Integrative taxonomic revision of the Camellia rhytidocarpa complex (Theaceae) synonymous status of C. lipingensis and C. zengii supported by morphological, anatomical, palynological, and molecular evidence

Integrative taxonomic revision of the Camellia rhytidocarpa complex (Theaceae) synonymous status of C. lipingensis and C. zengii supported by morphological, anatomical, palynological, and molecular evidence | 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 Integrative taxonomic revision of the Camellia rhytidocarpa complex (Theaceae) synonymous status of C. lipingensis and C. zengii supported by morphological, anatomical, palynological, and molecular evidence Weihao Gu, Mingtai An, Chao Yan, Xu Xiao, Zhaohui Ran, Zhi Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7021883/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jan, 2026 Read the published version in Botanical Studies → Version 1 posted 5 You are reading this latest preprint version Abstract Background The section Tuberculata ( Camellia L.), as a monophyletic group characterized by tuberculate fruits, exhibits persistent taxonomic ambiguities among its constituent species, exemplified by the unresolved delimitation of Camellia lipingensis , Camellia zengii , and Camellia rhytidocarpa . These three species are highly similar in terms of morphology, genetics, or ecology as a plant complex. Historical revisions have been hindered by the absence of key morphological characteristics in type specimens and the instability of morphological identification criteria, leading to unclear classification of species. This study, based on type locality specimens, morphology, and systematic molecular biology, systematically integrates macroscopic morphology, microscopic structure, and molecular systematics data for the first time, aiming to clarify the taxonomic relationships among the three species. Results Multidimensional evidence based on morphology, anatomy, palynology, and molecular systematics supports the merger of C. lipingensis and C. zengii into the synonym C. rhytidocarpa . Morphological analysis reveals continuous variation in key traits: leaves lanceolate (6.42–12.50 × 1.17–4.45 cm); floral parts with 6–9 rounded sepals, 3–5 hairy styles, and 2.2–4.1 cm long filaments; fruit subglobose (diameter 2.24–3.18 cm), ovary 3-4-loculed (1 seed per locule). Anatomical and pollen characteristics are conservative: leaf epidermal stomata are elliptical (39.9–41.2 × 31.4–36.7 µm), with a density of 62–86 per mm²; pollen is nearly spherical (polar axis 36.7–37.8 µm/equatorial axis 40.3–41.3 µm, P/E ratio 0.87–0.91). Molecular systematics confirmed that the three form a strongly supported monophyletic clade (ML/PP = 100/1.00), with consistent chloroplast genome structures (157,029, 157,029, 157,048 bp; GC 37.3%; containing 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes). Conclusions This integrative study consolidates C. lipingensis and C. zengii as conspecific synonyms of C. rhytidocarpa based on congruent morphological, anatomical, palynological, and molecular phylogenetic evidence. The taxonomic revision resolves persistent delimitation conflicts within sect. Tuberculata while establishing an empirical framework for: Phylogenetic reconstruction of Camellia lineages with overlapping morphological variation, Conservation prioritization of evolutionarily significant units in East Asian biodiversity hotspots, and Development of standardized species delimitation protocols for taxonomically complex plant groups. Section Tuberculata Species complex Taxonomic revision Integrative taxonomy Speices delimitation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Camellia sect. Tuberculata (Theaceae, Camellia L.) is distinguished from other Camellia groups by its uniquely "tuberculate fruit surface" making it the most morphologically peculiar fruit lineage in the genus (Chang and Ren, 1991; Min and Zhong, 1993; Chang, 1998). In the early 20th century, the renowned botanist, Prof. S.S. Chien (1939) discovered a taxon with tuberculate ovaries and pericarps during fieldwork in Jiading, Sichuan, China, and naming it as Camellia tuberculata S.S. Chien. Sealy (1958), later placed it in section Heterogenea . It was not until 1981 that Hung-Ta Chang established sect. Tuberculata based on the diagnostic "tuberculate-wrinkled fruit pericarp"that initially including 6 species, with 12 additional species reported over the next decade. In 1991, Chang subdivided the section into two subsections (subsect. Tuberculata Chang and subsect. Nudicarpa Chang) based on whether ovary pubescence. Tianlu Min (1993), a renowned Camellia taxonomist revised this classified process, and divided 18 species into 6 species, 6 varieties, and 1 form (Min and Zhong, 1993). It is noteworthy that the taxonomic treatments proposed by both researchers were primarily based on Herbarium museum specimens, with insufficient field investigations and a notable lack of multidisciplinary taxonomic evidence. As a result, the infraspecific classification within thesect. Tuberculata remains contentious. The plant species complex refers to a group of closely related taxa that exhibit high similarity in morphology, genetics, or ecology, with ambiguous taxonomic boundaries ( He et al., 2022 ), such complexes typically comprise multiple species or infraspecific units (e.g., subspecies, varieties), potentially involving hybridization, incomplete lineage sorting, or cryptic diversity, resulting in challenges for traditional morphological classification to accurately delineate species limits. C. lipingensis , C. zengii , and C. rhytidocarpa were all described by Hung-Ta Chang in 1984 (Chang and Ren, 1996). The type localities of the former two species are Wulong Mountain, Liping County, Guizhou Province, China. C. lipingensis is characterized by narrow lanceolate leaves, thickly leathery texture, glabrous styles, and 5 cm diameter flowers, and C. zengii exhibits villous styles, stamens 1.8-2.4 cm in length, 10 fractions, and sepals 1-4 cm in length. The type specimen of C. rhytidocarpa was collected from Tianping Mountain, Longsheng, Guangxi China, displaying distinctive features “lanceolate or oblong leaves measuring 8-12 cm in length, and 6-7 pairs of lateral veins that are not sunken”. In 1993, Tianlu Min proposed merging these three species into C. rhytidocarpa (Min et al., 1993) considered they share similar morphological traits “narrowly lanceolate leaves with caudate or acuminate apices, ovate sepals, and ovaries, sparsely pubescent styles with 3 lobes” and occurring in analogous habitats in theborder region of Hunan, Guizhou, Guangxi. Chang et al. (1996). However, challenged this classification, demonstrating that the three species are morphologically distinct in leaf shape, floral structures (sepals, petals), and style characteristics. Over the past three decades, taxonomic studies of Camellia have partially incorporated analyses of pollen micromorphology, cpDNA, and leaf epidermal features for the three species ((Jiang et al., 2010; Yan et al., 2024; Ran et al., 2024c). Nevertheless, the taxonomic delimitation within this species complex remains unresolved. In recent years, we have conducted extensive field investigations at the type localities of these three species, complemented by comprehensive morphological analyses (including both macro- and micro-morphological examinations of leaf epidermis and pollen) and molecular phylogenetic studies (cpDNA and ITS markers). By integrating these multidisciplinary lines of evidence to aim to resolve the taxonomic complexities within this species complex and clarify interspecific boundaries. Materials and Methods Observation and inspection of type specimens Type specimen examination was systematically conducted to clarify species boundaries and verify historical taxonomic foundations, utilizing digitized resources from the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE; http://pe.ibcas.ac.cn/peweb/), Sun Yat-sen University Herbarium (SYS; https://eco.sysu.edu.cn/platforms/museum), and the Chinese Virtual Herbarium (CVH; https://www.cvh.ac.cn/). Morphological parameters were rigorously measured using standardized techniques (Ran et al., 2024a), with type specimens of C. lipingensis , C. zengii , and C. rhytidocarpa sourced through original literature review and authoritative digital platforms including CVH and the National Specimen Resource Sharing Platform (NSII; http://www.nsii.org.cn/2017/home.php) (Wu et al., 2022; Seo et al., 2025). Comprehensive morphological observations and comparative analyses were performed on all examined specimens. Material collection The field materials used for experimental and morphological data measurements in this study were collected through systematic field surveys between 2022 and 2024. Fresh leaves and floral organs collected in the field were preserved via dry ice flash-freezing to ensure sample integrity (Xiao et al., 2024). Voucher specimens were processed following international herbarium standards and deposited in the Herbarium of the Forestry College, Guizhou University ( C. lipingensis : LZ-20231126-7; C. zengii : LZ-20231127-3; C. rhytidocarpa : LZ-20240114-4). Morphology Study This study systematically investigated plant morphological characteristics through integrated herbarium specimen examinations and field observations, documenting natural habitats and overall plant morphology before conducting standardized measurements of qualitative and quantitative traits from ≥10 voucher specimens per species. The comprehensive measurements included stem/branch bark coloration and texture, leaf characteristics (texture, color, dimensions, margin morphology, base traits, and venation patterns), floral organ features (petal size, number, color, sepal morphology, and style number), and fruit attributes (diameter, shape, pericarp rugosity, and maturation-related color changes), with all parameters measured in triplicate and averaged (Ran et al., 2024b). The standardized dataset (Table S1) was processed using Excel 2025 and subsequently analyzed through principal component analysis (PCA) of 35 morphological variables in R v4.4.2 (R Core Team, 2020). Leaf epidermis and pollen micromorphology Fresh leaves and floral buds of C. lipingensis , C. zengii , and C. rhytidocarpa were collected for micromorphological analyses. Leaf samples underwent dual processing protocols: 1) FAA fixation (70% ethanol:acetic acid:formaldehyde=90:5:5, v/v), distilled water rinsing, midvein dissection (0.5 cm²), sodium hypochlorite dissociation (30°C), and epidermal sectioning with acetate fuchsin staining for light microscopic examination of periclinal walls and stomatal apparatus (Jiang et al., 2022; Situngu et al., 2022); 2) Leaf tissue was gently rinsed with PBS. Tissue blocks were immediately fixed in EM fixative (2 h, RT), then stored at 4°C. Blocks were washed in 0.1 M PB (pH 7.4) ×3 (15 min each), post-fixed in 1% OsO₄ (in 0.1 M PB, pH 7.4; 1-2 h, RT), and washed again in PB ×3 (15 min each). Samples were dehydrated (ethanol series), critical point dried, mounted on aluminum stubs with carbon tabs, sputter-coated with Au (30 s), and imaged using SEM. (Zhang et al., 2021; Neilands et al., 2023). Concurrently, pollen grains (10/species) from FAA-fixed, ethanol-dehydrated anthers (Smith et al., 2021) were acetolyzed, released via microdissection, gold-coated, and analyzed by SEM (JSM-6490LV) for quantitative assessment of morphology, size (P/E axes), colpi, exine ornamentation, and wall sculpturing using ImageTool software (Ocaña et al., 2022; Pan et al., 2022). Total DNA extraction, sequencing, assembly, and annotation Fresh leaves of the three species collected during field surveys were subjected to total DNA extraction using the CTAB (cetyltrimethylammonium bromide) method (Chai et al., 2023; Xing et al., 2023). Following Illumina DNA library preparation standards, paired-end sequencing libraries were constructed with 350-bp insert fragments (Li et al., 2021). DNA quality and concentration were assessed by agarose gel electrophoresis. Qualified fragments were sequenced on the Illumina platform after random fragmentation, end repair, and adapter ligation (Zong et al., 2023). Raw reads generated via base-calling (CASAVA) were assembled using SPAdes v.3.15.2 and annotated with CPGAVAS and ORFFinder. Annotation results were validated by BLASTN/BLASTP alignments. tRNA genes were annotated using ARWEN v1.2 (Zheng et al., 2023; Gu et al., 2024). The complete chloroplast genome was finalized by online annotation, BLAST alignment, and manual curation against the Camellia rubituberculata Chang & Yu reference (MZ424202) (Xiao et al., 2025). Chloroplast genome maps were generated with OGDRAW (Yan et al., 2023; Ran et al., 2024c). Sequences were deposited in NCBI GenBank to obtain accession numbers. IR boundary expansion and contraction Chloroplast genome sequences of C. lipingensis , C. zengii , and C. rhytidocarpa were screened, Three complete chloroplast genomes were obtained from the samples. Sequences of Theaceae species were downloaded from the NCBI for Biotechnology Information database. IR boundary expansion/contraction analysis was performed, and comparative maps were generated using IRscope (https://irscope.shinyapps.io/irapp/) (Amiryousefi et al., 2018; Xiao et al., 2024). Phylogenetic analysis Chloroplast genomes of 24 Camellia species were retrieved from NCBI, with Apterosperma oblata (NC_035641) as the outgroup (Ran et al., 2024a). Sequence alignment used MAFFT v7, followed by maximum likelihood (ML) tree construction in IQ-TREE v1.6.12. Manual refinement in MEGA X selected optimal substitution models (Smith et al., 2022). MrModeltest v2.3 identified (TVM+F+I) as the best-fit model, and Bayesian inference (BI) trees were reconstructed using MrBayes v3.2.7 (Catalano et al., 2025). Phylogenies based on protein-coding genes and ITS were congruent with whole chloroplast genomes. Final trees were visualized with iTOL v5 (Letunic et al., 2021). Result The morphological characteristics of three species Systematic morphological comparisons revealed highly consistent frameworks among the three species, with only continuous or subtle phenotypic variations (Figure 1, Table 1). C. lipingensis and C. zengii shared brownish mottled bark exfoliating to green/grey-green surfaces, red-brown quadrangular branchlets, and leathery lanceolate leaves (vs. occasionally oblong in C. rhytidocarpa ) with acute apices and sparse serrations. Leaf dimensions (6.66-12.48 × 2.16-3.93 cm vs. 5.81-11.60 × 1.17-4.10 cm) and petiole lengths (0.12-1.10 cm vs. 0.30-0.91 cm) overlapped. Floral traits showed near-identical petal dimensions (3.44-5.20 × 1.30-2.00 cm vs. 3.47-4.71 × 1.34-2.21 cm), number (6-9 vs. 7-8), ovate sepals, and overlapping ovary/style pubescence. Fruit diameter (2.24-3.15 cm vs. 2.26-3.18 cm) and shape (subglobose) were consistent, with only minor pericarp thickness variation (0.45-0.60 cm vs. 0.43-0.52 cm), falling within intraspecific geographic variation. C .rhytidocarpa exhibits significant morphological homology with C. lipingensis despite minor variations. Its occasionally oblong leaves with acuminate apices show complete dimensional overlap (7.04-12.50 × 2.16-4.45 cm). Higher serration density may reflect local adaptation. Floral traits partially overlap: petal dimensions (3.20-4.28 × 1.25-1.90 cm), obtuse sepal apices falling within intraspecific polymorphism. Fruit diameter (2.19-3.07 cm) and shape (subglobose/oblate) align closely, though pericarp thickness is reduced (0.30-0.49 cm), suggesting developmental plasticity. Continuous trait gradients and microhabitat-induced adjustments indicate these variations lack taxonomic diagnostic value, supporting reclassification as geographical variants or ecotypes of a single taxon. Table 1 Comparison of the main morphological characteristics of three Species of sect. Tuberculata Species C. lipingensis C. zengii C. rhytidocarpa Trunk Brownish with mottled exfoliation, revealing glaucous green trunks after bark shedding Mottled brown with inconspicuously exfoliating, rugose bark Russet bark with conspicuous exfoliation, revealing glaucous green trunks Branches Annual branchlets tetragonally ribbed Annual branchlets tetragonally ribbed Annual branchlets tetragonally ribbed Leaf type Leathery, lanceolate, leaf tail acute Leathery, lanceolate, leaf tail acute Leathery, lanceolate or long elliptic, leaf tail acuminate Leaf length (cm) 6.66-12.48 5.81-11.60 6.42-12.50 Leaf width (cm) 2.16-3.93 1.17-4.10 2.16-4.21 Leaf margin Acute serrate Acute serrate Acute serrate Petiole length (cm) 0.12-1.10 0.30-0.91 0.43-1.30 Petal length (cm) 3.44-5.20 3.47-4.71 3.20-4.28 Petal width (cm) 1.30-2.00 1.34-2.21 1.25-1.90 Number of petals 6-9 7-8 6-8 Number of calyxes 6-9 7-8 6-8 Sepal Ovate, apex rounded Ovate, few rounded, apex rounded Rounded, apex rounded Ovaries 3-4 3-4 3-4 Style (female organ of flower) 3-5, pubescent 3-5, pubescent 3-5, pubescent Fruit diameter (cm) 2.24-3.15 2.26-3.18 2.24-3.09 Fruit shape Subglobular Subglobular Subglobular Shell thickness (cm) 0.45-0.60 0.43-0.52 0.30-0.49 In order to conduct a comprehensive classification value assessment of each morphological trait of the three species, we carried out principal component analysis. The PCA results segregated all samples into three clusters. C. lipingensis , C. zengii and C. rhytidocarpa exhibited extensive morphological overlap, with measured traits indicating a nested relationship among the three taxa (Figure 2). Leaf Epidermal Micromorphological Characteristics Microscopy revealed (Figure 3, Table 2) highly conserved yet continuously variable leaf micromorphology across the three species. C. lipingensis , C. zengii , and C. rhytidocarpa shared irregular epidermal cells with sinuous anticlinal walls on adaxial surfaces. Crucially, oil glands were absent in C. lipingensis and C. zengii , while sparse in C. rhytidocarpa . Abaxially, C. lipingensis and C. zengii exhibited sinuous anticlinal walls versus undulate walls in C. rhytidocarpa , though all species showed tight cell arrangement and anomocytic stomata with reniform guard cells and 3-5 similarly configured subsidiary cells. Table 2 Morphological characteristics of leaf epidermis of the three species Species Sshape of stomata Size of stomata (length/μm × width/μm) Length / Width Inner margin of outer rim Stomatal density (stomata/mm²) Type of stomatal apparatus C. lipingensis Long ellipse 40.8 - 40.9×32.5 - 36.5 1.12-1.25 Shallow waveform 68-86 Oval C∙ zengii Long ellipse 39.9 - 40.8×31.9 - 36.7 1.10-1.26 Shallow waveform 65-85 Oval C. rhytidocarpa Long ellipse 40.1-41.2×31.4-36.7 1.14-1.23 Shallow waveform 62-78 Oval C. lipingensis and C. zengii share elliptical stomata with fully overlapping dimensions: length (40.8-40.9 μm vs. 39.9-40.8 μm), width (32.5-36.5 μm vs. 31.9-36.7 μm), length-to-width ratio (1.12-1.25 vs. 1.10-1.26), and density (68-86 vs. 65-85 stomata / mm²). Both exhibit sinuous inner periclinal walls. C. lipingensis and C. rhytidocarpa show morphological continuity: stomatal length (40.1-41.2 μm vs. 40.8-40.9 μm) differs slightly, but width (31.4-36.7 μm vs. 32.5-36.5 μm) and L/W ratio (1.14-1.23 vs. 1.12-1.25) fall within intraspecific variation. Minor density differences occur (62-78 vs. 68-86 stomata / mm²) (Table S2). The microscopic morphological characteristics of pollen of three species Palynological analysis (Table 3, Figure 4) revealed near-identical pollen characteristics in C. lipingensis and C. zengii . Both species produce subspherical pollen with partially overlapping polar axis (36.7-36.8 μm vs. 36.8 μm) and equatorial axis lengths (40.3 μm vs. 41.3 μm). The P/E ratio (0.90 vs. 0.9) showed complete convergence, while colpus dimensions exhibited minimal divergence: length (26.3-26.4 μm vs. 26.3 μm), width (7.3 μm vs. 7.4 μm), and mean L/W ratio of 3.6. Identical granular exine ornamentation and muri-granula complexes further support morphological continuity. C. rhytidocarpa exhibits continuous clinal variation in pollen morphology versus C. lipingensis / C. zengii , yet maintains substantial overlap. Its oblate-spheroidal pollen shows slightly longer polar axes (37.2-37.8 μm vs. C. zengii : 36.8 μm), while equatorial axes (41.3-41.4 μm vs. 41.3-41.4 μm) and P/E ratios (0.9) display complete congruence. Identical granular exine ornamentation and muri width (0.5 μm vs. 0.5-0.6 μm) confirm undifferentiated ultrastructure. Table 3 Pollen morphological characteristics of three species Species The shape of pollen Polar axis (P) /μm Equatorial axis (E) /μm Polar axis/equatorial axis Germination groove /μm Length Width Length/Width (average value) C. lipingensis Near-spherical shape 36.7-36.8 40.3 0.9 26.3-26.4 7.3 3.6 C. zengii Near-spherical shape 36.8 41.3 0.9 26.3 7.4 3.6 C. rhytidocarpa Near-spherical shape 37.2-37.8 41.3-41.4 0.9 27.7-27.7 7.4 3.5 Chloroplast genomic characteristics Integrated molecular analyses (Table 4, Figure 5) revealed high chloroplast genome conservation among C. lipingensis , C. zengii , and C. rhytidocarpa . Genome sizes were 157,029 bp ( C. lipingensis / C. zengii ) versus 157,048 bp ( C. rhytidocarpa ), with identical GC content (37.3%). LSC/SSC/IR lengths were conserved in C. lipingensis and C. zengii (LSC 86,630 bp, SSC 18,281 bp, IR 52,118 bp), while C. rhytidocarpa showed minimal LSC expansion (86,648 bp) and stable SSC (18,282 bp). Regional GC content exhibited negligible variation: LSC 35.31-35.32%, SSC 30.61%, IR 42.94-42.97%. Gene inventories were identical: 132 total genes (87 protein-coding, 37 tRNA, 8 rRNA). Slight divergence occurred only in third-codon position GC content (29.40-29.47%). IR boundary analysis confirmed conserved gene positioning ( rpl22 , rpl2 , psbA ). The LSC expansion in C. rhytidocarpa suggests potential ecological adaptation. Overall genome stability supports close phylogenetic affinity, with subtle structural differences indicating incipient adaptive divergence. Table 4 The genome-wide characteristics of chloroplasts in three species Species lipingensis C. zengii rhytidocarpa Genome size (bp) 157029 157029 157048 GC (%) 37.3 37.3 37.3 LSC size (bp) 86630 86630 86648 SSC size (bp) 18281 18281 18282 IR size (bp) 52118 52118 52118 GC in LSC (%) 35.32 35.32 35.31 GC in SSC (%) 30.61 30.61 30.61 GC in IR (%) 42.94 42.94 42.97 GC in CDS (%) 37.61 37.61 37.61 1st position GC (%) 45.21 45.21 45.37 2nd position GC (%) 37.9 37.9 38.04 3rd position GC (%) 29.47 29.45 29.40 Length of CDS 79100 79100 79100 Number of genes 132 132 132 Number of CDS 87 87 87 Number of tRNA 37 37 37 Number of rRNA 8 8 8 IR boundary contraction/expansion analysis (Figure 6) confirmed conserved positioning and lengths of rpl22 , rpl2 , and psbA genes in C. lipingensis , C. zengii , and C. rhytidocarpa . Minimal interspecific divergence occurred, with slight LSC length variations. Comparative assessment with Camellia rubituberculata and Camellia anlungensis revealed distinct IR boundary initiation patterns: the focal species initiated at the trnH gene, whereas C. rubituberculata and C. anlungensis initiated at tRNA genes. All five species exhibited similar contraction/expansion trends. Despite conserved gene architecture, subtle interspecific differences indicate lineage-specific adaptations. These structural variations likely reflect ecological selection pressures, influencing genomic organization and functional expression. Evolutionary fine-tuning appears associated with niche specialization. Phylogenetic tree analysis Phylogenomic analyses (Figure 7) placed C. lipingensis , C. zengii , and C. rhytidocarpa within Clade I with maximal support (ML=100, BI=1.00). Specifically, C. lipingensis and C. zengii formed a monophyletic subclade I-1 (ML=100, BI=1.00), while C. rhytidocarpa occupied subclade I-2. Congruently, phylogenies based on protein-coding genes (Figure 8) and ITS sequences (Figure 9) consistently resolved all three species within sect . Tuberculata , clustering them on a shared branch with high statistical support. Taxonomic treatment The integrated evidence from morphology, anatomy, palynology, and molecular phylogenetics supports recognizing C. lipingensis , C. zengii , and C. rhytidocarpa as a single species. These taxa should be taxonomically subsumed under C. rhytidocarpa (Figure 10). Camellia rhytidocarpa Hung T. Chang & S. Y. Liang in Chang, Tax. Gen. Camellia: 49. 1981. syn. nov. Type : CHINA. Guangxi Zhuang Autonomous Region: Longsheng County, Tianpingshan Village, s.c. 700, 908 (holotype SCBI; isotype PE). = Camellia lipingensis Hung T. Chang, Acta Sci. Nat. Univ. Sunyatseni 23(2): 78. 1984. Type : CHINA. Guizhou Province: Liping County, Wulong Mountain, s.c. 808, 81064 (holotype SYS; isotype GZFI). = Camellia zengii Hung T. Chang, Acta Sci. Nat. Univ. Sunyatseni 23(2): 77. 1984. syn. nov. Type : CHINA. Guizhou Province: Liping County, Wulong Mountain, s.c. 8017, 8018 (holotype GZFI; isotypes SYS). Botanical Description: Small macrophanerophytes with glabrous, lustrous young branches. Leaves thickly coriaceous, lanceolate, 6-12 cm long × 2-4 cm wide; apex caudate-acuminate, base broadly cuneate to rounded; adaxially greenish and slightly lustrous when dry, abaxially fulvous and glabrous except for sparse villous hairs along midvein; lateral veins 5-10 pairs prominently raised on both surfaces, reticulation obscure; margin sharply serrate; petiole 5-11 mm. Flowers white, terminal, subsessile, to 5 cm diameter. Outer bracts 4, scarious, apex apiculate and pubescent. Inner bracts ovate, 1-1.4 cm long, apex acute, margin scarious and densely pubescent. Petals 10, oblong to ovate, 3.5 cm long. Stamens 3 cm long, outer filaments nearly free. Ovary 3-loculed, densely pubescent; styles 3, 3.5 cm long, pubescent. Capsule oblate, tuberculate, 2.5-3 cm diameter, 3-loculed; seeds 1 per locule, densely tomentose. Flowering August to September. Distribution and Habitat : C. lipingensis and C. zengii are endemic to Liping County, Guizhou Province. Their typical habitats include Wulong Mountain (Qiutuan Village), Cenba Village, and Huangtianbang, where they thrive in mountain ecosystems at 1000-1200 m elevation, predominantly on slope gradients of 15-45°. In contrast, C. rhytidocarpa is restricted to Tianping Mountain Village, Longsheng County, Guangxi Zhuang Autonomous Region. This species occupies gentle valleys, forest understories, and riparian zones within the same altitudinal range (1000-1200 m) (Figure 11). Discussion The taxonomic controversy surrounding C. lipingensis , C. zengii , and C. rhytidocarpa has long centered on intraspecific variation in floral and leaf morphology (Min et al., 1993; Chang, 1996). This study integrates morphological, anatomical, palynological, and molecular phylogenetic evidence with long-term field surveys and type specimen examinations to clarify their delimitation. Results demonstrate highly conserved traits—including leaf shape, petiole length, and fruit size—supporting their status as geographic variants within a single species. Such trait stability represents a phenotypic response to heterogeneous habitats in plant taxonomy (Williams, 2010). Barrio (2023) established that leaf morphology conservatism correlates strongly with local environmental selection pressures. Anatomical data confirm homologous stomatal apparatus parameters and anticlinal wall patterns across all three taxa, aligning with intraspecific micromorphological clines (Pinedo et al., 2016; Longhi et al., 2024). Observed foliar differences likely reflect adaptive adjustments to microenvironments (e.g., light, humidity gradients) rather than conclusive evidence for speciation. Type specimen verification and wild population sampling are essential for species delimitation (Xue et al., 2018; Xu et al., 2023). In this study, when systematically verifying the digital specimen banks (such as CVH, GBIF, etc.) and the type specimens in the collection, it was found that there were some information deficiencies in the existing specimens, such as the absence of complete morphological characteristics of flowers and fruits on the specimens. Such deficiencies have historically led to overreliance on fragmented characters, neglecting population-level trait continuity (Bebber et al., 2010). By integrating voucher specimens with field-collected data, we reconstructed morphological profiles and taxonomic status for these taxa within sect. Tuberculata , as corroborated by Bossa-Castro et al.’s (2024) finding that >30% of global herbarium specimens lack critical diagnostic traits, necessitating field validation. The diversity of pollen morphology is the result of plants adapting to different environmental and ecological conditions over the course of long-term evolution (Mander et al., 2021). The continuous variation in pollen polarisation ratio and outer wall ornamentation (coarse net-ridge-papilla composite structure) of C. lipingensis , C∙ zengii , and C. rhytidocarpa is consistent with intraspecific pollen polymorphism (Gamal, 2025), while the flattened spherical pollen and significantly reduced papilla density of C. rhytidocarpa point to an independent evolutionary path. This difference may reflect their adaptation to different pollination environments, with distribution in drier, hotter regions potentially driving the restructuring of pollen outer wall structures. Additionally, the regular differences in germination pore distribution further reinforce their taxonomic independence (Amstutz et al., 2024). Molecular phylogenetic evidence provides critical support for taxonomic revision (Wei et al., 2023; Abe et al., 2024; Qin et al., 2024). Chloroplast genome analyses reveal sequence similarity among C. lipingensis , C. zengii , and C. rhytidocarpa , with phylogenies placing them in a monophyletic clade (ML=100, BI=1.00). This aligns with Chang et al. (2021) on chloroplast genome conservation in closely related species. Despite sympatric distribution of C. lipingensis and C. zengii , nuclear ITS phylogeny confirms their genetic homogeneity, validating Wang et al. (2025) regarding multi-gene analysis reliability in taxonomy. Geographic isolation and niche differentiation drive interspecific divergence (Lindelof et al., 2020). C. lipingensis and C. zengii co-occur in similar mountainous habitats (1000-1200 m, 15-45° slopes) in Liping County, Guizhou, whereas C. rhytidocarpa is restricted to Longsheng County, Guangxi. Habitat heterogeneity likely drives morphological adaptations through natural selection. PCA results indicate slightly larger leaves in C. rhytidocarpa , but other traits align closely with the Guizhou taxa. Environmental variation elicits differential phenotypic responses within species — e.g., acuminate leaf apices may reduce transpiration in drier microhabitats (Liu et al., 2025). Molecular phylogenetic data showed that the chloroplast genomic differences among the three species were small, and it is possible that none of them reached the level of species-level differentiation within plant genera (Mondini et al., 2009). This is consistent with the (asynchrony of morphological-genetic differentiation) demonstrated by Zou et al. (2021) in Rhododendron L., which suggests that phenotypic plasticity may play a dominant role in the formation of morphological differences. Conclusion This study pioneers an integrated multidisciplinary analysis of C. lipingensis , C. zengii , and C. rhytidocarpa , systematically resolving their morphoanatomical traits, palynological patterns, and molecular phylogenetics. Results demonstrate discontinuous variation across multidimensional trait spectra with significant genetic differentiation, supporting the reclassification of C. lipingensis and C. zengii as synonyms subsumed under C. rhytidocarpa . This revision resolves long-standing synonymy issues in sect. Tuberculata and establishes a robust framework for germplasm conservation. The revealed morpho-molecular coevolution further provides a theoretical foundation for assessing intraspecific variation, genetic diversity patterns, and adaptive evolution within this complex. Abbreviations FAA Formalin-Acetic Acid-Alcohol PBS Phosphate-Buffered Saline SEM Scanning Electron Microscopy CTAB Cetyltrimethylammonium Bromide cpDNA Chloroplast DNA LSC Large Single-Copy region SSC Small Single-Copy region IR Inverted Repeat GC Guanine-Cytosine content CDS Coding DNA Sequence tRNA Transfer RNA rRNA Ribosomal RNA ML Maximum Likelihood BI Bayesian Inference PP Posterior Probability BS Bootstrap Support PCGs Protein-Coding Genes ITS Internal Transcribed Spacer PCA Principal Component Analysis NCBI National Center for Biotechnology Information GBIF Global Biodiversity Information Facility Declarations Author Contributions Weihao Gu : Formal analysis (lead); Methodology (lead); Writing - original draft (lead). Mingtai An : Formal analysis (equal); Validation (supporting). Chao Yan : Formal analysis (equal); Validation (supporting). Xu Xiao : Visualization (supporting). Zhaohui Ran : Validation (supporting). Zhi Li : Conceptualization (equal); Supervision (equal). Funding This work was supported by the National Natural Science Foundation of China (32400179), and the 2024 Guizhou Science and Technology Innovation Talent Team Construction Project: Wildlife Innovation Team of the Forestry college of Guizhou University (Qian ke he ren cai CXTD [2025] 053). Availability of data and materials GenBank accession numbers: PV761587, PV750797, PV750798, PV750799, PV750800, PV750801, PV750802. The Appendix Table for this article can be found online. Competing interests There is no competing interests to declare. Author details 1 College of Forestry, Guizhou University, Guiyang 550025, China. References Abe H, Ueno S, Matsuo A, Hirota SK, Miura H, Su M-H, et al. (2024) Evolutionary histories of Camellia japonica and Camellia rusticana . Ecology and Evolution 14: e70721. https://doi.org/10.1002/ece3.70721 Amiryousefi A, Hyvönen J, Poczai P (2018) IRscope: an online program to visualize the junction sites of chloroplast genomes. 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Journal of Systematics and Evolution 59:1244-1255.https://doi.org/10.1111/jse.12690 Supplementary Files TableS1.xlsx TableS2.xlsx Cite Share Download PDF Status: Published Journal Publication published 19 Jan, 2026 Read the published version in Botanical Studies → Version 1 posted Editorial decision: Major revision 19 Oct, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 09 Jul, 2025 Editor assigned by journal 02 Jul, 2025 First submitted to journal 01 Jul, 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. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7021883","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483225812,"identity":"678fc9b0-2909-48bc-9708-a4bd79127344","order_by":0,"name":"Weihao Gu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Weihao","middleName":"","lastName":"Gu","suffix":""},{"id":483225813,"identity":"3657cae0-b986-442a-b1a0-f7c97f56d0dd","order_by":1,"name":"Mingtai An","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Mingtai","middleName":"","lastName":"An","suffix":""},{"id":483225814,"identity":"afa7c1de-a576-408b-9a5d-fc7ce1307338","order_by":2,"name":"Chao Yan","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Yan","suffix":""},{"id":483225815,"identity":"09a53300-d9a0-4ddc-912c-b38d8bda7508","order_by":3,"name":"Xu Xiao","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Xiao","suffix":""},{"id":483225816,"identity":"7b8e32f0-a4ad-421b-9765-6502ca4a91a6","order_by":4,"name":"Zhaohui Ran","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhaohui","middleName":"","lastName":"Ran","suffix":""},{"id":483225817,"identity":"b9b9a443-c320-490a-9607-d6678ae58411","order_by":5,"name":"Zhi Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYDACdsb2Hx8gTAMitTAzNkjOIFELA4M0D0la5JuZG4xtftUlNrA3b5NgqLlDWAtjM2NDcm4fW2IDz7EyCYZjz4hwF9Avh3N7eBIbJHLMJIBswlrYmBkbmy17JBIb5N8QqYWHmbGZmeGHAdAWHiK1SDAztjH2NiQYt/GkFVskHCNCi3x7+zOGH3/qZPvZD2+88aGGCC1gwNgG9BSIkUCkBiD4Q7zSUTAKRsEoGIEAACHmMckoAG0gAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7094-1672","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-01 15:29:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7021883/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7021883/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40529-025-00489-5","type":"published","date":"2026-01-19T15:58:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86641418,"identity":"74d871ae-03e6-40b8-9a88-8ac34a9cb225","added_by":"auto","created_at":"2025-07-14 08:19:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":640490,"visible":true,"origin":"","legend":"\u003cp\u003eThe main morphological characteristics of the three species (A: \u003cem\u003eC. lipingensis\u003c/em\u003e; B:\u003cem\u003e C. zengii\u003c/em\u003e; C: \u003cem\u003eC. rhytidocarpa\u003c/em\u003e. 1: Wild habitat, 2: Flower, 3: Fruit, 4: Petals, 5: Calyx, 6: Mature fruit, 7: Seeds, 8: Filaments, 9: One year branches, 10: Front and back of the leaf.)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/f881e0c959c1829e1f3dc686.png"},{"id":86641413,"identity":"d0cf0084-f235-4ef4-8bd9-7d89aeb44327","added_by":"auto","created_at":"2025-07-14 08:19:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42184,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of the morphological characteristics of three species. (Note: Confidence ellipses indicate the 95% normal distribution range for each group.)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/eb62fe8e499f5a20afd16858.png"},{"id":86642553,"identity":"c4cf7cb6-e4b1-48da-9197-6eff55e1e73b","added_by":"auto","created_at":"2025-07-14 08:27:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":658438,"visible":true,"origin":"","legend":"\u003cp\u003eThe micromorphological characteristics of leaf epidermis of three species. (A: \u003cem\u003eC. lipingensis\u003c/em\u003e; B: \u003cem\u003eC. zengii\u003c/em\u003e; C: \u003cem\u003eC. rhytidocarpa\u003c/em\u003e. 1, 2: Adaxial surface; 3, 4: Hypodermis; 1, 3: 20×; 2, 4: 40×; 5, 6: Stomatal apparatus)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/60d7677e1135cf2ec73f2231.png"},{"id":86642544,"identity":"226515f8-6174-43bb-abe6-703c6ea075d1","added_by":"auto","created_at":"2025-07-14 08:27:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":388399,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscope scans of three species. (A: \u003cem\u003eC. lipingensis\u003c/em\u003e; B: \u003cem\u003eC. zengii\u003c/em\u003e; C: \u003cem\u003eC. rhytidocarpa\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/d46b1120fb41a17098a87cb7.png"},{"id":86641388,"identity":"b733ab23-2144-4073-b87d-4ed6684dc6e2","added_by":"auto","created_at":"2025-07-14 08:19:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":249623,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome maps of three species. (A: \u003cem\u003eC. lipingensis\u003c/em\u003e; B: \u003cem\u003eC. zengii\u003c/em\u003e; C:\u003cem\u003e C. rhytidocarpa\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/0aca1c0e7f779db36af6ef45.png"},{"id":86641397,"identity":"d7ce40bb-d0bb-4765-a198-7f06804bcfc3","added_by":"auto","created_at":"2025-07-14 08:19:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":150613,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the contraction and expansion of IR boundaries in five species\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/911af51a3535f4cc42a0dea9.png"},{"id":86643241,"identity":"2ba2acc9-076e-4976-a747-484999ced68c","added_by":"auto","created_at":"2025-07-14 08:35:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":97193,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on the chloroplast genome (Maximum Likelihood (ML) and Bayesian (BI) trees, BS ≥ 50% and PP ≥ 0.95 are indicated above branches as BS/PP)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/52dd900d804d66075d3b9741.png"},{"id":86641389,"identity":"35185a8c-fc5a-4d14-bedb-72b9b5432555","added_by":"auto","created_at":"2025-07-14 08:19:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":97576,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on the PCGs (Maximum Likelihood (ML) and Bayesian (BI) trees, BS ≥ 50% and PP ≥ 0.95 are indicated above branches as BS/PP)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/019106071786e5ae68feded6.png"},{"id":86641393,"identity":"5a8043fc-9883-420a-ae18-eca3882a55b4","added_by":"auto","created_at":"2025-07-14 08:19:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":83174,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on the ITS (Maximum Likelihood (ML) and Bayesian (BI) trees, BS ≥ 50% and PP ≥ 0.95 are indicated above branches as BS/PP)\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/1ad15eef37c046ee9eda71ec.png"},{"id":86641456,"identity":"400eb68a-cd8d-43da-b1fc-fc5b08e1dd89","added_by":"auto","created_at":"2025-07-14 08:20:00","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":386932,"visible":true,"origin":"","legend":"\u003cp\u003eType specimen information of the three species. (A: \u003cem\u003eC. lipingensis\u003c/em\u003e; B: \u003cem\u003eC. zengii\u003c/em\u003e; C:\u003cem\u003e C. rhytidocarpa. \u003c/em\u003eNote: Figure B was obtained from the National Specimen Platform (http://www.nsii.org.cn/2017/home.php) and was found to be a type specimen after verification.)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/9b1c31053f9a2c7a72fccf46.png"},{"id":86641391,"identity":"ec9fa882-3f78-4a32-b987-d90ee1b3e42c","added_by":"auto","created_at":"2025-07-14 08:19:57","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":159462,"visible":true,"origin":"","legend":"\u003cp\u003eMap of collection loci of \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC∙ zengii\u003c/em\u003e and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/3ad580356663d488c3633100.png"},{"id":101152555,"identity":"45413d9c-9b3f-4c6b-8179-43abbecb30ec","added_by":"auto","created_at":"2026-01-26 16:12:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4086568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/eeb324a6-6d92-40ff-afae-427b53e6b90d.pdf"},{"id":86641409,"identity":"bb025bd3-9ca1-445b-b74a-d8a47e841419","added_by":"auto","created_at":"2025-07-14 08:19:58","extension":"xlsx","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":32751,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/836c4d5794853c2bda8d9734.xlsx"},{"id":86642549,"identity":"008e7f83-ab7d-4b31-a310-d68ab51c440a","added_by":"auto","created_at":"2025-07-14 08:27:57","extension":"xlsx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":18248,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7021883/v1/ebe39b115714ac743a6470f3.xlsx"}],"financialInterests":"","formattedTitle":"Integrative taxonomic revision of the Camellia rhytidocarpa complex (Theaceae) synonymous status of C. lipingensis and C. zengii supported by morphological, anatomical, palynological, and molecular evidence","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eCamellia\u0026nbsp;\u003c/em\u003esect. \u003cem\u003eTuberculata\u003c/em\u003e (Theaceae, \u003cem\u003eCamellia\u003c/em\u003e L.) is distinguished from other \u003cem\u003eCamellia\u003c/em\u003e groups by its uniquely \"tuberculate fruit surface\" making it the most morphologically peculiar fruit lineage in the genus\u0026nbsp;(Chang and Ren, 1991; Min and Zhong, 1993; Chang, 1998). In the early 20th century, the renowned botanist, Prof. S.S. Chien (1939) discovered a taxon with tuberculate ovaries and pericarps during fieldwork in Jiading, Sichuan, China, and naming it as \u003cem\u003eCamellia\u003c/em\u003e \u003cem\u003etuberculata\u003c/em\u003e S.S. Chien.\u0026nbsp;Sealy (1958),\u0026nbsp;later placed it in section \u003cem\u003eHeterogenea\u0026nbsp;\u003c/em\u003e. It was not until 1981 that Hung-Ta Chang established sect. \u003cem\u003eTuberculata\u003c/em\u003e based on the diagnostic \"tuberculate-wrinkled fruit pericarp\"that initially including 6 species, with 12 additional species reported over the next decade. In 1991, Chang subdivided the section into two subsections (subsect. \u003cem\u003eTuberculata\u003c/em\u003e Chang and subsect. \u003cem\u003eNudicarpa\u003c/em\u003e Chang) based on whether ovary pubescence. Tianlu Min (1993), a renowned \u003cem\u003eCamellia\u0026nbsp;\u003c/em\u003etaxonomist revised this classified process, and divided 18 species into 6 species, 6 varieties, and 1 form\u0026nbsp;(Min and Zhong, 1993). It is noteworthy that the taxonomic treatments proposed by both researchers were primarily based on Herbarium museum specimens, with insufficient field investigations and a notable lack of multidisciplinary taxonomic evidence. As a result, the infraspecific classification within thesect. \u003cem\u003eTuberculata\u0026nbsp;\u003c/em\u003eremains contentious.\u003c/p\u003e\n\u003cp\u003eThe plant species complex refers to a group of closely related taxa that exhibit high similarity in morphology, genetics, or ecology, with ambiguous taxonomic boundaries (\u003cstrong\u003eHe\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;et al., 2022\u003c/strong\u003e), such complexes typically comprise multiple species or infraspecific units (e.g., subspecies, varieties), potentially involving hybridization, incomplete lineage sorting, or cryptic diversity, resulting in challenges for traditional morphological classification to accurately delineate species limits.\u0026nbsp;\u003cem\u003eC.\u0026nbsp;\u003c/em\u003e\u003cem\u003elipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e were all described by Hung-Ta Chang in 1984 (Chang and Ren, 1996). The type localities of the former two species are Wulong Mountain, Liping County, Guizhou Province, China. \u003cem\u003eC. lipingensis\u0026nbsp;\u003c/em\u003eis characterized by narrow lanceolate leaves, thickly leathery texture, glabrous styles, and 5 cm diameter flowers, and \u003cem\u003eC. zengii\u0026nbsp;\u003c/em\u003eexhibits villous styles, stamens 1.8-2.4 cm in length, 10 fractions, and sepals 1-4 cm in length. The type specimen of \u003cem\u003eC. rhytidocarpa\u003c/em\u003e was collected from Tianping Mountain, Longsheng, Guangxi China, displaying distinctive features “lanceolate or oblong leaves measuring 8-12 cm in length, and 6-7 pairs of lateral veins that are not sunken”. In 1993, Tianlu Min proposed merging these three species into \u003cem\u003eC. rhytidocarpa\u003c/em\u003e (Min et al., 1993)\u0026nbsp;considered they share similar morphological traits “narrowly lanceolate leaves with caudate or acuminate apices, ovate sepals, and ovaries, sparsely pubescent styles with 3 lobes” and occurring in analogous habitats in theborder region of Hunan, Guizhou, Guangxi. Chang et al. (1996). However, challenged this classification, demonstrating that the three species are morphologically distinct in leaf shape, floral structures (sepals, petals), and style characteristics. Over the past three decades, taxonomic studies of \u003cem\u003eCamellia\u0026nbsp;\u003c/em\u003ehave partially incorporated analyses of pollen micromorphology, cpDNA, and leaf epidermal features for the three species ((Jiang et al., 2010; Yan et al., 2024; Ran et al., 2024c). Nevertheless, the taxonomic delimitation within this species complex remains unresolved.\u003c/p\u003e\n\u003cp\u003eIn recent years, we have conducted extensive field investigations at the type localities of these three species, complemented by comprehensive morphological analyses (including both macro- and micro-morphological examinations of leaf epidermis and pollen) and molecular phylogenetic studies (cpDNA and ITS markers). By integrating these multidisciplinary lines of evidence to aim to resolve the taxonomic complexities within this species complex and clarify interspecific boundaries.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eObservation and inspection of type specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eType specimen examination was systematically conducted to clarify species boundaries and verify historical taxonomic foundations, utilizing digitized resources from the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE; http://pe.ibcas.ac.cn/peweb/), Sun Yat-sen University Herbarium (SYS; https://eco.sysu.edu.cn/platforms/museum), and the Chinese Virtual Herbarium (CVH; https://www.cvh.ac.cn/). Morphological parameters were rigorously measured using standardized techniques (Ran et al., 2024a), with type specimens of \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e sourced through original literature review and authoritative digital platforms including CVH and the National Specimen Resource Sharing Platform (NSII; http://www.nsii.org.cn/2017/home.php) (Wu et al., 2022; Seo et al., 2025). Comprehensive morphological observations and comparative analyses were performed on all examined specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe field materials used for experimental and morphological data measurements in this study were collected through systematic field surveys between 2022 and 2024. Fresh leaves and floral organs collected in the field were preserved via dry ice flash-freezing to ensure sample integrity (Xiao et al., 2024). Voucher specimens were processed following international herbarium standards and deposited in the Herbarium of the Forestry College, Guizhou University (\u003cem\u003eC. lipingensis\u003c/em\u003e: LZ-20231126-7; \u003cem\u003eC. zengii\u003c/em\u003e: LZ-20231127-3; \u003cem\u003eC. rhytidocarpa\u003c/em\u003e: LZ-20240114-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphology Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study systematically investigated plant morphological characteristics through integrated herbarium specimen examinations and field observations, documenting natural habitats and overall plant morphology before conducting standardized measurements of qualitative and quantitative traits from ≥10 voucher specimens per species. The comprehensive measurements included stem/branch bark coloration and texture, leaf characteristics (texture, color, dimensions, margin morphology, base traits, and venation patterns), floral organ features (petal size, number, color, sepal morphology, and style number), and fruit attributes (diameter, shape, pericarp rugosity, and maturation-related color changes), with all parameters measured in triplicate and averaged (Ran et al., 2024b). The standardized dataset (Table S1) was processed using Excel 2025 and subsequently analyzed through principal component analysis (PCA) of 35 morphological variables in R v4.4.2 (R Core Team, 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeaf epidermis and pollen micromorphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh leaves and floral buds of \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e were collected for micromorphological analyses. Leaf samples underwent dual processing protocols: 1) FAA fixation (70% ethanol:acetic acid:formaldehyde=90:5:5, v/v), distilled water rinsing, midvein dissection (0.5 cm²), sodium hypochlorite dissociation (30°C), and epidermal sectioning with acetate fuchsin staining for light microscopic examination of periclinal walls and stomatal apparatus (Jiang et al., 2022; Situngu et al., 2022); 2) Leaf tissue was gently rinsed with PBS. Tissue blocks were immediately fixed in EM fixative (2 h, RT), then stored at 4°C. Blocks were washed in 0.1 M PB (pH 7.4) ×3 (15 min each), post-fixed in 1% OsO₄ (in 0.1 M PB, pH 7.4; 1-2 h, RT), and washed again in PB ×3 (15 min each). Samples were dehydrated (ethanol series), critical point dried, mounted on aluminum stubs with carbon tabs, sputter-coated with Au (30 s), and imaged using SEM. (Zhang et al., 2021; Neilands et al., 2023).\u003c/p\u003e\n\u003cp\u003eConcurrently, pollen grains (10/species) from FAA-fixed, ethanol-dehydrated anthers (Smith et al., 2021) were acetolyzed, released via microdissection, gold-coated, and analyzed by SEM (JSM-6490LV) for quantitative assessment of morphology, size (P/E axes), colpi, exine ornamentation, and wall sculpturing using ImageTool software (Ocaña et al., 2022; Pan et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal DNA extraction, sequencing, assembly, and annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh leaves of the three species collected during field surveys were subjected to total DNA extraction using the CTAB (cetyltrimethylammonium bromide) method (Chai et al., 2023; Xing et al., 2023). Following Illumina DNA library preparation standards, paired-end sequencing libraries were constructed with 350-bp insert fragments (Li et al., 2021). DNA quality and concentration were assessed by agarose gel electrophoresis. Qualified fragments were sequenced on the Illumina platform after random fragmentation, end repair, and adapter ligation (Zong et al., 2023).\u003c/p\u003e\n\u003cp\u003eRaw reads generated via base-calling (CASAVA) were assembled using SPAdes v.3.15.2 and annotated with CPGAVAS and ORFFinder. Annotation results were validated by BLASTN/BLASTP alignments. tRNA genes were annotated using ARWEN v1.2 (Zheng et al., 2023; Gu et al., 2024). The complete chloroplast genome was finalized by online annotation, BLAST alignment, and manual curation against the \u003cem\u003eCamellia\u003c/em\u003e \u003cem\u003erubituberculata \u003c/em\u003eChang \u0026amp; Yu reference (MZ424202) (Xiao et al., 2025). Chloroplast genome maps were generated with OGDRAW (Yan et al., 2023; Ran et al., 2024c). Sequences were deposited in NCBI GenBank to obtain accession numbers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIR boundary expansion and contraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChloroplast genome sequences of \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e were screened, Three complete chloroplast genomes were obtained from the samples. Sequences of Theaceae species were downloaded from the NCBI for Biotechnology Information database. IR boundary expansion/contraction analysis was performed, and comparative maps were generated using IRscope (https://irscope.shinyapps.io/irapp/) (Amiryousefi et al., 2018; Xiao et al., 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChloroplast genomes of 24 \u003cem\u003eCamellia\u003c/em\u003e species were retrieved from NCBI, with \u003cem\u003eApterosperma oblata\u003c/em\u003e (NC_035641) as the outgroup (Ran et al., 2024a). Sequence alignment used MAFFT v7, followed by maximum likelihood (ML) tree construction in IQ-TREE v1.6.12. Manual refinement in MEGA X selected optimal substitution models (Smith et al., 2022). MrModeltest v2.3 identified (TVM+F+I) as the best-fit model, and Bayesian inference (BI) trees were reconstructed using MrBayes v3.2.7 (Catalano et al., 2025). Phylogenies based on protein-coding genes and ITS were congruent with whole chloroplast genomes. Final trees were visualized with iTOL v5 (Letunic et al., 2021).\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eThe morphological characteristics of three species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystematic morphological comparisons revealed highly consistent frameworks among the three species, with only continuous or subtle phenotypic variations (Figure 1, Table 1). \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e shared brownish mottled bark exfoliating to green/grey-green surfaces, red-brown quadrangular branchlets, and leathery lanceolate leaves (vs. occasionally oblong in \u003cem\u003eC. rhytidocarpa\u003c/em\u003e) with acute apices and sparse serrations. Leaf dimensions (6.66-12.48 \u0026times; 2.16-3.93 cm vs. 5.81-11.60 \u0026times; 1.17-4.10 cm) and petiole lengths (0.12-1.10 cm vs. 0.30-0.91 cm) overlapped. Floral traits showed near-identical petal dimensions (3.44-5.20 \u0026times; 1.30-2.00 cm vs. 3.47-4.71 \u0026times; 1.34-2.21 cm), number (6-9 vs. 7-8), ovate sepals, and overlapping ovary/style pubescence. Fruit diameter (2.24-3.15 cm vs. 2.26-3.18 cm) and shape (subglobose) were consistent, with only minor pericarp thickness variation (0.45-0.60 cm vs. 0.43-0.52 cm), falling within intraspecific geographic variation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC .rhytidocarpa\u003c/em\u003e exhibits significant morphological homology with \u003cem\u003eC. lipingensis\u003c/em\u003e despite minor variations. Its occasionally oblong leaves with acuminate apices show complete dimensional overlap (7.04-12.50 \u0026times; 2.16-4.45 cm). Higher serration density may reflect local adaptation. Floral traits partially overlap: petal dimensions (3.20-4.28 \u0026times; 1.25-1.90 cm), obtuse sepal apices falling within intraspecific polymorphism. Fruit diameter (2.19-3.07 cm) and shape (subglobose/oblate) align closely, though pericarp thickness is reduced (0.30-0.49 cm), suggesting developmental plasticity. Continuous trait gradients and microhabitat-induced adjustments indicate these variations lack taxonomic diagnostic value, supporting reclassification as geographical variants or ecotypes of a single taxon.\u003c/p\u003e\n\u003cp\u003eTable 1 Comparison of the main morphological characteristics of three Species of sect. \u003cem\u003eTuberculata\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. lipingensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. zengii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. rhytidocarpa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTrunk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eBrownish with mottled exfoliation, revealing glaucous green trunks after bark shedding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eMottled brown with inconspicuously exfoliating, rugose bark\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eRusset bark with conspicuous exfoliation, revealing glaucous green trunks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eBranches\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eAnnual branchlets tetragonally ribbed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eAnnual branchlets tetragonally ribbed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eAnnual branchlets tetragonally ribbed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eLeaf type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eLeathery, lanceolate, leaf tail acute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eLeathery, lanceolate, leaf tail acute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eLeathery, lanceolate or long elliptic, leaf tail acuminate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eLeaf length (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e6.66-12.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e5.81-11.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e6.42-12.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eLeaf width (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e2.16-3.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e1.17-4.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e2.16-4.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eLeaf margin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eAcute serrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eAcute serrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eAcute serrate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003ePetiole length (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e0.12-1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e0.30-0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.43-1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003ePetal length (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e3.44-5.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e3.47-4.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e3.20-4.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003ePetal width (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e1.30-2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e1.34-2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e1.25-1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eNumber of petals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e6-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e7-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e6-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eNumber of calyxes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e6-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e7-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e6-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eSepal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eOvate, apex rounded\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eOvate, few rounded, apex rounded\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eRounded, apex rounded\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eOvaries\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e3-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e3-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e3-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eStyle (female organ of flower)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e3-5, pubescent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e3-5, pubescent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e3-5, pubescent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eFruit diameter (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e2.24-3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e2.26-3.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e2.24-3.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eFruit shape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSubglobular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eSubglobular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eSubglobular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eShell thickness (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e0.45-0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e0.43-0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.30-0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn order to conduct a comprehensive classification value assessment of each morphological trait of the three species, we carried out principal component analysis. The PCA results segregated all samples into three clusters.\u003cem\u003e\u0026nbsp;C. lipingensis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;C. zengii\u003c/em\u003e and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e exhibited extensive morphological overlap, with measured traits indicating a nested relationship among the three taxa (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeaf Epidermal Micromorphological Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroscopy revealed (Figure 3, Table 2) highly conserved yet continuously variable leaf micromorphology across the three species. \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e shared irregular epidermal cells with sinuous anticlinal walls on adaxial surfaces. Crucially, oil glands were absent in \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e, while sparse in \u003cem\u003eC. rhytidocarpa\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAbaxially, \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u0026nbsp;\u003c/em\u003eexhibited sinuous anticlinal walls versus undulate walls in \u003cem\u003eC. rhytidocarpa\u003c/em\u003e, though all species showed tight cell arrangement and anomocytic stomata with reniform guard cells and 3-5 similarly configured subsidiary cells.\u003c/p\u003e\n\u003cp\u003eTable 2 Morphological characteristics of leaf epidermis of the three species\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eSshape of stomata\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eSize of stomata (length/\u0026mu;m\u0026thinsp;\u0026times;\u0026thinsp;width/\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eLength\u0026thinsp;/\u0026thinsp;Width\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eInner margin of outer rim\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eStomatal density (stomata/mm\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eType of stomatal apparatus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. lipingensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eLong ellipse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e40.8 - 40.9\u0026times;32.5 - 36.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e1.12-1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eShallow waveform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e68-86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOval\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cem\u003eC∙ zengii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eLong ellipse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e39.9 - 40.8\u0026times;31.9 - 36.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e1.10-1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eShallow waveform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e65-85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOval\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. rhytidocarpa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eLong ellipse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e40.1-41.2\u0026times;31.4-36.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e1.14-1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eShallow waveform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e62-78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOval\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e share elliptical stomata with fully overlapping dimensions: length (40.8-40.9 \u0026mu;m vs. 39.9-40.8 \u0026mu;m), width (32.5-36.5 \u0026mu;m vs. 31.9-36.7 \u0026mu;m), length-to-width ratio (1.12-1.25 vs. 1.10-1.26), and density (68-86 vs. 65-85 stomata / mm\u0026sup2;). Both exhibit sinuous inner periclinal walls.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e show morphological continuity: stomatal length (40.1-41.2 \u0026mu;m vs. 40.8-40.9 \u0026mu;m) differs slightly, but width (31.4-36.7 \u0026mu;m vs. 32.5-36.5 \u0026mu;m) and L/W ratio (1.14-1.23 vs. 1.12-1.25) fall within intraspecific variation. Minor density differences occur (62-78 vs. 68-86 stomata / mm\u0026sup2;) (Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe microscopic morphological characteristics of pollen of three species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePalynological analysis (Table 3, Figure 4) revealed near-identical pollen characteristics in \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e. Both species produce subspherical pollen with partially overlapping polar axis (36.7-36.8 \u0026mu;m vs. 36.8 \u0026mu;m) and equatorial axis lengths (40.3 \u0026mu;m vs. 41.3 \u0026mu;m). The P/E ratio (0.90 vs. 0.9) showed complete convergence, while colpus dimensions exhibited minimal divergence: length (26.3-26.4 \u0026mu;m vs. 26.3 \u0026mu;m), width (7.3 \u0026mu;m vs. 7.4 \u0026mu;m), and mean L/W ratio of 3.6. Identical granular exine ornamentation and muri-granula complexes further support morphological continuity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. rhytidocarpa\u003c/em\u003e exhibits continuous clinal variation in pollen morphology versus \u003cem\u003eC. lipingensis\u003c/em\u003e/\u003cem\u003eC. zengii\u003c/em\u003e, yet maintains substantial overlap. Its oblate-spheroidal pollen shows slightly longer polar axes (37.2-37.8 \u0026mu;m vs. \u003cem\u003eC. zengii\u003c/em\u003e: 36.8 \u0026mu;m), while equatorial axes (41.3-41.4 \u0026mu;m vs. 41.3-41.4 \u0026mu;m) and P/E ratios (0.9) display complete congruence. Identical granular exine ornamentation and muri width (0.5 \u0026mu;m vs. 0.5-0.6 \u0026mu;m) confirm undifferentiated ultrastructure.\u003c/p\u003e\n\u003cp\u003eTable 3 Pollen morphological characteristics of three species\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 17px;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003eThe shape of pollen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003ePolar axis (P) /\u0026mu;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003eEquatorial axis (E) /\u0026mu;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003ePolar axis/equatorial axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 33px;\"\u003e\n \u003cp\u003eGermination groove /\u0026mu;m\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eLength\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eWidth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eLength/Width (average value)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. lipingensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eNear-spherical shape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e36.7-36.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e40.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e26.3-26.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. zengii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eNear-spherical shape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e36.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e41.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e26.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. rhytidocarpa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eNear-spherical shape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e37.2-37.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e41.3-41.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e27.7-27.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eChloroplast genomic characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntegrated molecular analyses (Table 4, Figure 5) revealed high chloroplast genome conservation among \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;C. rhytidocarpa\u003c/em\u003e. Genome sizes were 157,029 bp (\u003cem\u003eC. lipingensis\u0026nbsp;\u003c/em\u003e/ \u003cem\u003eC. zengii\u003c/em\u003e) versus 157,048 bp (\u003cem\u003eC. rhytidocarpa\u003c/em\u003e), with identical GC content (37.3%). LSC/SSC/IR lengths were conserved in \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e (LSC 86,630 bp, SSC 18,281 bp, IR 52,118 bp), while \u003cem\u003eC. rhytidocarpa\u003c/em\u003e showed minimal LSC expansion (86,648 bp) and stable SSC (18,282 bp).\u003c/p\u003e\n\u003cp\u003eRegional GC content exhibited negligible variation: LSC 35.31-35.32%, SSC 30.61%, IR 42.94-42.97%. Gene inventories were identical: 132 total genes (87 protein-coding, 37 tRNA, 8 rRNA). Slight divergence occurred only in third-codon position GC content (29.40-29.47%).\u003c/p\u003e\n\u003cp\u003eIR boundary analysis confirmed conserved gene positioning (\u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e). The LSC expansion in \u003cem\u003eC. rhytidocarpa\u003c/em\u003e suggests potential ecological adaptation. Overall genome stability supports close phylogenetic affinity, with subtle structural differences indicating incipient adaptive divergence.\u003c/p\u003e\n\u003cp\u003eTable 4 The genome-wide characteristics of chloroplasts in three species\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"512\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 179px;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003col start=\"100\"\u003e\n \u003cli\u003e\u003cem\u003elipingensis\u003c/em\u003e\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. zengii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003col start=\"100\"\u003e\n \u003cli\u003e\u003cem\u003erhytidocarpa\u003c/em\u003e\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenome size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e157029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e157029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e157048\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLSC size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e86630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e86630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e86648\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSSC size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18282\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIR size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52118\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC in LSC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC in SSC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC in IR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC in CDS (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1st position GC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2nd position GC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3rd position GC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e29.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e29.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e29.40\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLength of CDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e79100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e79100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e79100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNumber of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNumber of CDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNumber of tRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNumber of rRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIR boundary contraction/expansion analysis (Figure 6) confirmed conserved positioning and lengths of \u003cem\u003erpl22\u003c/em\u003e,\u003cem\u003e\u0026nbsp;rpl2\u003c/em\u003e, and \u003cem\u003epsbA\u003c/em\u003e genes in \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e. Minimal interspecific divergence occurred, with slight LSC length variations. Comparative assessment with \u003cem\u003eCamellia rubituberculata\u003c/em\u003e and \u003cem\u003eCamellia anlungensis\u003c/em\u003e revealed distinct IR boundary initiation patterns: the focal species initiated at the \u003cem\u003etrnH\u003c/em\u003e gene, whereas \u003cem\u003eC. rubituberculata\u003c/em\u003e and \u003cem\u003eC. anlungensis\u003c/em\u003e initiated at tRNA genes. All five species exhibited similar contraction/expansion trends. Despite conserved gene architecture, subtle interspecific differences indicate lineage-specific adaptations. These structural variations likely reflect ecological selection pressures, influencing genomic organization and functional expression. Evolutionary fine-tuning appears associated with niche specialization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic tree analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenomic analyses (Figure 7) placed \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e within Clade I with maximal support (ML=100, BI=1.00). Specifically, \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e formed a monophyletic subclade I-1 (ML=100, BI=1.00), while \u003cem\u003eC. rhytidocarpa\u003c/em\u003e occupied subclade I-2. Congruently, phylogenies based on protein-coding genes (Figure 8) and ITS sequences (Figure 9) consistently resolved all three species within sect\u003cem\u003e. Tuberculata\u003c/em\u003e, clustering them on a shared branch with high statistical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTaxonomic treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe integrated evidence from morphology, anatomy, palynology, and molecular phylogenetics supports recognizing\u003cem\u003e\u0026nbsp;C. lipingensis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;C. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e as a single species. These taxa should be taxonomically subsumed under \u003cem\u003eC. rhytidocarpa\u003c/em\u003e (Figure 10).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCamellia rhytidocarpa\u003c/em\u003e Hung T. Chang \u0026amp; S. Y. Liang in Chang, Tax. Gen. Camellia: 49. 1981. syn. nov.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eType\u003c/strong\u003e: CHINA. Guangxi Zhuang Autonomous Region: Longsheng County, Tianpingshan Village, s.c. 700, 908 (holotype SCBI; isotype PE).\u003c/p\u003e\n\u003cp\u003e= \u003cem\u003eCamellia lipingensis\u003c/em\u003e Hung T. Chang, Acta Sci. Nat. Univ. Sunyatseni 23(2): 78. 1984.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eType\u003c/strong\u003e: CHINA. Guizhou Province: Liping County, Wulong Mountain, s.c. 808, 81064 (holotype SYS; isotype GZFI).\u003c/p\u003e\n\u003cp\u003e= \u003cem\u003eCamellia zengii\u003c/em\u003e Hung T. Chang, Acta Sci. Nat. Univ. Sunyatseni 23(2): 77. 1984. syn. nov.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eType\u003c/strong\u003e: CHINA. Guizhou Province: Liping County, Wulong Mountain, s.c. 8017, 8018 (holotype GZFI; isotypes SYS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBotanical Description:\u0026nbsp;\u003c/strong\u003eSmall macrophanerophytes with glabrous, lustrous young branches. Leaves thickly coriaceous, lanceolate, 6-12 cm long \u0026times; 2-4 cm wide; apex caudate-acuminate, base broadly cuneate to rounded; adaxially greenish and slightly lustrous when dry, abaxially fulvous and glabrous except for sparse villous hairs along midvein; lateral veins 5-10 pairs prominently raised on both surfaces, reticulation obscure; margin sharply serrate; petiole 5-11 mm. Flowers white, terminal, subsessile, to 5 cm diameter. Outer bracts 4, scarious, apex apiculate and pubescent. Inner bracts ovate, 1-1.4 cm long, apex acute, margin scarious and densely pubescent. Petals 10, oblong to ovate, 3.5 cm long. Stamens 3 cm long, outer filaments nearly free. Ovary 3-loculed, densely pubescent; styles 3, 3.5 cm long, pubescent. Capsule oblate, tuberculate, 2.5-3 cm diameter, 3-loculed; seeds 1 per locule, densely tomentose. Flowering August to September.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution and Habitat\u003c/strong\u003e: \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e are endemic to Liping County, Guizhou Province. Their typical habitats include Wulong Mountain (Qiutuan Village), Cenba Village, and Huangtianbang, where they thrive in mountain ecosystems at 1000-1200 m elevation, predominantly on slope gradients of 15-45\u0026deg;. In contrast, \u003cem\u003eC. rhytidocarpa\u003c/em\u003e is restricted to Tianping Mountain Village, Longsheng County, Guangxi Zhuang Autonomous Region. This species occupies gentle valleys, forest understories, and riparian zones within the same altitudinal range (1000-1200 m) (Figure 11).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe taxonomic controversy surrounding \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e has long centered on intraspecific variation in floral and leaf morphology (Min et al., 1993; Chang, 1996). This study integrates morphological, anatomical, palynological, and molecular phylogenetic evidence with long-term field surveys and type specimen examinations to clarify their delimitation. Results demonstrate highly conserved traits\u0026mdash;including leaf shape, petiole length, and fruit size\u0026mdash;supporting their status as geographic variants within a single species. Such trait stability represents a phenotypic response to heterogeneous habitats in plant taxonomy (Williams, 2010).\u003c/p\u003e\n\u003cp\u003eBarrio (2023)\u0026nbsp;established that leaf morphology conservatism correlates strongly with local environmental selection pressures. Anatomical data confirm homologous stomatal apparatus parameters and anticlinal wall patterns across all three taxa, aligning with intraspecific micromorphological clines (Pinedo et al., 2016; Longhi et al., 2024). Observed foliar differences likely reflect adaptive adjustments to microenvironments (e.g., light, humidity gradients) rather than conclusive evidence for speciation.\u003c/p\u003e\n\u003cp\u003eType specimen verification and wild population sampling are essential for species delimitation (Xue et al., 2018; Xu et al., 2023). In this study, when systematically verifying the digital specimen banks (such as CVH, GBIF, etc.) and the type specimens in the collection, it was found that there were some information deficiencies in the existing specimens, such as the absence of complete morphological characteristics of flowers and fruits on the specimens. Such deficiencies have historically led to overreliance on fragmented characters, neglecting population-level trait continuity (Bebber et al., 2010). By integrating voucher specimens with field-collected data, we reconstructed morphological profiles and taxonomic status for these taxa within sect. \u003cem\u003eTuberculata\u003c/em\u003e, as corroborated by Bossa-Castro et al.\u0026rsquo;s (2024)\u0026nbsp;finding that \u0026gt;30% of global herbarium specimens lack critical diagnostic traits, necessitating field validation.\u003c/p\u003e\n\u003cp\u003eThe diversity of pollen morphology is the result of plants adapting to different environmental and ecological conditions over the course of long-term evolution (Mander et al., 2021). The continuous variation in pollen polarisation ratio and outer wall ornamentation (coarse net-ridge-papilla composite structure) of \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC∙ zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e is consistent with intraspecific pollen polymorphism (Gamal, 2025), while the flattened spherical pollen and significantly reduced papilla density of \u003cem\u003eC. rhytidocarpa\u003c/em\u003e point to an independent evolutionary path. This difference may reflect their adaptation to different pollination environments, with distribution in drier, hotter regions potentially driving the restructuring of pollen outer wall structures. Additionally, the regular differences in germination pore distribution further reinforce their taxonomic independence (Amstutz et al., 2024).\u003c/p\u003e\n\u003cp\u003eMolecular phylogenetic evidence provides critical support for taxonomic revision (Wei et al., 2023; Abe et al., 2024; Qin et al., 2024). Chloroplast genome analyses reveal sequence similarity among \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e, with phylogenies placing them in a monophyletic clade (ML=100, BI=1.00). This aligns with Chang et al. (2021) on chloroplast genome conservation in closely related species. Despite sympatric distribution of \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e, nuclear ITS phylogeny confirms their genetic homogeneity, validating Wang et al. (2025) regarding multi-gene analysis reliability in taxonomy.\u003c/p\u003e\n\u003cp\u003eGeographic isolation and niche differentiation drive interspecific divergence (Lindelof et al., 2020). \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e co-occur in similar mountainous habitats (1000-1200 m, 15-45\u0026deg; slopes) in Liping County, Guizhou, whereas \u003cem\u003eC. rhytidocarpa\u003c/em\u003e is restricted to Longsheng County, Guangxi. Habitat heterogeneity likely drives morphological adaptations through natural selection. PCA results indicate slightly larger leaves in \u003cem\u003eC. rhytidocarpa\u003c/em\u003e, but other traits align closely with the Guizhou taxa. Environmental variation elicits differential phenotypic responses within species \u0026mdash; e.g., acuminate leaf apices may reduce transpiration in drier microhabitats (Liu et al., 2025).\u003c/p\u003e\n\u003cp\u003eMolecular phylogenetic data showed that the chloroplast genomic differences among the three species were small, and it is possible that none of them reached the level of species-level differentiation within plant genera (Mondini et al., 2009). This is consistent with the (asynchrony of morphological-genetic differentiation) demonstrated by Zou et al. (2021) in \u003cem\u003eRhododendron\u003c/em\u003e L., which suggests that phenotypic plasticity may play a dominant role in the formation of morphological differences.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study pioneers an integrated multidisciplinary analysis of \u003cem\u003eC. lipingensis\u003c/em\u003e, \u003cem\u003eC. zengii\u003c/em\u003e, and \u003cem\u003eC. rhytidocarpa\u003c/em\u003e, systematically resolving their morphoanatomical traits, palynological patterns, and molecular phylogenetics. Results demonstrate discontinuous variation across multidimensional trait spectra with significant genetic differentiation, supporting the reclassification of \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e as synonyms subsumed under \u003cem\u003eC. rhytidocarpa\u003c/em\u003e. This revision resolves long-standing synonymy issues in sect. \u003cem\u003eTuberculata\u003c/em\u003e and establishes a robust framework for germplasm conservation. The revealed morpho-molecular coevolution further provides a theoretical foundation for assessing intraspecific variation, genetic diversity patterns, and adaptive evolution within this complex.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFormalin-Acetic Acid-Alcohol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate-Buffered Saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eScanning Electron Microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTAB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCetyltrimethylammonium Bromide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecpDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChloroplast DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLarge Single-Copy region\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSmall Single-Copy region\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInverted Repeat\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGuanine-Cytosine content\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoding DNA Sequence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003etRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransfer RNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRibosomal RNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eML\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMaximum Likelihood\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBayesian Inference\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePosterior Probability\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBootstrap Support\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCGs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProtein-Coding Genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eITS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInternal Transcribed Spacer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrincipal Component Analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNCBI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNational Center for Biotechnology Information\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGBIF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlobal Biodiversity Information Facility\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeihao Gu\u003c/strong\u003e: Formal analysis (lead); Methodology (lead); Writing - original draft (lead). \u003cstrong\u003eMingtai An\u003c/strong\u003e: Formal analysis (equal); Validation (supporting). \u003cstrong\u003eChao Yan\u003c/strong\u003e: Formal analysis (equal); Validation (supporting). \u003cstrong\u003eXu Xiao\u003c/strong\u003e: Visualization (supporting). \u003cstrong\u003eZhaohui Ran\u003c/strong\u003e: Validation (supporting). \u003cstrong\u003eZhi Li\u003c/strong\u003e: Conceptualization (equal); Supervision (equal).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32400179), and the 2024 Guizhou Science and Technology Innovation Talent Team Construction Project: Wildlife Innovation Team of the Forestry college of Guizhou University (Qian ke he ren cai CXTD [2025] 053).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenBank accession numbers: PV761587, PV750797, PV750798, PV750799, PV750800, PV750801, PV750802. The Appendix Table for this article can be found online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Forestry, Guizhou University, Guiyang 550025, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbe H, Ueno S, Matsuo A, Hirota SK, Miura H, Su M-H, et al. 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(2021) Joint effect of phylogenetic relatedness and trait selection on the elevational distribution of\u003cem\u003eRhododendron\u003c/em\u003especies.\u003cem\u003eJournal of Systematics and Evolution\u003c/em\u003e59:1244-1255.https://doi.org/10.1111/jse.12690\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Section Tuberculata, Species complex, Taxonomic revision, Integrative taxonomy, Speices delimitation","lastPublishedDoi":"10.21203/rs.3.rs-7021883/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7021883/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe section \u003cem\u003eTuberculata\u003c/em\u003e (\u003cem\u003eCamellia\u003c/em\u003e L.), as a monophyletic group characterized by tuberculate fruits, exhibits persistent taxonomic ambiguities among its constituent species, exemplified by the unresolved delimitation of \u003cem\u003eCamellia lipingensis\u003c/em\u003e, \u003cem\u003eCamellia zengii\u003c/em\u003e, and \u003cem\u003eCamellia rhytidocarpa\u003c/em\u003e. These three species are highly similar in terms of morphology, genetics, or ecology as a plant complex. Historical revisions have been hindered by the absence of key morphological characteristics in type specimens and the instability of morphological identification criteria, leading to unclear classification of species. This study, based on type locality specimens, morphology, and systematic molecular biology, systematically integrates macroscopic morphology, microscopic structure, and molecular systematics data for the first time, aiming to clarify the taxonomic relationships among the three species.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eMultidimensional evidence based on morphology, anatomy, palynology, and molecular systematics supports the merger of \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e into the synonym \u003cem\u003eC. rhytidocarpa\u003c/em\u003e. Morphological analysis reveals continuous variation in key traits: leaves lanceolate (6.42\u0026ndash;12.50 \u0026times; 1.17\u0026ndash;4.45 cm); floral parts with 6\u0026ndash;9 rounded sepals, 3\u0026ndash;5 hairy styles, and 2.2\u0026ndash;4.1 cm long filaments; fruit subglobose (diameter 2.24\u0026ndash;3.18 cm), ovary 3-4-loculed (1 seed per locule). Anatomical and pollen characteristics are conservative: leaf epidermal stomata are elliptical (39.9\u0026ndash;41.2 \u0026times; 31.4\u0026ndash;36.7 \u0026micro;m), with a density of 62\u0026ndash;86 per mm\u0026sup2;; pollen is nearly spherical (polar axis 36.7\u0026ndash;37.8 \u0026micro;m/equatorial axis 40.3\u0026ndash;41.3 \u0026micro;m, P/E ratio 0.87\u0026ndash;0.91). Molecular systematics confirmed that the three form a strongly supported monophyletic clade (ML/PP\u0026thinsp;=\u0026thinsp;100/1.00), with consistent chloroplast genome structures (157,029, 157,029, 157,048 bp; GC 37.3%; containing 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis integrative study consolidates \u003cem\u003eC. lipingensis\u003c/em\u003e and \u003cem\u003eC. zengii\u003c/em\u003e as conspecific synonyms of \u003cem\u003eC. rhytidocarpa\u003c/em\u003e based on congruent morphological, anatomical, palynological, and molecular phylogenetic evidence. The taxonomic revision resolves persistent delimitation conflicts within sect. \u003cem\u003eTuberculata\u003c/em\u003e while establishing an empirical framework for: Phylogenetic reconstruction of \u003cem\u003eCamellia\u003c/em\u003e lineages with overlapping morphological variation, Conservation prioritization of evolutionarily significant units in East Asian biodiversity hotspots, and Development of standardized species delimitation protocols for taxonomically complex plant groups.\u003c/p\u003e","manuscriptTitle":"Integrative taxonomic revision of the Camellia rhytidocarpa complex (Theaceae) synonymous status of C. lipingensis and C. zengii supported by morphological, anatomical, palynological, and molecular evidence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 08:19:52","doi":"10.21203/rs.3.rs-7021883/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-10-19T22:52:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-14T07:37:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-10T02:30:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-02T07:11:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Botanical Studies","date":"2025-07-01T11:28:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bb085c9-6873-46dd-a7eb-1b042bf8d39e","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:09:17+00:00","versionOfRecord":{"articleIdentity":"rs-7021883","link":"https://doi.org/10.1186/s40529-025-00489-5","journal":{"identity":"botanical-studies","isVorOnly":false,"title":"Botanical Studies"},"publishedOn":"2026-01-19 15:58:56","publishedOnDateReadable":"January 19th, 2026"},"versionCreatedAt":"2025-07-14 08:19:52","video":"","vorDoi":"10.1186/s40529-025-00489-5","vorDoiUrl":"https://doi.org/10.1186/s40529-025-00489-5","workflowStages":[]},"version":"v1","identity":"rs-7021883","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7021883","identity":"rs-7021883","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}