Construction of a Core Collection of Notopterygii Rhizoma et Radix Based on Molecular Phylogeography

Construction of a Core Collection of Notopterygii Rhizoma et Radix Based on Molecular Phylogeography | 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 Construction of a Core Collection of Notopterygii Rhizoma et Radix Based on Molecular Phylogeography Lucun Yang, Jinping Li, Shengbin Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7238023/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Nov, 2025 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Background As both an endangered medicinal species (China's Category III protected plant) and an authentic traditional Chinese medicine, Notopterygium incisum Ting ex H. T. Chang and N. franchetii Boiss . require urgent conservation of their germplasm resources. While core collections offer an efficient solution for preserving genetic diversity, no such resource currently exists for these species despite their ecological and pharmacological importance. Methods In this study, three chloroplast DNA regions ( rbcL , matK , and trnS - trnG ) and the nuclear ribosomal ITS sequence were employed as molecular markers to conduct phylogeographic analyses of N. incisum and N. franchetii , and their core collections were constructed through stratified sampling of evolutionary significant haplotypes. Results Network analyses revealed complete cpDNA differentiation between species (12 vs. 10 haplotypes separated by ≥5 mutations), while ITS data showed limited historical introgression. Wild populations exhibited strong genetic structure ( G ST : 0.673-0.713) with ancestral haplotypes (cpDNA Hap_2/Hap_13; ITS Hap_3-Hap_4/Hap_23), whereas cultivated accessions showed 3.2× higher haplotype diversity but reduced differentiation ( G ST : 0.077-0.094). Demographic tests (Tajima's D = -1.39 to -2.15, P<0.01) and growth indices (G=856-2901) confirmed post-glacial expansions. Conclusion Using integrated cpDNA and ITS markers, we established optimized core collections for both species ( N. incisum : 50-103 accessions; N. franchetii : 30-40 accessions) that effectively preserved genetic diversity. The dual-marker approach resolved cultivated populations' paradoxical genetic patterns (higher diversity but lower differentiation) and provides a conservation model for medicinal plants facing anthropogenic pressures. Notopterygium incisum N. franchetii Notopterygii Rhizoma et Radix haplotype germplasm resources genetic structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plant germplasm resources serve as fundamental materials for genetic research and crop improvement. Worldwide efforts to conserve these resources in germplasm banks face growing challenges, including escalating management costs and difficulties in identifying unique genetic materials from increasingly large collections. The concept of a core collection—a minimal yet maximally representative subset preserving the genetic and geographic diversity of entire populations—was introduced by Frankel and Brown (1984) to address these issues [ 1 ]. While core collections have been established for numerous agricultural and forestry species [ 2 – 5 ], their application to medicinal plants remains limited despite rising conservation needs. Notopterygii Rhizoma et Radix, derived from the endangered species N. incisum and N. forbesii [ 6 – 7 ], is a critically important traditional Chinese medicine with a millennia-long history of use [ 8 ]. Recognized in multiple editions of the Chinese Pharmacopoeia, it holds cultural significance in Tibetan, Qiang, Uyghur, and Mongolian medicine. However, wild populations have sharply declined due to habitat destruction and overharvesting [ 9 ], prompting its designation as a third-class protected species in China and a Near-Threatened (NT) species in the China Biodiversity Red List [ 10 – 11 ]. Cultivation has become the primary source for commercial supply [ 12 – 15 ], yet unmanaged hybridization between wild and cultivated populations has led to genetic erosion, loss of superior traits, and increased susceptibility to pests and diseases [ 16 ]. Establishing a core germplasm collection for Notopterygium is thus urgent to safeguard genetic diversity, support breeding programs, and ensure sustainable cultivation. Molecular phylogeography, grounded in coalescent theory, provides a powerful framework for constructing core collections of medicinal plants by elucidating the historical biogeographic processes that shape contemporary genetic patterns [ 17 – 18 ]. Integrating molecular techniques with analytical tools such as nested clade analysis (NCA) enables the reconstruction of species histories (including dispersal and migration events) through analysis of neutral genetic variation[ 19 – 20 ], the construction of gene trees using maternally inherited chloroplast markers, the discrimination between contemporary processes like gene flow and historical events such as population fragmentation or range expansions, and the reconstruction of population structures and evolutionary timelines[ 21 ]. For medicinal plants specifically, phylogeographic analysis offers unique advantages in revealing geographical genetic differentiation among wild populations, tracing the origins of cultivated populations, and identifying domestication-induced genetic admixture, thereby facilitating maximal preservation of genetic diversity with minimal germplasm redundancy. Medicinal plant germplasm necessitates differentiated conservation strategies for species possessing both wild and cultivated populations (e.g., Scutellaria baicalensis , Salvia miltiorrhiza ) versus exclusively cultivated species lacking wild progenitors (e.g., Coptis chinensis , Panax ginseng ). Phylogeography facilitates the identification of genetically distinct wild populations, characterized by distinct haplotypes and large genetic distances, for priority collection in species with extant wild relatives. For exclusively cultivated species, systematic screening of germplasm effectively identifies unique genotypes while minimizing the collection of genetically redundant material. In our study of Notopterygium species, an integrated marker system combining nuclear ITS with chloroplast sequences enabled maternal lineage tracking, assessment of biparental inheritance patterns, and coalescent-based demographic modeling. For the strictly wild N. incisum , conservation focused on preserving phylogenetically distinct haplotypes across their native ranges. For N. forbesii , which exhibits both wild and cultivated populations, ancestry analysis differentiated indigenous wild genotypes from admixed cultivated lineages. This dual-strategy approach successfully preserved ≥ 85% of observed genetic diversity, maintained population evolutionary potential, and established a valuable genetic reservoir for future breeding. The methodology demonstrates particular efficacy for alpine medicinal plants like Notopterygium and serves as a transferable model for other mountain-dwelling species characterized by complex wild-cultivated relationships. Results Haplotype network of N. incisum and N. franchetii Sequence analysis of three chloroplast DNA regions ( rbcL , matK , and trnS-trnG ) identified 12 haplotypes in N. incisum (Hap_1-Hap_12) and 10 in N. franchetii (Hap_13-Hap_22), with NETWORK analysis revealing complete genetic isolation between the species (Fig. 1). The network showed no shared haplotypes and a minimum of five mutational steps separating the two species' haplotype clusters, indicating an absence of gene flow. N. incisum exhibited a star-like topology centered on the ancestral Hap_2 (n = 78), which diverged into seven derived haplotypes through one to two mutations, while N. franchetii displayed stepwise differentiation from Hap_13 into multiple descendant haplotypes. Parallel analysis of ITS sequences identified 20 haplotypes in N. incisum (Hap_1-Hap_20) and 10 in N. franchetii (Hap_21-Hap_30), with networks showing deeper divergence (≥ 20 mutational steps) and a single shared haplotype, suggesting limited historical introgression. In N. incisum , the ITS network was structured around two central haplotypes, Hap_3 (n = 139) and Hap_4 (n = 192), from which all others were derived, whereas N. franchetii exhibited a radial pattern centered on Hap_23 (n = 119) (Fig. 2). These findings demonstrate strong species differentiation with distinct evolutionary histories, supported by both chloroplast and nuclear markers. Haplotype Distributions Our multilocus analysis revealed distinct yet complementary patterns of genetic diversity between chloroplast and nuclear markers in Notopterygium populations. Chloroplast DNA (cpDNA) markers demonstrated remarkable geographical conservation in both N. incisum and N. franchetii (Fig. 3–4, Table 1 ). For N. incisum , three dominant cpDNA haplotypes accounted for 86% of all occurrences, with the ancestral Hap_2 distributed across 19 populations (61% of total), accompanied by Hap_6 (11 populations, 35%) and Hap_1 (6 populations, 19%). The remaining seven populations (23%) developed unique cpDNA variants (Hap_8–12), suggesting localized microevolutionary processes. N. franchetii exhibited parallel conservation patterns, where ancestral haplotypes Hap_13 (15 populations, 50%) and Hap_14 (14 populations, 47%) showed panmictic distribution. Wild populations displayed particularly strong cpDNA conservation (90% monomorphic), while cultivated accessions showed significantly higher haplotype diversity, with only DTR and WYR populations retaining both ancestral haplotypes. Table 1 The haplotype distribution of different provenances of N. incisum and N. franchetii Species Populations cpDNA ITS No. Haplotype distribution No. Haplotype distribution Notopterygium incisum LD 7 Hap_2(4), Hap_6(3) 19 Hap_4(19) HZ 5 Hap_6(5) 15 Hap_4(15) MY 9 Hap_6(9) 16 Hap_4(15), Hap_13(1) BM 8 Hap_2(8) 20 Hap_3(20) JZ 7 Hap_2(6), Hap_10(1) 14 Hap_3(4), Hap_5 (1), Hap_6(4), Hap_11(1), Hap_19(4) DR 5 Hap_2(5) 11 Hap_3(10), Hap_7 (1) YS 7 Hap_11(7) 14 Hap_3(14) CD 6 Hap_2(3), Hap_6(3) 10 Hap_3(10) NQ 5 Hap_2(5) 20 Hap_3(3), Hap_4(17) ML 6 Hap_2(3), Hap_6(3) 8 Hap_4(8) SD 5 Hap_6(5) 19 Hap_4(18), Hap_18(1) YZH 8 Hap_1(4), Hap_2(1), Hap_7(1), Hap_9(2) 16 Hap_4(16) HZU 5 Hap_2(4), Hap_6(1) 17 Hap_4(17) ZN 5 Hap_2(5) 13 Hap_4(13) LT 6 Hap_2(5), Hap_7(1) 11 Hap_4(8), Hap_10(3) MQ 6 Hap_1(3), Hap_6(3) 11 Hap_3(2), Hap_4(8), Hap_6(1) LQ 5 Hap_1(5) 11 Hap_4(11) REG 8 Hap_1(8) 14 Hap_1(1), Hap_2 (4), Hap_3(7), Hap_4(1), Hap_11(1) HOY 8 Hap_2(5), Hap_3(1), Hap_4(2) 16 Hap_3(3), Hap_4 (6), Hap_11(2), Hap_12(5) AB 6 Hap_2(2), Hap_5(3), Hap_6(1) 12 Hap_3(4), Hap_5 (4), Hap_6(2), Hap_12(2) MEK 7 Hap_2(5), Hap_7(1), Hap_12(1) 14 Hap_2(1), Hap_3 (11), Hap_14(1), Hap_16(1) JC 10 Hap_2(9), Hap_6(1) 14 Hap_2(1), Hap_3 (9), Hap_4(4), Hap_6(2) DF 8 Hap_1(3), Hap_2(5) 19 Hap_3(11), Hap_4 (3), Hap_9(5) GZ 10 Hap_2(9), Hap_7(1) 16 Hap_3(15), Hap_17 (1) LH 8 Hap_6(8) 15 Hap_3(12), Hap_4 (2), Hap_15(1) DB 9 Hap_1(8), Hap_2(1) 10 Hap_3(2), Hap_4 (6), Hap_6(2) XJ 7 Hap_2(1), Hap_7(1), Hap_8(5) 15 Hap_2(1), Hap_3 (11), Hap_8(1), Hap_20(2) N. franchetii ZK 10 Hap_13(8), Hap_14(2) 10 Hap_21(6), Hap_22 (1), Hap_23(3) MH 10 Hap_13(10) 10 Hap_23(10) LD 10 Hap_13(6), Hap_14(3), Hap_17(1) 10 Hap_23(10) PA 10 Hap_13(10) 10 Hap_23(10) DT 9 Hap_13(9) 10 Hap_23(10) HZ 10 Hap_13(9), Hap_14(1) 10 Hap_23(10) HZH 10 Hap_13(8), Hap_14(2) 10 Hap_23(10) HZU 10 Hap_14(8), Hap_19(2) 7 Hap_23(7) MX 10 Hap_15(9), Hap_16(1) 10 Hap_24(10) LT 9 Hap_13(8), Hap_14(1) 2 Hap_26(2) ZN 9 Hap_13(9) 3 Hap_23(1), Hap_26(2) LQ 10 Hap_14(10) 10 Hap_23(7), Hap_26(2), Hap_30(1) Cultivated N. franchetii LDR 10 Hap_13(1), Hap_14(5), Hap_17(2), Hap_20(1), Hap_21(1) 10 Hap_21(1), Hap_23(7), Hap_26(1), Hap_27(1) HZ1R 10 Hap_13(3), Hap_14(6), Hap_20(1) 6 Hap_23(5), Hap_26(1) HZ2R 10 Hap_13(5), Hap_14(3), Hap_20(2) 5 Hap_23(5) HZ3R 10 Hap_14(7), Hap_20(2), Hap_22(1) 9 Hap_23(6), Hap_26(1), Hap_28(1), Hap_29(1) DTR 10 Hap_13(8), Hap_14(2) 5 Hap_23(5) MXR 10 Hap_13(2), Hap_14(4), Hap_17(1), Hap_18(1), Hap_19(1) 10 Hap_4(4), Hap_23(5), Hap_25(1) WYR 9 Hap_13(4), Hap_14(5) 10 Hap_4(1), Hap_23(8), Hap_29(1) Nuclear ITS markers provided higher resolution of population structure while confirming the core patterns observed in cpDNA (Fig. 5–6, Table 1 ). N. incisum exhibited a dual-origin genetic architecture, with co-dominant ancestral haplotypes Hap_3 and Hap_4 (each in 17 populations, 55%) and nine populations (29%) containing unique variants (Hap_11–20). This pattern suggests either ancestral hybridization or prolonged incomplete lineage sorting. In N. franchetii , the predominant ITS haplotype (Hap_23) occurred in 17 populations (57%), with three wild populations (10%) showing unique variants. Cultivated accessions displayed 3.2-fold higher haplotype diversity than wild counterparts, with only HZ2R and DTR populations maintaining Hap_23 monomorphism. The concordant patterns between marker systems, particularly the elevated diversity in cultivated populations and conserved haplotypes in wild populations, provide robust evidence for distinct evolutionary trajectories shaped by natural versus anthropogenic selection pressures. Genetic Diversity and Structure The genetic diversity and population structure of N. incisum and N. franchetii were assessed using cpDNA and ITS markers (Table 2 ). In both markers, the total genetic diversity ( h T ) showed the following order: N. incisum > cultivated N. franchetii > wild N. franchetii (cpDNA: 0.726 > 0.659 > 0.584; ITS: 0.651 > 0.517 > 0.383). For within-population diversity ( h S ), cultivated N. franchetii exhibited the highest values followed by N. incisum and then wild N. franchetii in both markers (cpDNA: 0.597 > 0.308 > 0.1912; ITS: 0.353 > 0.302 > 0.148). Table 2 The genetic structure of N. incisum and N. franchetii Species cpDNA ITS h T h S G ST N ST h T h S G ST N ST N. incisum 0.726 ± 0.0466 0.308 ± 0.0604 0.575 ± 0.0851 0.512 ± 0.0878 0.651 ± 0.0396 0.302 ± 0.0558 0.536 ± 0.0654 0.494 ± 0.0554 N. franchetii 0.584 ± 0.1029 0.191 ± 0.0572 0.673 ± 0.1042 0.618 ± 0.1031 0.383 ± 0.1008 0.148 ± 0.0780 0.713 ± 0.1405 0.942 ± 0.0550 Cultivated N. franchetii 0.659 ± 0.0536 0.597 ± 0.0651 0.094 ± 0.0613 0.040 ± 0.0756 0.517 ± 0.1403 0.353 ± 0.1001 0.077 ± 0.0106 0.301 ± 0.0305 Population differentiation analysis yielded particularly noteworthy results. Wild N. franchetii populations exhibited strong genetic differentiation, with high G ST (cpDNA: 0.673; ITS: 0.713) and N ST (cpDNA: 0.618; ITS: 0.924) values. In striking contrast, cultivated N. franchetii showed significantly reduced differentiation (cpDNA G ST : 0.094; ITS G ST : 0.077; cpDNA N ST : 0.040; ITS N ST : 0.301), suggesting homogenization among cultivated groups. N. incisum displayed intermediate levels of population differentiation, with G ST (cpDNA: 0.575; ITS: 0.536) and N ST (cpDNA: 0.512; ITS: 0.494) values consistently lower than those of wild N. franchetii but higher than cultivated populations. Population Dynamics History Using cpDNA sequence data, we conducted multiple statistical analyses to reconstruct the demographic histories of the two Notopterygium species (see Table 3 and Supplementary Figure S1 ). The mismatch distributions for N. incisum and N. franchetii displayed unimodal curves, accompanied by significantly negative Tajima’s D and Fu’s F S values. These findings strongly support historical population expansions in both species. Further evidence comes from their substantial population growth indices ( N. incisum : G = 856; N. franchetii : G = 2901.736), which further corroborate rapid demographic expansion events. Table 3 Results of cpDNA mismatch distribution and neutrality tests for N. incisum and N. franchetii Species mismatch distribution Neutrality tests θ 0 θ 1 SSD( P -value) Rag( P -value) G Tajima’s D Fu and Li’s F_ Fu’s F S N. incisum 3.25 11.56 0.4988(0.08) 0.04238(0.03) 856 -1.39084 -0.27104 -9.402 N. franchetii 0.0 9999 0.1015(0.014) 0.1057(0.001) 2901.736 -1.17776 -0.39033 -3.365 Selection of the Core Collection Comparative analysis of chloroplast (cpDNA) and nuclear (ITS) markers revealed distinct patterns of genetic diversity between cultivated and wild populations of N. incisum and N. franchetii . Cultivated N. franchetii exhibited substantial genetic admixture across both marker systems, reflecting the consequences of unregulated introduction and cultivation practices. In contrast, wild populations maintained more structured genetic backgrounds, characterized by predominant ancestral haplotypes (cpDNA Hap_2 and ITS Hap_3/Hap_4 in N. incisum ; cpDNA Hap_13/Hap_14 and ITS Hap_23 in N. franchetii ) with clear geographical distributions. This contrast informed our strategy to establish core collections preferentially from wild populations, which retain distinct genetic lineages and demonstrate significant inter-provenance differentiation. Targeted sampling focused on three key components: dominant haplotypes across multiple provenances, locally endemic variants (e.g., cpDNA Hap_8–12 and ITS Hap_11–20 in N. incisum ), and rare haplotypes to ensure comprehensive genetic representation. For cpDNA markers, seed collections for N. incisum included: Hap_2 from the JZ population, Hap_2 and Hap_9 from the YZH population, Hap_3 and Hap_4 from the HOY population, Hap_5 and Hap_6 from the AB population, Hap_7, Hap_8, and Hap_12 from the XJ population, Hap_10 from the BM population, and Hap_11 from the YS population. For N. franchetii , cpDNA-based collections comprised: Hap_13 and Hap_17 from the LD population, Hap_14 and Hap_19 from the HZU population, and Hap_15 and Hap_16 from the MX population. For ITS markers, sampling of N. incisum included: Hap_3 and Hap_4 from the NQ and MQ populations, Hap_1, Hap_2 and Hap_11 from the REG population, Hap_5, Hap_6 and Hap_19 from the JZ population, Hap_13 from the MY population, Hap_18 from the SD population, Hap_6 and Hap_12from the AB population, Hap_14 and Hap_16 from the MEK population, Hap_8 and Hap_20 from the XJ population. N. franchetii collections focused on: Hap_23 from ZN and ZK populations, Hap_26 from ZN and LQ populations, and Hap_30 from LQ population. This comprehensive sampling strategy ensured representation of both conserved and variable regions across the two genomes. The established core collections demonstrated marked improvements in genetic diversity parameters compared to original populations (Table 4 ). Both marker systems showed consistent trends, with within-population diversity ( h S ) increasing by 38–42% and total diversity ( h T ) by 29–33%. Notably, differentiation coefficients ( G ST ) decreased substantially, with reductions of 0.128–0.148 for cpDNA and 0.135–0.140 for ITS markers. These improvements were achieved through strategic sampling from key populations, including N. incisum sources for cpDNA Hap_2 (JZ), Hap_5 + Hap_6 (AB), and ITS Hap_3 + Hap_4 (HOY), as well as N. franchetii sources for cpDNA Hap_13 + Hap_17 (LD) and ITS Hap_23 (multiple populations). The parallel results from both marker systems not only validate the robustness of this conservation approach but also highlight its value for maintaining genetic resources for future breeding programs. The successful integration of cpDNA and ITS data provides a model for developing core collections in other medicinal plant species with similar cultivation histories and conservation needs. Table 4 The genetic structure of the core collection of N. incisum and N. forbesii Species cpDNA ITS h T h S G ST h T h S G ST N. incisum 0.853 ± 0.0698 0.489 ± 0.1006 0.427 ± 0.1374 0.739 ± 0.0231 0.456 ± 0.0336 0.437 ± 0.0322 N. franchetii 0.924 ± 0.0251 0.432 ± 0.1110 0.533 ± 0.1787 0.425 ± 0.0344 0.298 ± 0.0216 0.621 ± 0.0124 Discussion As a nationally protected endangered medicinal plant species (Category III) in China, Notopterygium requires urgent strategic conservation of its germplasm resources. This study developed the first core collection for Notopterygium species through integrated analysis of chloroplast DNA (cpDNA) and nuclear ITS markers, establishing a scientifically validated framework for both conservation and sustainable utilization of these valuable medicinal resources The advantages of molecular phylogeography in constructing core collection The conservation of medicinal plant germplasm presents unique challenges that fundamentally differ from crop germplasm management, necessitating distinct methodological approaches [ 23 – 30 ]. Conventional strategies employed for crops are often inadequate for medicinal species due to several intrinsic factors: (1) the frequent lack of well-established germplasm repositories, (2) complex evolutionary histories, and (3) widespread occurrence of cryptic genetic diversity. In this context, molecular phylogeography integrating both chloroplast DNA (cpDNA) and nuclear ITS markers has emerged as the most effective approach for core germplasm collection, providing comprehensive solutions to key conservation challenges [ 22 ]. The primary scientific challenge in medicinal plant conservation is germplasm admixture - populations with distinct genetic backgrounds often lack distinguishable morphological characteristics. This morphological uniformity significantly compromises the accuracy of germplasm identification. The molecular phylogeography approach based on a dual cpDNA-ITS marker system offers the following core advantages: (1) the maternal inheritance pattern of cpDNA enables tracing of historical migration routes, (2) the biparental inheritance of ITS markers facilitates detection of hybridization and gene flow events, (3) integrated analysis provides complete assessment of population genetic structure, and (4) enables precise quantification of genetic differentiation at various levels. These capabilities enable precise targeting and preservation of evolutionarily significant genetic variation, establishing molecular phylogeography as an indispensable methodology for developing scientifically robust core collections of medicinal plants. Genetic structure of N. incisum and N. franchetii The phylogenetic geographical pattern of N. incisum exhibits a "star-like" structure, wherein most haplotypes are connected to a central haplotype via relatively short branches. This relatively straightforward pattern, combined with significantly negative values of Tajima’s D and Fu’s F S (e.g., Tajima’s D = -1.39084, P < 0.01; Fu’s FS = -9.402, P < 0.01), strongly suggests that the population underwent a recent expansion during periods of favorable climatic conditions [ 31 – 35 ]. Such negative values are indicative of an excess of rare mutations, a hallmark of demographic expansion or positive selection. However, due to insufficient time for the establishment of more complex genetic structures following this expansion, the haplotype network remains relatively simple and radiating. This "star-like" haplotype phylogenetic geographic pattern, often associated with population expansions, has also been reported in other species [ 31 , 32 , 36 – 38 ]. The haplotypes of cultivated and wild N. franchetii were compared. Some haplotypes were found in the wild population, while others were found in the cultivated population, indicating that some haplotypes of wild N. franchetii were lost in the cultivated population. Meanwhile, the total genetic diversity ( h T ) of the wild population was 0.584 (cpDNA) and 0.383 (ITS), and that of the cultivated population was 0.659 (cpDNA) and 0.517 (ITS) (Table 2 ). The h T of the cultivated population was slightly higher than that of the wild population based on cpDNA data, but the ITS data showed a more pronounced difference. This suggests that N. franchetii did not experience the genetic bottleneck phenomenon that most cultivated crops do during the cultivation process, indicating that the initial population size of cultivated N. franchetii was large and it was not subject to much artificial selection. However, although the total genetic diversity of cultivated N. franchetii increased slightly (cpDNA) or showed mixed trends (ITS), its genetic structure changed significantly. The distribution range of shared haplotypes between the cultivated and wild populations in the cultivated population was much larger than that in the wild population, and the number of haplotypes in each cultivated population was significantly higher than that in the wild population. The genetic diversity within the population ( h S ) of the cultivated population was 0.597 (cpDNA) and 0.353 (ITS), and that of the wild population was 0.191 (cpDNA) and 0.148 (ITS). The h S of the cultivated population was significantly higher, which led to a significant decrease in the coefficient of gene differentiation among cultivated populations ( G ST , 0.094 for cpDNA and 0.077 for ITS in cultivated populations, compared to 0.673 for cpDNA and 0.713 for ITS in wild populations). This indicates a very weak genetic structure in the cultivated populations, that is, a decrease in genetic diversity among populations and an increase in genetic diversity within populations. This suggests that there was an increase in gene flow among cultivated populations during the cultivation process, and the germplasm of cultivated N. franchetii was severely mixed, which will lead to outbreeding depression and germplasm degradation of the cultivated species. Construction of core collection of N. incisum and N. franchetii The establishment of a core collection serves as a strategic approach to efficiently conserve and utilize genetic diversity. In this study, we employed a phylogeographic approach based on chloroplast (cpDNA) and nuclear (ITS) marker analyses to construct core collections for N. incisum and N. franchetii . From original germplasms of 187 ( N. incisum ) and 186 ( N. franchetii ) accessions, we selected 50 and 30 core accessions, respectively, achieving sampling rates of 26.74% and 25.64%. These proportions were carefully determined to balance representativeness and efficiency, as supported by previous studies indicating that 5–10% of a core collection can capture over 70% of genetic variation in the entire germplasm, with medicinal plants typically requiring 10–40% sampling depending on species-specific diversity. The core collections demonstrated significant improvements in genetic diversity parameters, with N. incisum showing retention rates of 117.49% for total diversity ( h T ), 158.77% for within-population diversity ( h S ), and 74.26% for differentiation ( G ST ), while N. franchetii exhibited even greater retention rates of 158.22% ( h T ), 226.18% ( h S ), and 79.20% ( G ST ). These results confirm that the core collections effectively minimized redundancy while preserving and even enhancing genetic variation, with the parallel trends from both cpDNA and ITS markers validating the robustness of our approach. Our sampling strategy prioritized wild populations due to their structured genetic backgrounds and distinct ancestral haplotypes, such as cpDNA Hap_2 and ITS Hap_3/Hap_4 in N. incisum and cpDNA Hap_13/Hap_14 and ITS Hap_23 in N. franchetii . Targeted sampling ensured the inclusion of dominant haplotypes across multiple provenances, locally endemic variants, and rare haplotypes, leading to reduced differentiation coefficients ( G ST ) by 0.128–0.148 for cpDNA and 0.135–0.140 for ITS markers. While core collections are powerful tools for germplasm management, they cannot fully capture a species' entire genetic diversity, necessitating dynamic updates as new germplasm becomes available. Moreover, given the geographical authenticity of medicinal plants and the habitat-dependent synthesis of bioactive compounds, future efforts should integrate genetic diversity with agronomic traits and phytochemical profiles to ensure comprehensive conservation and breeding utility. This study lays the foundation for molecular-based core collections of N. incisum and N. franchetii , with subsequent research needed to incorporate phenotypic and biochemical data for more robust germplasm management. Conclusion Through comprehensive genetic analyses, we established core collections for both species with optimized sampling efficiency. For N. incisum , the core collection comprised 50 accessions (26.74%) based on cpDNA markers and 103 accessions (40.74%) using ITS data. Similarly, N. franchetii core collections contained 30 (25.64%, cpDNA) and 40 accessions (21.05%, ITS), respectively. This dual-marker approach leveraged complementary inheritance patterns - the rapidly evolving, maternally inherited chloroplast DNA provided clear differentiation of germplasm lineages, while biparentally inherited ITS markers revealed finer-scale population structure. The integrated phylogeographic framework enabled precise quantification of genetic differentiation and informed targeted selection of evolutionarily distinct germplasm, establishing an effective methodology for medicinal plant conservation. Materials and methods Plant materials A total of 730 samples of Notopterygii Rhizoma et Radix, comprising 540 specimens of N. incisum and 190 specimens of N. franchetii , were collected from diverse locations. The precise collection sites were recorded using a single-plant GPS system (Table S1 , Fig. 7). No authorization was required for sample collection. Representative voucher specimens from each population were authenticated by Professor Yubi Zhou at the Northwest Institute of Plateau Biology, Chinese Academy of Sciences. These specimens are deposited in the Qinghai-Tibetan Plateau Museum of Biology (QPMB), with voucher numbers HNWP2014001–HNWP2014027 for N. incisum and HNWP2024001–HNWP2024019 for N. franchetii . Leaves were randomly collected from each sample, ensuring that individual samples were separated by at least fifty meters. The freshly collected leaves were immediately dried in silica gel within ziplock bags. Institutional, governmental, and international rules are followed in all aspects of our experimental study, including the gathering of plant samples. DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from individual samples using an improved CTAB protocol [ 47 ]. DNA concentration and quality were assessed via 0.8% agarose gel electrophoresis against lambda DNA standards and confirmed by UV spectroscopy. Samples were diluted to approximately 10 ng/µL in 0.1% TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) for subsequent analyses. Chloroplast DNA (cpDNA) Sequencing: A total of 730 samples from 46 populations were sequenced for three cpDNA regions ( trnS-trnG , matK , and rbcL ) (Table S1 , Fig. 7). This dataset incorporated 540 samples (27 populations) from a prior study [ 48 , 49 ] and 190 newly collected individuals (19 populations). Primer pairs (Table S4 ) [ 50 ] were selected based on previous Notopterygium research. PCR amplification was performed in 25-µL reactions containing 2 µL DNA template (10–50 ng/µL), 12.5 µL PCR Master Mix, 0.75 µL of each primer (20 µM), and 9 µL ddH₂O. Thermocycling conditions are detailed in Supplementary Table S2 . Nuclear ITS Sequencing: For the internal transcribed spacer (ITS) region, 730 samples from the same 46 populations were sequenced (Table S1 , Fig. 7). This included 540 samples (27 populations) from a previous study [ 51 ] and 190 newly collected individuals (19 populations). PCR amplification used identical reaction components and volumes as described for cpDNA, employing Xi’an Runde (China) PCR Master Mix. All high-quality PCR products were bidirectionally sequenced using the amplification primers and an ABI 3730 XL genetic analyzer (Applied Biosystems, Foster City, CA, USA). The chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) regions of N. incisum and N. franchetii were successfully sequenced and assembled. All obtained sequences, as detailed in Table 1 , have been deposited in the GenBank database under their respective accession numbers. DNA Analysis Sequence assembly was performed using ContigExpress (Informax), followed by multiple alignment with ClustalX v1.81 [ 52 ]. To characterize population genetic structure, we conducted hierarchical analyses using complementary approaches. Genetic diversity parameters - including within-population diversity ( h S ), total diversity ( h T ), and differentiation indices ( G ST and N ST ) - were computed using PERMUT v1.0 [ 53 ], with N ST incorporating both haplotype frequencies and mutational distances for enhanced sensitivity to recent differentiation. Demographic history analyses were performed using Arlequin v3.5 [ 54 ], including: (1) neutrality tests (Tajima's D, Fu's FS, and Fu & Li's F*) to detect population size changes, and (2) mismatch distribution analysis to examine population expansion patterns. Model validity was assessed through the sum of squared deviations (SSD) between observed and expected distributions and Harpending's raggedness index (Rag), with significance tested by 10,000 parametric bootstrap replicates. For haplotype analysis, we constructed median-joining networks of both cpDNA and ITS sequences using NETWORK v5.0.0[ 55 ] (Polzin and Daneshmand, 2003) to visualize mutational relationships among haplotypes. The spatial distribution of haplotypes was mapped using ArcGIS v10.2 based on georeferenced occurrence data [ 56 ], with kernel density estimation applied to identify regional distribution patterns. Frequency distribution histograms were generated to compare haplotype compositions between wild and cultivated populations. Construction of Core Collections According to previous studies, the size of a core collection typically represents 5–30% of the initial population samples [ 39 , 57 – 61 ]. In this study, molecular phylogeography was employed to construct the core germplasm of Notopterygii Rhizoma et Radix. Through comparative analysis of genetic diversity and genetic structure between wild and cultivated populations, we elucidated the geographic origins of major cultivated genotypes or haplotypes and assessed the degree of germplasm confounding in these wild populations. The core collection can be effectively constructed by collecting and preserving wild populations characterized by single genotype or haplotype distributions and significant genetic distances. Declarations Ethics approval and consent to participate This study including the collection of plant samples complies with relevant institutional, national, and international guidelines and legislation. All the necessary permissions have been granted for this research. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical trial number Not applicable. Funding This study was supported by the Open Project of Qinghai Key Laboratory of Qinghai-Tibet Plateau Biological Resources. Author Contribution YL conceived the study, designed the methodology, and drafted the manuscript. LJ performed the statistical analyses. LS was responsible for sample collection and contributed to manuscript editing. All authors revised the manuscript critically for intellectual content and approved the final version. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets generated and/or analyzed during the current study are available in the GenBank repository (The accession numbers for trnS-trnG in *N. incisum* are PV239459–PV239464, and for rbcL are PV261945–PV261947. Meanwhile, the accession numbers for trnS-trnG in *N. franchetii* are PV239448–PV239458, and for rbcL are PV261942–PV261944) References Frankel OH, Brown AHD. 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Supplementary Files FigureS1.tif Figure S1 Mismatch distribution of Notopterygium incisum and N. franchetii in the overall populations based on cpDNA dataset. TableS1.docx TableS2.docx Cite Share Download PDF Status: Published Journal Publication published 27 Nov, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 26 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers invited by journal 08 Sep, 2025 Editor assigned by journal 08 Sep, 2025 Editor invited by journal 18 Aug, 2025 Submission checks completed at journal 18 Aug, 2025 First submitted to journal 18 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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18:49:43","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49484428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMismatch distribution of \u003cem\u003eNotopterygium incisum\u003c/em\u003e \u003cem\u003eand N. franchetii\u003c/em\u003e in the overall populations based on cpDNA dataset.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7238023/v1/9b048a63705db56ecf0b501f.tif"},{"id":91372222,"identity":"03cb2e15-a011-4e44-9769-4590069427db","added_by":"auto","created_at":"2025-09-15 18:57:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28133,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7238023/v1/3c12d241b1a2f5750edbdb2c.docx"},{"id":91372686,"identity":"140752cb-4a77-487e-9967-9b459e918010","added_by":"auto","created_at":"2025-09-15 19:05:41","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15777,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7238023/v1/de77356bc1a6e60807995046.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Construction of a Core Collection of Notopterygii Rhizoma et Radix Based on Molecular Phylogeography","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant germplasm resources serve as fundamental materials for genetic research and crop improvement. Worldwide efforts to conserve these resources in germplasm banks face growing challenges, including escalating management costs and difficulties in identifying unique genetic materials from increasingly large collections. The concept of a core collection\u0026mdash;a minimal yet maximally representative subset preserving the genetic and geographic diversity of entire populations\u0026mdash;was introduced by Frankel and Brown (1984) to address these issues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While core collections have been established for numerous agricultural and forestry species [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], their application to medicinal plants remains limited despite rising conservation needs.\u003c/p\u003e\u003cp\u003eNotopterygii Rhizoma et Radix, derived from the endangered species \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. forbesii\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], is a critically important traditional Chinese medicine with a millennia-long history of use [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recognized in multiple editions of the Chinese Pharmacopoeia, it holds cultural significance in Tibetan, Qiang, Uyghur, and Mongolian medicine. However, wild populations have sharply declined due to habitat destruction and overharvesting [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], prompting its designation as a third-class protected species in China and a Near-Threatened (NT) species in the China Biodiversity Red List [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cultivation has become the primary source for commercial supply [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], yet unmanaged hybridization between wild and cultivated populations has led to genetic erosion, loss of superior traits, and increased susceptibility to pests and diseases [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Establishing a core germplasm collection for Notopterygium is thus urgent to safeguard genetic diversity, support breeding programs, and ensure sustainable cultivation.\u003c/p\u003e\u003cp\u003eMolecular phylogeography, grounded in coalescent theory, provides a powerful framework for constructing core collections of medicinal plants by elucidating the historical biogeographic processes that shape contemporary genetic patterns [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Integrating molecular techniques with analytical tools such as nested clade analysis (NCA) enables the reconstruction of species histories (including dispersal and migration events) through analysis of neutral genetic variation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the construction of gene trees using maternally inherited chloroplast markers, the discrimination between contemporary processes like gene flow and historical events such as population fragmentation or range expansions, and the reconstruction of population structures and evolutionary timelines[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For medicinal plants specifically, phylogeographic analysis offers unique advantages in revealing geographical genetic differentiation among wild populations, tracing the origins of cultivated populations, and identifying domestication-induced genetic admixture, thereby facilitating maximal preservation of genetic diversity with minimal germplasm redundancy.\u003c/p\u003e\u003cp\u003eMedicinal plant germplasm necessitates differentiated conservation strategies for species possessing both wild and cultivated populations (e.g., \u003cem\u003eScutellaria baicalensis\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e) versus exclusively cultivated species lacking wild progenitors (e.g., \u003cem\u003eCoptis chinensis\u003c/em\u003e, \u003cem\u003ePanax ginseng\u003c/em\u003e). Phylogeography facilitates the identification of genetically distinct wild populations, characterized by distinct haplotypes and large genetic distances, for priority collection in species with extant wild relatives. For exclusively cultivated species, systematic screening of germplasm effectively identifies unique genotypes while minimizing the collection of genetically redundant material.\u003c/p\u003e\u003cp\u003eIn our study of \u003cem\u003eNotopterygium\u003c/em\u003e species, an integrated marker system combining nuclear ITS with chloroplast sequences enabled maternal lineage tracking, assessment of biparental inheritance patterns, and coalescent-based demographic modeling. For the strictly wild \u003cem\u003eN. incisum\u003c/em\u003e, conservation focused on preserving phylogenetically distinct haplotypes across their native ranges. For \u003cem\u003eN. forbesii\u003c/em\u003e, which exhibits both wild and cultivated populations, ancestry analysis differentiated indigenous wild genotypes from admixed cultivated lineages. This dual-strategy approach successfully preserved\u0026thinsp;\u0026ge;\u0026thinsp;85% of observed genetic diversity, maintained population evolutionary potential, and established a valuable genetic reservoir for future breeding. The methodology demonstrates particular efficacy for alpine medicinal plants like \u003cem\u003eNotopterygium\u003c/em\u003e and serves as a transferable model for other mountain-dwelling species characterized by complex wild-cultivated relationships.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eHaplotype network of\u003c/b\u003e \u003cb\u003eN. incisum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eN. franchetii\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSequence analysis of three chloroplast DNA regions (\u003cem\u003erbcL\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, and \u003cem\u003etrnS-trnG\u003c/em\u003e) identified 12 haplotypes in \u003cem\u003eN. incisum\u003c/em\u003e (Hap_1-Hap_12) and 10 in \u003cem\u003eN. franchetii\u003c/em\u003e (Hap_13-Hap_22), with NETWORK analysis revealing complete genetic isolation between the species (Fig.\u0026nbsp;1). The network showed no shared haplotypes and a minimum of five mutational steps separating the two species' haplotype clusters, indicating an absence of gene flow. \u003cem\u003eN. incisum\u003c/em\u003e exhibited a star-like topology centered on the ancestral Hap_2 (n\u0026thinsp;=\u0026thinsp;78), which diverged into seven derived haplotypes through one to two mutations, while \u003cem\u003eN. franchetii\u003c/em\u003e displayed stepwise differentiation from Hap_13 into multiple descendant haplotypes. Parallel analysis of ITS sequences identified 20 haplotypes in \u003cem\u003eN. incisum\u003c/em\u003e (Hap_1-Hap_20) and 10 in \u003cem\u003eN. franchetii\u003c/em\u003e (Hap_21-Hap_30), with networks showing deeper divergence (\u0026ge;\u0026thinsp;20 mutational steps) and a single shared haplotype, suggesting limited historical introgression. In \u003cem\u003eN. incisum\u003c/em\u003e, the ITS network was structured around two central haplotypes, Hap_3 (n\u0026thinsp;=\u0026thinsp;139) and Hap_4 (n\u0026thinsp;=\u0026thinsp;192), from which all others were derived, whereas \u003cem\u003eN. franchetii\u003c/em\u003e exhibited a radial pattern centered on Hap_23 (n\u0026thinsp;=\u0026thinsp;119) (Fig.\u0026nbsp;2). These findings demonstrate strong species differentiation with distinct evolutionary histories, supported by both chloroplast and nuclear markers.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHaplotype Distributions\u003c/h2\u003e\u003cp\u003eOur multilocus analysis revealed distinct yet complementary patterns of genetic diversity between chloroplast and nuclear markers in Notopterygium populations. Chloroplast DNA (cpDNA) markers demonstrated remarkable geographical conservation in both \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e (Fig.\u0026nbsp;3\u0026ndash;4, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For \u003cem\u003eN. incisum\u003c/em\u003e, three dominant cpDNA haplotypes accounted for 86% of all occurrences, with the ancestral Hap_2 distributed across 19 populations (61% of total), accompanied by Hap_6 (11 populations, 35%) and Hap_1 (6 populations, 19%). The remaining seven populations (23%) developed unique cpDNA variants (Hap_8\u0026ndash;12), suggesting localized microevolutionary processes. \u003cem\u003eN. franchetii\u003c/em\u003e exhibited parallel conservation patterns, where ancestral haplotypes Hap_13 (15 populations, 50%) and Hap_14 (14 populations, 47%) showed panmictic distribution. Wild populations displayed particularly strong cpDNA conservation (90% monomorphic), while cultivated accessions showed significantly higher haplotype diversity, with only DTR and WYR populations retaining both ancestral haplotypes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe haplotype distribution of different provenances of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePopulations\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003ecpDNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"47\" rowspan=\"48\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eITS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eNo.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eHaplotype distribution\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eNo.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eHaplotype distribution\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"26\" rowspan=\"27\"\u003e\u003cp\u003e\u003cem\u003eNotopterygium incisum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(4), Hap_6(3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(19)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_6(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(15)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMY\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_6(9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(15), Hap_13(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(20)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(6), Hap_10(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(4), Hap_5 (1), Hap_6(4), Hap_11(1), Hap_19(4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(10), Hap_7 (1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_11(7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(14)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(3), Hap_6(3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(3), Hap_4(17)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eML\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(3), Hap_6(3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_6(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(18), Hap_18(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYZH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_1(4), Hap_2(1), Hap_7(1), Hap_9(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(16)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(4), Hap_6(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(17)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(13)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(5), Hap_7(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(8), Hap_10(3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_1(3), Hap_6(3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(2), Hap_4(8), Hap_6(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_1(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(11)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eREG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_1(8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_1(1), Hap_2 (4), Hap_3(7), Hap_4(1), Hap_11(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHOY\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(5), Hap_3(1), Hap_4(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(3), Hap_4 (6), Hap_11(2), Hap_12(5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(2), Hap_5(3), Hap_6(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(4), Hap_5 (4), Hap_6(2), Hap_12(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMEK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(5), Hap_7(1), Hap_12(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_2(1), Hap_3 (11), Hap_14(1), Hap_16(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(9), Hap_6(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_2(1), Hap_3 (9), Hap_4(4), Hap_6(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_1(3), Hap_2(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(11), Hap_4 (3), Hap_9(5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(9), Hap_7(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(15), Hap_17 (1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_6(8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(12), Hap_4 (2), Hap_15(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_1(8), Hap_2(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_3(2), Hap_4 (6), Hap_6(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXJ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_2(1), Hap_7(1), Hap_8(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_2(1), Hap_3 (11), Hap_8(1), Hap_20(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e\u003cp\u003e\u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(8), Hap_14(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_21(6), Hap_22 (1), Hap_23(3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(6), Hap_14(3), Hap_17(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(9), Hap_14(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(8), Hap_14(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_14(8), Hap_19(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_15(9), Hap_16(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_24(10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(8), Hap_14(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_26(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(1), Hap_26(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_14(10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(7), Hap_26(2), Hap_30(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003e\u003cem\u003eCultivated N. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(1), Hap_14(5), Hap_17(2), Hap_20(1), Hap_21(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_21(1), Hap_23(7), Hap_26(1), Hap_27(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZ1R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(3), Hap_14(6), Hap_20(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(5), Hap_26(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZ2R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(5), Hap_14(3), Hap_20(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHZ3R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_14(7), Hap_20(2), Hap_22(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(6), Hap_26(1), Hap_28(1), Hap_29(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDTR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(8), Hap_14(2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_23(5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMXR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(2), Hap_14(4), Hap_17(1), Hap_18(1), Hap_19(1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(4), Hap_23(5), Hap_25(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWYR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHap_13(4), Hap_14(5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHap_4(1), Hap_23(8), Hap_29(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNuclear ITS markers provided higher resolution of population structure while confirming the core patterns observed in cpDNA (Fig.\u0026nbsp;5\u0026ndash;6, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eN. incisum\u003c/em\u003e exhibited a dual-origin genetic architecture, with co-dominant ancestral haplotypes Hap_3 and Hap_4 (each in 17 populations, 55%) and nine populations (29%) containing unique variants (Hap_11\u0026ndash;20). This pattern suggests either ancestral hybridization or prolonged incomplete lineage sorting. In \u003cem\u003eN. franchetii\u003c/em\u003e, the predominant ITS haplotype (Hap_23) occurred in 17 populations (57%), with three wild populations (10%) showing unique variants. Cultivated accessions displayed 3.2-fold higher haplotype diversity than wild counterparts, with only HZ2R and DTR populations maintaining Hap_23 monomorphism. The concordant patterns between marker systems, particularly the elevated diversity in cultivated populations and conserved haplotypes in wild populations, provide robust evidence for distinct evolutionary trajectories shaped by natural versus anthropogenic selection pressures.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenetic Diversity and Structure\u003c/h3\u003e\n\u003cp\u003eThe genetic diversity and population structure of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e were assessed using cpDNA and ITS markers (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In both markers, the total genetic diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e) showed the following order: \u003cem\u003eN. incisum\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;cultivated \u003cem\u003eN. franchetii\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;wild \u003cem\u003eN. franchetii\u003c/em\u003e (cpDNA: 0.726\u0026thinsp;\u0026gt;\u0026thinsp;0.659\u0026thinsp;\u0026gt;\u0026thinsp;0.584; ITS: 0.651\u0026thinsp;\u0026gt;\u0026thinsp;0.517\u0026thinsp;\u0026gt;\u0026thinsp;0.383). For within-population diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e), cultivated \u003cem\u003eN. franchetii\u003c/em\u003e exhibited the highest values followed by \u003cem\u003eN. incisum\u003c/em\u003e and then wild \u003cem\u003eN. franchetii\u003c/em\u003e in both markers (cpDNA: 0.597\u0026thinsp;\u0026gt;\u0026thinsp;0.308\u0026thinsp;\u0026gt;\u0026thinsp;0.1912; ITS: 0.353\u0026thinsp;\u0026gt;\u0026thinsp;0.302\u0026thinsp;\u0026gt;\u0026thinsp;0.148).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe genetic structure of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003ecpDNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"4\" rowspan=\"5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003eITS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eS\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003csub\u003e\u003cb\u003eST\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eST\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eS\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003csub\u003e\u003cb\u003eST\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eST\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN. incisum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.726\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.308\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0604\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.575\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0851\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.512\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0878\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.651\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.302\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0558\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.536\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0654\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.494\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0554\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.584\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.191\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0572\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.673\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.618\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.383\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.148\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0780\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.713\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1405\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.942\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0550\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCultivated N. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.659\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0536\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.597\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0651\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.094\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0613\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.040\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0756\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.517\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1403\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.353\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.301\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0305\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePopulation differentiation analysis yielded particularly noteworthy results. Wild \u003cem\u003eN. franchetii\u003c/em\u003e populations exhibited strong genetic differentiation, with high \u003cem\u003eG\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e (cpDNA: 0.673; ITS: 0.713) and \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e (cpDNA: 0.618; ITS: 0.924) values. In striking contrast, cultivated \u003cem\u003eN. franchetii\u003c/em\u003e showed significantly reduced differentiation (cpDNA \u003cem\u003eG\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e: 0.094; ITS \u003cem\u003eG\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e: 0.077; cpDNA \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e: 0.040; ITS \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e: 0.301), suggesting homogenization among cultivated groups. \u003cem\u003eN. incisum\u003c/em\u003e displayed intermediate levels of population differentiation, with \u003cem\u003eG\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e (cpDNA: 0.575; ITS: 0.536) and \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e (cpDNA: 0.512; ITS: 0.494) values consistently lower than those of wild \u003cem\u003eN. franchetii\u003c/em\u003e but higher than cultivated populations.\u003c/p\u003e\n\u003ch3\u003ePopulation Dynamics History\u003c/h3\u003e\n\u003cp\u003eUsing cpDNA sequence data, we conducted multiple statistical analyses to reconstruct the demographic histories of the two \u003cem\u003eNotopterygium\u003c/em\u003e species (see Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The mismatch distributions for \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e displayed unimodal curves, accompanied by significantly negative Tajima\u0026rsquo;s \u003cem\u003eD\u003c/em\u003e and Fu\u0026rsquo;s \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e values. These findings strongly support historical population expansions in both species. Further evidence comes from their substantial population growth indices (\u003cem\u003eN. incisum\u003c/em\u003e: G\u0026thinsp;=\u0026thinsp;856; \u003cem\u003eN. franchetii\u003c/em\u003e: G\u0026thinsp;=\u0026thinsp;2901.736), which further corroborate rapid demographic expansion events.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of cpDNA mismatch distribution and neutrality tests for \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003emismatch distribution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003eNeutrality tests\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eθ\u003c/b\u003e\u003csub\u003e\u003cb\u003e0\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eθ\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSSD(\u003c/b\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003e-value)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eRag(\u003c/b\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003e-value)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eTajima\u0026rsquo;s\u0026nbsp;D\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eFu and Li\u0026rsquo;s F_\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eFu\u0026rsquo;s\u003c/b\u003e\u0026nbsp;\u003cb\u003eF\u003c/b\u003e\u003csub\u003e\u003cb\u003eS\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN. incisum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.4988(0.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.04238(0.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e856\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-1.39084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-0.27104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-9.402\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1015(0.014)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.1057(0.001)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2901.736\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-1.17776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-0.39033\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-3.365\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eSelection of the Core Collection\u003c/h3\u003e\n\u003cp\u003eComparative analysis of chloroplast (cpDNA) and nuclear (ITS) markers revealed distinct patterns of genetic diversity between cultivated and wild populations of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e. Cultivated \u003cem\u003eN. franchetii\u003c/em\u003e exhibited substantial genetic admixture across both marker systems, reflecting the consequences of unregulated introduction and cultivation practices. In contrast, wild populations maintained more structured genetic backgrounds, characterized by predominant ancestral haplotypes (cpDNA Hap_2 and ITS Hap_3/Hap_4 in \u003cem\u003eN. incisum\u003c/em\u003e; cpDNA Hap_13/Hap_14 and ITS Hap_23 in \u003cem\u003eN. franchetii\u003c/em\u003e) with clear geographical distributions. This contrast informed our strategy to establish core collections preferentially from wild populations, which retain distinct genetic lineages and demonstrate significant inter-provenance differentiation. Targeted sampling focused on three key components: dominant haplotypes across multiple provenances, locally endemic variants (e.g., cpDNA Hap_8\u0026ndash;12 and ITS Hap_11\u0026ndash;20 in \u003cem\u003eN. incisum\u003c/em\u003e), and rare haplotypes to ensure comprehensive genetic representation.\u003c/p\u003e\u003cp\u003eFor cpDNA markers, seed collections for \u003cem\u003eN. incisum\u003c/em\u003e included: Hap_2 from the JZ population, Hap_2 and Hap_9 from the YZH population, Hap_3 and Hap_4 from the HOY population, Hap_5 and Hap_6 from the AB population, Hap_7, Hap_8, and Hap_12 from the XJ population, Hap_10 from the BM population, and Hap_11 from the YS population. For \u003cem\u003eN. franchetii\u003c/em\u003e, cpDNA-based collections comprised: Hap_13 and Hap_17 from the LD population, Hap_14 and Hap_19 from the HZU population, and Hap_15 and Hap_16 from the MX population.\u003c/p\u003e\u003cp\u003eFor ITS markers, sampling of \u003cem\u003eN. incisum\u003c/em\u003e included: Hap_3 and Hap_4 from the NQ and MQ populations, Hap_1, Hap_2 and Hap_11 from the REG population, Hap_5, Hap_6 and Hap_19 from the JZ population, Hap_13 from the MY population, Hap_18 from the SD population, Hap_6 and Hap_12from the AB population, Hap_14 and Hap_16 from the MEK population, Hap_8 and Hap_20 from the XJ population. \u003cem\u003eN. franchetii\u003c/em\u003e collections focused on: Hap_23 from ZN and ZK populations, Hap_26 from ZN and LQ populations, and Hap_30 from LQ population. This comprehensive sampling strategy ensured representation of both conserved and variable regions across the two genomes.\u003c/p\u003e\u003cp\u003eThe established core collections demonstrated marked improvements in genetic diversity parameters compared to original populations (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both marker systems showed consistent trends, with within-population diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e) increasing by 38\u0026ndash;42% and total diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e) by 29\u0026ndash;33%. Notably, differentiation coefficients (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e) decreased substantially, with reductions of 0.128\u0026ndash;0.148 for cpDNA and 0.135\u0026ndash;0.140 for ITS markers. These improvements were achieved through strategic sampling from key populations, including \u003cem\u003eN. incisum\u003c/em\u003e sources for cpDNA Hap_2 (JZ), Hap_5\u0026thinsp;+\u0026thinsp;Hap_6 (AB), and ITS Hap_3\u0026thinsp;+\u0026thinsp;Hap_4 (HOY), as well as \u003cem\u003eN. franchetii\u003c/em\u003e sources for cpDNA Hap_13\u0026thinsp;+\u0026thinsp;Hap_17 (LD) and ITS Hap_23 (multiple populations). The parallel results from both marker systems not only validate the robustness of this conservation approach but also highlight its value for maintaining genetic resources for future breeding programs. The successful integration of cpDNA and ITS data provides a model for developing core collections in other medicinal plant species with similar cultivation histories and conservation needs.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe genetic structure of the core collection of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. forbesii\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003ecpDNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eITS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eS\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003csub\u003e\u003cb\u003eST\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003csub\u003e\u003cb\u003eS\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003csub\u003e\u003cb\u003eST\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN. incisum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.853\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.489\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.427\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1374\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.739\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.456\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0336\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.437\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0322\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN. franchetii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.924\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0251\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.432\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.533\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.425\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0344\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.298\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0216\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.621\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0124\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a nationally protected endangered medicinal plant species (Category III) in China, Notopterygium requires urgent strategic conservation of its germplasm resources. This study developed the first core collection for Notopterygium species through integrated analysis of chloroplast DNA (cpDNA) and nuclear ITS markers, establishing a scientifically validated framework for both conservation and sustainable utilization of these valuable medicinal resources\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eThe advantages of molecular phylogeography in constructing core collection\u003c/h2\u003e\u003cp\u003eThe conservation of medicinal plant germplasm presents unique challenges that fundamentally differ from crop germplasm management, necessitating distinct methodological approaches [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Conventional strategies employed for crops are often inadequate for medicinal species due to several intrinsic factors: (1) the frequent lack of well-established germplasm repositories, (2) complex evolutionary histories, and (3) widespread occurrence of cryptic genetic diversity. In this context, molecular phylogeography integrating both chloroplast DNA (cpDNA) and nuclear ITS markers has emerged as the most effective approach for core germplasm collection, providing comprehensive solutions to key conservation challenges [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe primary scientific challenge in medicinal plant conservation is germplasm admixture - populations with distinct genetic backgrounds often lack distinguishable morphological characteristics. This morphological uniformity significantly compromises the accuracy of germplasm identification. The molecular phylogeography approach based on a dual cpDNA-ITS marker system offers the following core advantages: (1) the maternal inheritance pattern of cpDNA enables tracing of historical migration routes, (2) the biparental inheritance of ITS markers facilitates detection of hybridization and gene flow events, (3) integrated analysis provides complete assessment of population genetic structure, and (4) enables precise quantification of genetic differentiation at various levels. These capabilities enable precise targeting and preservation of evolutionarily significant genetic variation, establishing molecular phylogeography as an indispensable methodology for developing scientifically robust core collections of medicinal plants.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenetic structure of\u003c/b\u003e \u003cb\u003eN. incisum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eN. franchetii\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe phylogenetic geographical pattern of \u003cem\u003eN. incisum\u003c/em\u003e exhibits a \"star-like\" structure, wherein most haplotypes are connected to a central haplotype via relatively short branches. This relatively straightforward pattern, combined with significantly negative values of Tajima\u0026rsquo;s D and Fu\u0026rsquo;s \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e (e.g., Tajima\u0026rsquo;s \u003cem\u003eD\u003c/em\u003e = -1.39084, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fu\u0026rsquo;s \u003cem\u003eFS\u003c/em\u003e = -9.402, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), strongly suggests that the population underwent a recent expansion during periods of favorable climatic conditions [\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Such negative values are indicative of an excess of rare mutations, a hallmark of demographic expansion or positive selection. However, due to insufficient time for the establishment of more complex genetic structures following this expansion, the haplotype network remains relatively simple and radiating. This \"star-like\" haplotype phylogenetic geographic pattern, often associated with population expansions, has also been reported in other species [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe haplotypes of cultivated and wild \u003cem\u003eN. franchetii\u003c/em\u003e were compared. Some haplotypes were found in the wild population, while others were found in the cultivated population, indicating that some haplotypes of wild \u003cem\u003eN. franchetii\u003c/em\u003e were lost in the cultivated population. Meanwhile, the total genetic diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e) of the wild population was 0.584 (cpDNA) and 0.383 (ITS), and that of the cultivated population was 0.659 (cpDNA) and 0.517 (ITS) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e of the cultivated population was slightly higher than that of the wild population based on cpDNA data, but the ITS data showed a more pronounced difference. This suggests that \u003cem\u003eN. franchetii\u003c/em\u003e did not experience the genetic bottleneck phenomenon that most cultivated crops do during the cultivation process, indicating that the initial population size of cultivated \u003cem\u003eN. franchetii\u003c/em\u003e was large and it was not subject to much artificial selection. However, although the total genetic diversity of cultivated \u003cem\u003eN. franchetii\u003c/em\u003e increased slightly (cpDNA) or showed mixed trends (ITS), its genetic structure changed significantly. The distribution range of shared haplotypes between the cultivated and wild populations in the cultivated population was much larger than that in the wild population, and the number of haplotypes in each cultivated population was significantly higher than that in the wild population. The genetic diversity within the population (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e) of the cultivated population was 0.597 (cpDNA) and 0.353 (ITS), and that of the wild population was 0.191 (cpDNA) and 0.148 (ITS). The \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e of the cultivated population was significantly higher, which led to a significant decrease in the coefficient of gene differentiation among cultivated populations (\u003cem\u003eG\u003c/em\u003e\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e, 0.094 for cpDNA and 0.077 for ITS in cultivated populations, compared to 0.673 for cpDNA and 0.713 for ITS in wild populations). This indicates a very weak genetic structure in the cultivated populations, that is, a decrease in genetic diversity among populations and an increase in genetic diversity within populations. This suggests that there was an increase in gene flow among cultivated populations during the cultivation process, and the germplasm of cultivated \u003cem\u003eN. franchetii\u003c/em\u003e was severely mixed, which will lead to outbreeding depression and germplasm degradation of the cultivated species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of core collection of\u003c/b\u003e \u003cb\u003eN. incisum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eN. franchetii\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe establishment of a core collection serves as a strategic approach to efficiently conserve and utilize genetic diversity. In this study, we employed a phylogeographic approach based on chloroplast (cpDNA) and nuclear (ITS) marker analyses to construct core collections for \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e. From original germplasms of 187 (\u003cem\u003eN. incisum\u003c/em\u003e) and 186 (\u003cem\u003eN. franchetii\u003c/em\u003e) accessions, we selected 50 and 30 core accessions, respectively, achieving sampling rates of 26.74% and 25.64%. These proportions were carefully determined to balance representativeness and efficiency, as supported by previous studies indicating that 5\u0026ndash;10% of a core collection can capture over 70% of genetic variation in the entire germplasm, with medicinal plants typically requiring 10\u0026ndash;40% sampling depending on species-specific diversity. The core collections demonstrated significant improvements in genetic diversity parameters, with \u003cem\u003eN. incisum\u003c/em\u003e showing retention rates of 117.49% for total diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e), 158.77% for within-population diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e), and 74.26% for differentiation (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e), while \u003cem\u003eN. franchetii\u003c/em\u003e exhibited even greater retention rates of 158.22% (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e), 226.18% (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e), and 79.20% (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e). These results confirm that the core collections effectively minimized redundancy while preserving and even enhancing genetic variation, with the parallel trends from both cpDNA and ITS markers validating the robustness of our approach.\u003c/p\u003e\u003cp\u003eOur sampling strategy prioritized wild populations due to their structured genetic backgrounds and distinct ancestral haplotypes, such as cpDNA Hap_2 and ITS Hap_3/Hap_4 in \u003cem\u003eN. incisum\u003c/em\u003e and cpDNA Hap_13/Hap_14 and ITS Hap_23 in \u003cem\u003eN. franchetii\u003c/em\u003e. Targeted sampling ensured the inclusion of dominant haplotypes across multiple provenances, locally endemic variants, and rare haplotypes, leading to reduced differentiation coefficients (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e) by 0.128\u0026ndash;0.148 for cpDNA and 0.135\u0026ndash;0.140 for ITS markers. While core collections are powerful tools for germplasm management, they cannot fully capture a species' entire genetic diversity, necessitating dynamic updates as new germplasm becomes available. Moreover, given the geographical authenticity of medicinal plants and the habitat-dependent synthesis of bioactive compounds, future efforts should integrate genetic diversity with agronomic traits and phytochemical profiles to ensure comprehensive conservation and breeding utility. This study lays the foundation for molecular-based core collections of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e, with subsequent research needed to incorporate phenotypic and biochemical data for more robust germplasm management.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThrough comprehensive genetic analyses, we established core collections for both species with optimized sampling efficiency. For \u003cem\u003eN. incisum\u003c/em\u003e, the core collection comprised 50 accessions (26.74%) based on cpDNA markers and 103 accessions (40.74%) using ITS data. Similarly, \u003cem\u003eN. franchetii\u003c/em\u003e core collections contained 30 (25.64%, cpDNA) and 40 accessions (21.05%, ITS), respectively. This dual-marker approach leveraged complementary inheritance patterns - the rapidly evolving, maternally inherited chloroplast DNA provided clear differentiation of germplasm lineages, while biparentally inherited ITS markers revealed finer-scale population structure. The integrated phylogeographic framework enabled precise quantification of genetic differentiation and informed targeted selection of evolutionarily distinct germplasm, establishing an effective methodology for medicinal plant conservation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003eA total of 730 samples of Notopterygii Rhizoma et Radix, comprising 540 specimens of \u003cem\u003eN. incisum\u003c/em\u003e and 190 specimens of \u003cem\u003eN. franchetii\u003c/em\u003e, were collected from diverse locations. The precise collection sites were recorded using a single-plant GPS system (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;7). No authorization was required for sample collection. Representative voucher specimens from each population were authenticated by Professor Yubi Zhou at the Northwest Institute of Plateau Biology, Chinese Academy of Sciences. These specimens are deposited in the Qinghai-Tibetan Plateau Museum of Biology (QPMB), with voucher numbers HNWP2014001\u0026ndash;HNWP2014027 for \u003cem\u003eN. incisum\u003c/em\u003e and HNWP2024001\u0026ndash;HNWP2024019 for \u003cem\u003eN. franchetii\u003c/em\u003e. Leaves were randomly collected from each sample, ensuring that individual samples were separated by at least fifty meters. The freshly collected leaves were immediately dried in silica gel within ziplock bags. Institutional, governmental, and international rules are followed in all aspects of our experimental study, including the gathering of plant samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDNA extraction, PCR amplification and sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from individual samples using an improved CTAB protocol [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. DNA concentration and quality were assessed via 0.8% agarose gel electrophoresis against lambda DNA standards and confirmed by UV spectroscopy. Samples were diluted to approximately 10 ng/\u0026micro;L in 0.1% TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) for subsequent analyses.\u003c/p\u003e\u003cp\u003eChloroplast DNA (cpDNA) Sequencing: A total of 730 samples from 46 populations were sequenced for three cpDNA regions (\u003cem\u003etrnS-trnG\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, and \u003cem\u003erbcL\u003c/em\u003e) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;7). This dataset incorporated 540 samples (27 populations) from a prior study [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and 190 newly collected individuals (19 populations). Primer pairs (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] were selected based on previous \u003cem\u003eNotopterygium\u003c/em\u003e research. PCR amplification was performed in 25-\u0026micro;L reactions containing 2 \u0026micro;L DNA template (10\u0026ndash;50 ng/\u0026micro;L), 12.5 \u0026micro;L PCR Master Mix, 0.75 \u0026micro;L of each primer (20 \u0026micro;M), and 9 \u0026micro;L ddH₂O. Thermocycling conditions are detailed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eNuclear ITS Sequencing: For the internal transcribed spacer (ITS) region, 730 samples from the same 46 populations were sequenced (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;7). This included 540 samples (27 populations) from a previous study [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and 190 newly collected individuals (19 populations). PCR amplification used identical reaction components and volumes as described for cpDNA, employing Xi\u0026rsquo;an Runde (China) PCR Master Mix. All high-quality PCR products were bidirectionally sequenced using the amplification primers and an ABI 3730 XL genetic analyzer (Applied Biosystems, Foster City, CA, USA). The chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) regions of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e were successfully sequenced and assembled. All obtained sequences, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, have been deposited in the GenBank database under their respective accession numbers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDNA Analysis\u003c/h2\u003e\u003cp\u003eSequence assembly was performed using ContigExpress (Informax), followed by multiple alignment with ClustalX v1.81 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. To characterize population genetic structure, we conducted hierarchical analyses using complementary approaches. Genetic diversity parameters - including within-population diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e), total diversity (\u003cem\u003eh\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e), and differentiation indices (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e and \u003cem\u003eN\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e) - were computed using PERMUT v1.0 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], with \u003cem\u003eN\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e incorporating both haplotype frequencies and mutational distances for enhanced sensitivity to recent differentiation.\u003c/p\u003e\u003cp\u003eDemographic history analyses were performed using Arlequin v3.5 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], including: (1) neutrality tests (Tajima's D, Fu's FS, and Fu \u0026amp; Li's F*) to detect population size changes, and (2) mismatch distribution analysis to examine population expansion patterns. Model validity was assessed through the sum of squared deviations (SSD) between observed and expected distributions and Harpending's raggedness index (Rag), with significance tested by 10,000 parametric bootstrap replicates.\u003c/p\u003e\u003cp\u003eFor haplotype analysis, we constructed median-joining networks of both cpDNA and ITS sequences using NETWORK v5.0.0[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] (Polzin and Daneshmand, 2003) to visualize mutational relationships among haplotypes. The spatial distribution of haplotypes was mapped using ArcGIS v10.2 based on georeferenced occurrence data [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], with kernel density estimation applied to identify regional distribution patterns. Frequency distribution histograms were generated to compare haplotype compositions between wild and cultivated populations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of Core Collections\u003c/h2\u003e\u003cp\u003eAccording to previous studies, the size of a core collection typically represents 5\u0026ndash;30% of the initial population samples [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan additionalcitationids=\"CR58 CR59 CR60\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In this study, molecular phylogeography was employed to construct the core germplasm of Notopterygii Rhizoma et Radix. Through comparative analysis of genetic diversity and genetic structure between wild and cultivated populations, we elucidated the geographic origins of major cultivated genotypes or haplotypes and assessed the degree of germplasm confounding in these wild populations. The core collection can be effectively constructed by collecting and preserving wild populations characterized by single genotype or haplotype distributions and significant genetic distances.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e This study including the collection of plant samples complies with relevant institutional, national, and international guidelines and legislation. All the necessary permissions have been granted for this research.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by the Open Project of Qinghai Key Laboratory of Qinghai-Tibet Plateau Biological Resources.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYL conceived the study, designed the methodology, and drafted the manuscript. LJ performed the statistical analyses. LS was responsible for sample collection and contributed to manuscript editing. All authors revised the manuscript critically for intellectual content and approved the final version.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets generated and/or analyzed during the current study are available in the GenBank repository (The accession numbers for trnS-trnG in *N. incisum* are PV239459\u0026ndash;PV239464, and for rbcL are PV261945\u0026ndash;PV261947. Meanwhile, the accession numbers for trnS-trnG in *N. franchetii* are PV239448\u0026ndash;PV239458, and for rbcL are PV261942\u0026ndash;PV261944)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFrankel OH, Brown AHD. Current plant genetic resoureesacritical appraisal. Genetics: newforntiers. Vo1. IV. 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Sci Hortic. 2023;319:112192.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Notopterygium incisum, N. franchetii, Notopterygii Rhizoma et Radix, haplotype, germplasm resources, genetic structure","lastPublishedDoi":"10.21203/rs.3.rs-7238023/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7238023/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs both an endangered medicinal species (China's Category III protected plant) and an authentic traditional Chinese medicine,\u0026nbsp;\u003cem\u003eNotopterygium incisum\u003c/em\u003e Ting ex H. T. Chang\u003cem\u003e and N. franchetii \u003c/em\u003eBoiss\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003erequire urgent conservation of their germplasm resources. While core collections offer an efficient solution for preserving genetic diversity, no such resource currently exists for these species despite their ecological and pharmacological importance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, three chloroplast DNA regions (\u003cem\u003erbcL\u003c/em\u003e,\u003cem\u003e matK\u003c/em\u003e, and\u003cem\u003e trnS\u003c/em\u003e-\u003cem\u003etrnG\u003c/em\u003e) and the nuclear ribosomal ITS sequence were employed as molecular markers to conduct phylogeographic analyses of \u003cem\u003eN. incisum\u003c/em\u003e and \u003cem\u003eN. franchetii\u003c/em\u003e, and their core collections were constructed through stratified sampling of evolutionary significant haplotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNetwork analyses revealed complete cpDNA differentiation between species (12 vs. 10 haplotypes separated by ≥5 mutations), while ITS data showed limited historical introgression. Wild populations exhibited strong genetic structure (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e: 0.673-0.713) with ancestral haplotypes (cpDNA Hap_2/Hap_13; ITS Hap_3-Hap_4/Hap_23), whereas cultivated accessions showed 3.2× higher haplotype diversity but reduced differentiation (\u003cem\u003eG\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e: 0.077-0.094).\u0026nbsp;Demographic tests (Tajima's D = -1.39 to -2.15, P\u0026lt;0.01) and growth indices (G=856-2901) confirmed post-glacial expansions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing integrated cpDNA and ITS markers, we established optimized core collections for both species (\u003cem\u003eN. incisum\u003c/em\u003e: 50-103 accessions; \u003cem\u003eN. franchetii\u003c/em\u003e: 30-40 accessions) that effectively preserved genetic diversity. The dual-marker approach resolved cultivated populations' paradoxical genetic patterns (higher diversity but lower differentiation) and provides a conservation model for medicinal plants facing anthropogenic pressures.\u003c/p\u003e","manuscriptTitle":"Construction of a Core Collection of Notopterygii Rhizoma et Radix Based on Molecular Phylogeography","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 18:49:36","doi":"10.21203/rs.3.rs-7238023/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-26T10:17:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T01:15:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T15:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310390785479877701590693341590807922658","date":"2025-09-10T11:42:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31075019252952802736633319074885493893","date":"2025-09-08T10:42:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128177093037425544321862950569842280103","date":"2025-09-08T10:34:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T10:16:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T05:23:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-18T13:13:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-18T09:03:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-08-18T09:00:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"631093ac-ca04-413f-82cc-22617b9af6ab","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-7238023","link":"https://doi.org/10.1186/s12870-025-07715-z","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-11-27 15:58:15","publishedOnDateReadable":"November 27th, 2025"},"versionCreatedAt":"2025-09-15 18:49:36","video":"","vorDoi":"10.1186/s12870-025-07715-z","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07715-z","workflowStages":[]},"version":"v1","identity":"rs-7238023","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7238023","identity":"rs-7238023","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}