two clusters. The PCoA of the nSSR data showed that \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e formed distinct clusters, with all the sampled individuals of both \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e dispersed between them, indicating mixed origins. However, compared with \u003cem\u003eL. ningxiaense\u003c/em\u003e, \u003cem\u003eL. qingshuiheense\u003c/em\u003e had more genetic components from \u003cem\u003eL. ruthenicum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacteristics of the cp genomes of the four\u003c/b\u003e \u003cb\u003eLycium\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe length of the 27 cp genomes from the four \u003cem\u003eLycium\u003c/em\u003e species ranged from 154,830 bp in \u003cem\u003eL. ruthenicum\u003c/em\u003e to 155,148 bp in \u003cem\u003eL. barbarum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All the cp genomes exhibited characteristic quadripartite circular structures, consisting of a large single copy (LSC) region and a small single copy (SSC) region, which were separated by two inverted repeat (IR) regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The lengths of the LSC regions varied from 85,831 to 85,991 bp, while the lengths of the SSC and IR regions ranged from 18,190 to 18,209 bp and from 25,395 to 25,458 bp, respectively (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The GC content of the cp genomes was found to be 37.9%, whereas the GC content within the IR regions ranged between 43.14% and 43.19%. This is notably higher than that observed in the LSC and SSC regions, which were measured at 35.92%-35.98% and 32.31%-32.35%, respectively (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, the total length, LSC length, SSC length, IR length, and the GC content of the entire genome and that of individual regions of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were similar (closer to \u003cem\u003eL. ruthenicum\u003c/em\u003e than to \u003cem\u003eL. barbarum\u003c/em\u003e). Additionally, all cp genomes contained 132 annotated genes, including 87 protein-coding genes (PCGs), 8 rRNA genes, and 37 tRNA genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eChloroplast genome genes annotation of four \u003cem\u003eLycium\u003c/em\u003e species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClassification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene family\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003eProtein-coding genes (87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsa\u003c/em\u003eA, B, C, I, J\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsb\u003c/em\u003eA, B, C, D, E, F, H, I, J, K, L, M, N, T, Z\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCytochrome b/f complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epet\u003c/em\u003eA, B*, D*, G, L, N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatp\u003c/em\u003eA, B, E, F*, H, I\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNADH plastoquinone oxidoreductase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003endh\u003c/em\u003eA**, B**(x2), C, D, E, F, G, H, I, J, K\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eribulose\u0026minus;1,5-bisphosphate carboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbc\u003c/em\u003eL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall subunit ofribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps\u003c/em\u003e11, 12**(x2), 14, 15, 16*, 18, 19, 2, 3, 4, 7(x2), 8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit ofribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl\u003c/em\u003e14, 16*, 2*(x2), 20, 22, 23(x2), 32, 33, 36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA dependent RNA polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpo\u003c/em\u003eA, B, C1*, C2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003emat\u003c/em\u003eK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetyl-CoAcarboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eacc\u003c/em\u003eD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCytochrome C heme attachment protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccs\u003c/em\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChloroplast envelop membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecem\u003c/em\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClp protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclp\u003c/em\u003eP\u003cem\u003e**\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHypothetical chloroplast conserved open reading frame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eycf\u003c/em\u003e1(x2), 2(x2), 3**, 4, 15(x2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erRNA genes (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erRNA genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn\u003c/em\u003e16(x2), 23(x2), 4.5(x2), 5(x2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etRNA genes (37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etRNA genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eA(UGC)*(x2), C(GCA), D(GUC), E(UUC), F(GAA), fM(CAU), G(GCC), G(UCC)*, H(GUG, I(CAU)(x2), I(GAU)*(x2), K(UUU)*, L(CAA)(x2), L(UAA)*, L(UAG), M(CAU), N(GUU)(x2), P(UGG), Q(UUG), (ACG)(x2), (UCU), S(GCU), S(GGA), S(UGA), T(GGU), T(UGU), V(GAC)(x2), V(UAC)*, W(CCA), Y(GUA)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e* represents a gene with one intron, ** represents a gene with two introns, (x2) repeat genes\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis of the four\u003c/b\u003e \u003cb\u003eLycium\u003c/b\u003e \u003cb\u003especies based on their cp genomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe utilized \u003cem\u003eL. chinese\u003c/em\u003e as an outgroup to elucidate the phylogenetic relationships among the four species based on their cp genomes. The final concatenated dataset comprised 79 plastid genes and 67,422 sites (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In the phylogenetic trees, most nodes generated from both the maximum likelihood (ML) and Bayesian inference (BI) analyses exhibited nearly congruent topologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Figs. S2, S3, S4). A total of 27 individuals from four \u003cem\u003eLycium\u003c/em\u003e species were divided into two typical branches. Clade 1, comprised \u003cem\u003eL. barbarum\u003c/em\u003e (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1); and Clade 2, consisted of \u003cem\u003eL. qingshuiheense\u003c/em\u003e, \u003cem\u003eL. ningxiaense\u003c/em\u003e, and \u003cem\u003eL. ruthenicumon\u003c/em\u003e (BS; PP\u0026thinsp;=\u0026thinsp;100%, 1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Figs. S2, S3, S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the hybrid origins of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e, focusing on species delimitation and their genetic and morphological characteristics in relation to \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e. Our results provide evidence for their hybrid origin, as both \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e exhibited intermediate morphological traits and genetic profiles, which is consistent with the admixture of the two parental species.\u003c/p\u003e \u003cp\u003ePrevious research indicates that the morphological analysis is highly effective for distinguishing between closely related species [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Specifically, it is used to differentiate between overall changes in gross morphology, even if the individual morphology of different species overlaps [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Morphologically, \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e displayed leaf size and shape, corolla size, and fruit color that fall between those of \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; S1). The intermediate traits observed in our study support the hypothesis of hybridization, which is in line with previous research showing that hybrid species often exhibit characteristics blending those of their parent species [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Genetic analyses using SSR markers and cp genome sequencing further supported this conclusion, as both species showed genetic profiles indicative of hybridization, with alleles closely resembling those of the parental species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterspecific hybridization is a common phenomenon in nature [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and hybrid swarms or hybrid zones provide valuable insights into interspecific gene flow and hybrid fitness [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Although the origins of hybrids have been explored in numerous plant groups, such as \u003cem\u003ePopulus\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], \u003cem\u003eRhododendron\u003c/em\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and \u003cem\u003eMimulus\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], little attention has been paid to natural hybrids occurring in the \u003cem\u003eLycium\u003c/em\u003e genus. The current study fills this knowledge gap by providing evidence of hybridizations between the distantly related species \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e in the origin of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e. Our reasons for this conclusion are as follows. First, SSR marker data showed clear genetic admixture as alleles for both putative parental species were present in each individual of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e, which strongly suggested their hybrid origin. Moreover, most populations of the \u003cem\u003eL. ningxiaense\u003c/em\u003e comprised F1 hybrids, with some individuals showing evidence of backcrossing with each of the two parent species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This predominance of F1 hybrids is rare, as hybrid swarms typically contain a mix of F1s, F2s, and backcrosses [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This phenomenon of F1s appearing in \u003cem\u003eL. ningxiaense\u003c/em\u003e is possibly due to strong reproductive isolation between the parent species, limiting gene flow and preventing the generation of hybrids beyond the F1 generation. Field observations further supported our hypothesis, as populations of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were relatively small and produced few seeds. Moreover, it remains unknown whether these seeds germinate. We also failed to find young seedlings from the habitat of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e, which seems to support the conclusion that the populations of \u003cem\u003eL. ningxiaense\u003c/em\u003e mainly comprise F1s. The presence of these hybrid populations consisting mainly of F1s suggests that the new and recent hybridizations between two parental species may have continuously produced more F1s to repopulate the \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e hybrid zones and have maintained the unique F1 hybrid zones. Although they are hybrids, \u003cem\u003eL. qingshuiheense\u003c/em\u003e show a different genetic pattern than that of \u003cem\u003eL. ningxiaense\u003c/em\u003e due to backcross or interspecific genetic infiltration.\u003c/p\u003e \u003cp\u003eFurther, comparative analysis of the cp genomes in the current study further supported the hybrid hypothesis. The four \u003cem\u003eLycium\u003c/em\u003e species contained a total of 132 annotated genes, which comprised 87 protein-coding genes (PCGs), 37 tRNA genes, and 8 RNA genes. The size of the chloroplast genomes varied from 154,830 to 155,148 bp (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This cp genome result was consistent with Zhang et al.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], suggesting that the cp genomes of \u003cem\u003eLycium\u003c/em\u003e were conserved in structure. Among the four species, cp genome composition and structure, including basic characteristics, GC content, and gene number of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were identical (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In addition, all individuals of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e clustered on a branch with \u003cem\u003eL. ruthenicum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that \u003cem\u003eL. ruthenicum\u003c/em\u003e should be the maternal parent of the hybrid. The geographical overlap between \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e also supports the hybrid origin of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e, as these species coexist in the same region, providing the opportunity for interspecific hybridization. Thus, the genetic and morphological evidence obtained in this study strongly suggests that \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e are natural crosses of \u003cem\u003eL. ruthenicum\u003c/em\u003e and \u003cem\u003eL. barbarum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study examined four \u003cem\u003eLycium\u003c/em\u003e species using a combination of morphological and molecular approaches and cp genome sequence data. These findings suggest that individuals of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e are, hybrids of \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e. However, further research investigating chromosomal and genomic data is necessary to clarify species formation and highlight local adaptation processes. This includes examining factors such as chromosome number, population history, and gene flow to accurately ascertain the true identity of the hybrid group. Only through rigorous analyses can appropriate taxonomic revisions be conducted for \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eField survey and sample collection\u003c/h2\u003e\n \u003cp\u003eBefore the field investigations, we scrutinized all the specimens of the four species in the primary herbaria: PE (Herbarium, Institute of Botany, CAS, Beijing, China), KUN (Herbarium, Kunming Institute of Botany, CAS, Kunming, China), WUK (Herbarium, College of Life Sciences, Northwest A\u0026amp;F University, Yangling, China), and IBSC (South China Botanical Garden Herbarium, Guangzhou, China), along with the digital images of the specimens from the Chinese Virtual Herbarium database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cvh.ac.cn/\u003c/span\u003e\u003c/span\u003e), including that of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e held at PE. We explored two populations in the localities from where \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were previously collected and obtained specimens from 10 individual plants. As \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e are exclusively found in central Ningxia, we collected specimens from 57 individuals from eight populations of \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthnicum\u003c/em\u003e distributed in Ningxia (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). To avoid misidentification, we conducted a thorough verification of the typical morphological characteristics of leaves, flowers, and fruits during the specimen collection process. For all accessible populations of these four species in the field, healthy leaves were collected from mature plants that were situated at least 50 m apart. We obtained healthy fresh leaves from each tree and promptly dried them in silica gel for DNA extraction. In addition, for each population, branchlets with flowers or fruits were collected from each mature plant for the preparation of voucher specimens. Subsequently, these specimens were identified by Dr. Lei Zhang ([email protected]) and stored in the Herbarium of North Minzu University, Yinchuan, China. We recorded the geographic coordinates, including latitude and longitude, as well as the elevation for each sampled population (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In total, 67 individuals and 35 specimens from 10 representative populations of four species was available for population genetic and morphological analyses.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetailed information for the four \u003cem\u003eLycium\u003c/em\u003e species used for data analysis in this study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLatitude\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLongitude\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eElevation(m)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCollection site\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCollection number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHerbarium deposit*\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\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cem\u003eLycium barbarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.3385\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.4062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLingwu, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.1822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.7945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShizuishan, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.4717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.9586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZhongwei, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.2757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.3236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZhongwei, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.8431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.1341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuyuan, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.5575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.2893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWuzhong, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2023119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eLycium ruthenicum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.2318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103.2303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYinchuan, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.2757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.3236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZhongwei, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLycium ningxiaense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.2756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.3240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZhongwei, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLycium qingshuiheense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.2757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.3236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZhongwei, NingXia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ezlnmu2022307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNMU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e*Abbreviations: NMU, Herbarium of North MinZu University.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMorphological analysis\u003c/h3\u003e\n\u003cp\u003eTo obtain comprehensive information on the four species, 90 specimens from different individuals, including 31 specimens collected during the study and 59 specimens from herbaria collections were analyzed. The sampling process included selecting specimens and addressed the distributional ranges of the four species found in northern China (Table S3). Ten morphological traits related to leaves, flowers, and fruits were measured, focusing on traits that have been using to distinguish species in previous taxonomic treatments [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Two or three mature leaves, flowers, and fruits from each of the 98 specimens were selected for measurement using ImageJ version v1.53t [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Final morphometric analyses were conducted on four leaf traits (petiole length and leaf length, width, and area), five flower traits (flower length, corolla radius, tube length, corolla lobe length, and angle between the corolla and tube), and fruit color. Subsequently, PCoA was conducted using the R package ggrepel version 0.9.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggrepel.slowkow.com\u003c/span\u003e\u003c/span\u003e) and visualized using ggplot2 [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. The variability in each quantitative trait were shown in boxplots generated by the R program [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. ANOVA analysis was performed for each quantitative trait using SPSS version 19 [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGenetic analysis\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA was extracted from the dried leaves using the hexadecyltrimethylammonium bromide method [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. We used 10 nSSR primers (Table S4) to genotype our samples [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. Polymerase chain reactions (PCRs) were performed using a 25 \u0026micro;L reaction mix comprising 2.5 \u0026micro;L of 10\u0026times; Taq buffer, 0.5 mM of each dNTP, 50\u0026ndash;100 ng of diluted genomic DNA, 0.5 \u0026micro;L of each primer, and 0.5 units of \u003cem\u003eTaq\u003c/em\u003e polymerase (Vazyme Biotech, Nanjing, China). The cycling conditions were as follows: a single cycle at 95\u0026deg;C for 5 min, followed by 36 cycles of 95\u0026deg;C for 45 s, 55\u0026deg;C for 40 s, and 72\u0026deg;C for 80 s, with a final extension cycle at 72\u0026deg;C for 10 min. The amplified products were analyzed on an ABI 3830xl DNA analyzer (Applied Biosystems, Inc., Foster City, CA, USA) at Nuohe Biological Technology (Tianjin, China).\u003c/p\u003e\n\u003cp\u003eWe analyzed the genetic composition and structure of the four \u003cem\u003eLycium\u003c/em\u003e species populations (Table S5) using STRUCTURE v2.3.4 [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e] with Bayesian hybrid mixture computation. Ten replicates were performed for each genetic cluster, with K values spanning from 1 to 10 [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. Each run comprised 1,000,000 Markov chain Monte Carlo repetitions after a burn-in period of 500,000 iterations. We determined the most likely number of clusters using a combination of the Evanno [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e] and Pritchard [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e] methods performed using Structure Harvester software [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. For the SSR data, we conducted a PCoA using the \u003cem\u003eape\u003c/em\u003e package in R [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003ecp genome assembly and annotation\u003c/h2\u003e\n \u003cp\u003eAt least two gigabases of 2\u0026times; 150 bp short read data were generated for each sample. After filtering out reads with quality scores\u0026thinsp;\u0026lt;\u0026thinsp;7 and \u0026gt;\u0026thinsp;10% ambiguous nucleotides, clean reads were generated to ensure high-quality data for subsequent analysis. All of the remaining reads were assembled using NOVOPlasty version 2.7.2 [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e] software with k-mer\u0026thinsp;=\u0026thinsp;39, read length\u0026thinsp;=\u0026thinsp;150, and insert size\u0026thinsp;=\u0026thinsp;350. The contigs were aligned into sequences in Geneious version 9.1.8 software [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]. Finally, the CPGs were annotated using Plann version 1.1 [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e] software based on the \u003cem\u003eL. chinense\u003c/em\u003e cp genome as a reference. Additionally, The circular gene map images of the cp genome were created by OGDRAW version 1.2 [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e], and all cp genomes annotated for the first time were submitted to GenBank [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e\n \u003cp\u003eIn our study, two datasets of 27 cp genomes were used to reconstruct phylogenetic relationships: the whole chloroplast genome sequences (WPs) and the aligned protein-coding sequences (CDSs). Using Perl scripts to extract PCGs from GenBank formatted files and exclude possible pseudogenes, 79 PCGs were retained across all species. Each PCG was aligned using MAFFT v.7 [\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e], and the aligned sequences were assembled into a supermatrix. Independent phylogenetic analyses were conducted for each dataset (CDSs and WPs) utilizing both maximum likelihood (ML) and Bayesian inference (BI) methodologies. RAxML version 8.1.24 [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e] was employed to perform ML analyses was obtained using the rapid hill-climbing algorithm (i.e., the option \u0026ldquo;-f d\u0026rdquo;) with 1,000 bootstrap replicates under the gamma distribution (GTR\u0026thinsp;+\u0026thinsp;\u0026Gamma;) model. The optimal model (GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G) was identified using jModeltest software, and BI analysis was performed out using MrBayes version 3.2.6 [\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e]. Furthermore, we used FigTree version 1.4.2 [\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e] software to visualize all phylogeny topology results.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll sequence data has been uploaded to the NCBI Genbank database https://www.ncbi.nlm.nih.gov. All specimens have been deposited in public herbaria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ningxia Natural Science Foundation (2022AAC05063, 2021AAC02005), the National Natural Science Foundation of China (32260049, 32260409) and the graduate innovation project of North Minzu University (YCX24409). The reviewers and editors are sincerely acknowledged.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZL and ZGQ conceived and designed the experiments, and manuscript preparation. ZCP and ZED assisted with laboratory and herbarium work. ZED assisted with laboratory work and experimental design. WYQ undertook the experiments and analyses. WYQ and ZGQ assisted with manuscript preparation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang ZY, Lu AM. Solanaceae In: Wu Z, Raven P (Eds) Flora of China. Volume 17. Science Press, Beijing, China. 1994. pp:301-4.\u003c/li\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. 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University of Edinburgh, Edinburgh, UK. 2012.\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, morphological analyses, simple sequence repeat (SSR), chloroplast genome, hybridization identification","lastPublishedDoi":"10.21203/rs.3.rs-5874276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5874276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eInterspecific hybridization is a common natural phenomenon that plays a crucial role in the formation and maintenance of species. In recent years, the origins of hybrids have been extensively investigated across various plant groups. \u003cem\u003eLycium\u003c/em\u003ehas received relatively little attention regarding the occurrence of natural hybrids, despite its economic and ecological significance. In this study, hybridization of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e was identified based on morphological traits, nuclear simple sequence repeat (SSR) markers, and chloroplast genomic data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe selected 90 specimens of four species across their distributional ranges to examine 12 morphological traits and characterize clusters with distinct phenological traits. Similarly, we genotyped 67 individuals from four species across 10 populations using 10 SSR markers. The morphological analyses revealed a clear division between individuals of \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003ewhile most individuals of \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003ewere distributed between \u003cem\u003eL. barbarum\u003c/em\u003e and \u003cem\u003eL. ruthenicum\u003c/em\u003e. The genetic clusters were consistent with the morphological clusters. However, most of the \u003cem\u003eL. ningxiaense\u003c/em\u003e samples comprised F1 hybrids, while \u003cem\u003eL. qingshuiheense\u003c/em\u003eshowed a distinct genetic pattern compared to that of \u003cem\u003eL. ningxiaense\u003c/em\u003e due to backcross or interspecific genetic infiltration. Among the four \u003cem\u003eLycium\u003c/em\u003especies, the composition and structureal features of chloroplast genomes from \u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e were identical and clustered on a branch with \u003cem\u003eL. ruthenicum\u003c/em\u003e in the phylogenetic tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eCombining the morphological, SSR markers, and cp genomes analysis, we affirmed that\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eL. qingshuiheense\u003c/em\u003e and \u003cem\u003eL. ningxiaense\u003c/em\u003e are hybrids originating from crossing \u003cem\u003eL. ruthenicum\u003c/em\u003e (♀) and \u003cem\u003eL. barbarum\u003c/em\u003e (♂). This study elucidated the origin of two \u003cem\u003eLycium\u003c/em\u003especies, which contributes to our understanding of the evolutionary mechanisms underlying interspecific hybridization in \u003cem\u003eLycium\u003c/em\u003e. Furthermore, it provides a scientific foundation for the development, utilization, and improvement of \u003cem\u003eLycium\u003c/em\u003e varieties.\u003c/p\u003e","manuscriptTitle":"Hybridization of Lycium qingshuiheense and L. ningxiaense revealed using morphological, SSR markers, and chloroplast genomic data","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 10:46:35","doi":"10.21203/rs.3.rs-5874276/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-09T05:50:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T15:12:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T15:01:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84289899829437232716002680727152904979","date":"2025-03-31T14:41:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54266359378190641546177112295848828045","date":"2025-03-28T23:38:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-26T07:04:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T08:21:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-03-22T02:46:13+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 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:07:13+00:00","versionOfRecord":{"articleIdentity":"rs-5874276","link":"https://doi.org/10.1186/s12864-025-12150-3","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-10-28 15:58:10","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-03-27 10:46:35","video":"","vorDoi":"10.1186/s12864-025-12150-3","vorDoiUrl":"https://doi.org/10.1186/s12864-025-12150-3","workflowStages":[]},"version":"v1","identity":"rs-5874276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5874276","identity":"rs-5874276","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}