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Complete chloroplast genomes and phylogenetic analysis of 7 Murraya species in China
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Ziyuan Chena, Yan Jina, Yuyang Zhaoa, Chao Jianga, *, Yuan Yuanb, *
Science of Traditional Chinese Medicine | 2026, 4(1) : 62 - 72
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Science of Traditional Chinese Medicine | 2026, 4(1): 62-72
• Original Research •
Complete chloroplast genomes and phylogenetic analysis of 7 Murraya species in China
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Ziyuan Chena, Yan Jina, Yuyang Zhaoa, Chao Jianga, *, Yuan Yuanb, *
Affiliations
  • aState Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
  • bExperimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China
Published: 2026-03-25 doi: 10.1097/st9.0000000000000091
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Background:

Murraya, a genus of shrubs and trees in the Rutaceae family, consists of approximately 9 species in China with significant medicinal and horticultural value. However, the phylogeny and taxonomy of Murraya species remain controversial, particularly with respect to Murraya exotica and M. paniculata.

Objective:

This study aimed to provide insights into the taxonomy, phylogeny, and identification of Murraya.

Methods:

In this study, the chloroplast (CP) genomes of 7 Murraya species were sequenced, assembled, and subjected to comparative and phylogenetic analyses.

Results:

The CP genomes of Murraya ranged from 158,573 to 160,817 bp in length and encoded 112 unique genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Similar to other angiosperms, the inverted repeat regions of the CP genomes exhibited lower sequence divergence than the single-copy regions, and coding regions were more conserved than noncoding regions. Comparative analysis identified several highly variable regions (eg, matK, ycf1, ndhI-ndhA, trnH-GUG-psbA, rpl32-trnL) that could serve as molecular markers for species identification in Murraya. Among these, the ycf1 gene was validated as a useful marker for distinguishing M. exotica from M. paniculata. Positive selection was detected in 10 genes, including rbcL, psaJ, ndhD, ndhF, rpl2, rpl20, ycf1, accD, ccsA, and rpl32. Phylogenetic analysis based on CP genomes supported the recognition of M. exotica and M. paniculata as independent species. Moreover, the phylogenetic trees indicated that Murraya is not monophyletic, with sect. Bergera showing a closer relationship to Clausena. Molecular dating results suggested that the diversification of M. paniculata, M. alata, and M. exotica occurred approximately 9.11 Mya (95% highest posterior density: 4.90-13.87 Mya).

Conclusion:

These findings provide valuable CP genome data for clarifying the phylogenetic relationships between M. exotica and M. paniculata, and for advancing the study of DNA markers and the evolutionary history of Murraya.

Chloroplast genome  /  Comparative analysis  /  Murraya  /  Murraya exotica  /  Murraya paniculata  /  Phylogenetic analysis
Ziyuan Chen, Yan Jin, Yuyang Zhao, Chao Jiang, Yuan Yuan. Complete chloroplast genomes and phylogenetic analysis of 7 Murraya species in China[J]. Science of Traditional Chinese Medicine, 2026 , 4 (1) : 62 -72 . DOI: 10.1097/st9.0000000000000091
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1.　Introduction
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Murraya, comprising about 17 species, is a genus in the family Rutaceae distributed across East, South, and Southeast Asia, as well as Australia and the Southwest Pacific Islands. Murraya species possess medicinal, horticultural, and economic value. According to the Chinese Pharmacopoeia (2020 Edition), Murrayae Folium et Cacumen (MFC) is derived from Murraya exotica or M. paniculata, and is used in the treatment of stomachache, rheumatism, arthralgia, and toothache.[1]
The circumscription of species within Murraya has long been controversial. Based on morphological taxonomy and chemotaxonomy, some scholars divided the genus Murraya into 2 sections. Species with white-gray branches were classified as sect. Murraya, which includes M. exotica, M. paniculata, M. alata, and others.[2] Sect. Bergera comprises species with darkbrown branches, such as M. euchrestifolia, M. kwangsiensis, M. kwangsiensis var. macrophylla, and M. koenigii (Fig. 1).[3] Some researchers further proposed elevating the 2 sections to the rank of genera, thereby restoring the genus Bergera Koenig ex L. (1771).[4-6] Currently, molecular phylogenetic and morphological evidence generally supports revising the 2 sections as separate genera.[7,8]
At the species level, the taxonomic status of M. exotica L. and M. paniculata (L.) Jack remains controversial and has been revised several times. Due to the lack of type specimens and the presence of morphological intermediates in cultivation, some scholars have suggested that the 2 species should be synonymized or treated as a single species.[9-11] Others have maintained them as distinct species based on morphological and phytochemical differences.[12] The primary morphological distinction lies in the leaflets: M. exotica has elliptic-obovate or obovate leaflet blades, whereas M. paniculata exhibits suborbicular to ovate or elliptic leaflets. Additionally, the 2 species differ significantly in chemical composition.[13] The main constituents of M. exotica leaves are coumarins,[14,15] while M. paniculata leaves are rich in polymethoxylated flavones.[16,17] In the current market, M. paniculata serves as the main source of Murrayae Folium et Cacumen (MFC), whereas M. exotica is primarily used as a horticultural plant in southern China. Therefore, clarifying the relationship between M. exotica and M. paniculata is essential to ensure the safety and efficacy of MFC in clinical applications.
Molecular systematic studies have resolved some of the taxonomic issues within the genus Murraya. Analyses based on the nuclear ribosomal DNA ITS region and partial chloroplast (CP) DNA sequences indicate that M. koenigii, M. kwangsiensis, and M. microphylla are more closely related to Clausena than to other Murraya species.[18-20] Inter-simple sequence repeats, directed amplification of minisatellite DNA, and random amplified polymorphic DNA profiles also suggest the presence of distinct genotypes in M. exotica and M. paniculata.[21] Nevertheless, the relationship between M. exotica and M. paniculata remains unclear. In this study, we sequenced and assembled the complete CP genomes of M. exotica, M. paniculata, M. alata, M. euchrestifolia, M. kwangsiensis, M. kwangsiensis var. macrophylla, and M. microphylla from different regions. We analyzed genome features, long repeat sequences, and simple sequence repeats (SSRs), and conducted comparative and phylogenetic analyses based on the CP genomes. This work provides new insights into the taxonomy and phylogeny of Murraya.
2.　Materials and methods
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2.1.　 Plant material, DNA extraction, and CP genome sequencing
Thirty-two samples representing 7 Murraya species were collected from the Herbarium of the Institute of Botany, China Academy of Chinese Medical Sciences (PE), and the voucher specimens were deposited at the same herbarium (Supplemental Table S1, https://links.lww.com/STCM/A68). All samples were identified by Yan Jin. No specific permissions were required for the collection of Murraya. In addition, 6 complete CP genomes of Rutaceae were obtained from GenBank: M. koenigii (NC_032684.1), Citrus medica (NC_050939.1), C. maxima (MN782007.1), Merrillia caloxylon (NC_032688.1), C. excavata (NC_032685.1), and Zanthoxylum armatum (NC_050250.1). Total genomic DNA was extracted from 20 to 50 mg of dried leaves using the DNAsecure Plant Kit (Tiangen, Beijing, China). For each accession, 5.0 Gb of raw data were generated using the Illumina NovaSeq 6000 platform (Novogene, Beijing, China) with 150 bp paired-end reads.
2.2.　 CP genome assembly and annotation and feature analyses
Raw sequencing data were filtered using fastp version 0.19.7. The complete CP genomes of the 32 Murraya samples were then assembled from the clean data using GetOrganelle v1.6.2d[22] with the following parameters: maximum extension rounds = 30, max-reads = 2 × 107, and SPAdes k-mer values = 45, 65, 85, 115. Genome annotation was performed on 33 CP genomes (including M. koenigii, NC_032684.1) using PGA v3 and the online program GeSeq[23] (https://chlorobox.mpimp-golm.mpg.de/geseq.html), with Amborella trichopoda (curated by PGA) and M. paniculata (NC_052700.1) serving as reference genomes. The search identity threshold was set to 85%. Annotation results were manually reviewed and corrected. Circular CP genome maps were generated using OGDRAW[24] (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html).
2.3.　 Repeat analysis
REPuter[25] (https://bibiserv.cebitec.uni-bielefeld.de/reputer) was used to identify forward, reverse, complement, and palindromic repeats in the CP genomes. The parameters were set as follows: hamming distance = 3 and minimum repeat size = 30 bp. SSRs were identified using MISA[26] with the following thresholds: 8 repeats for mononucleotide SSRs, 4 repeats for dinucleotide and trinucleotide SSRs, and 3 repeats for tetranucleotide, pentanucleotide, and hexanucleotide SSRs.
2.4.　 Genomic comparison and divergence analysis
IRscope[27] (https://irscope.shinyapps.io/irapp/) was used to visualize the expansion and contraction of the inverted repeat (IR) regions in the CP genomes. To compare Murraya CP genomes, mVISTA[28] (https://genome.lbl.gov/vista/mvista/submit.shtml) was employed to analyze and visualize sequence divergence, using the annotation of M. exotica as the reference. MAVUE V2.4.0[29] was used to assess CP genome collinearity. Nucleotide diversity (π) of gene and intergenic regions was calculated using DnaSP v6.[30]
2.5.　 Positive selection analysis
Selective pressure was analyzed for 78 shared protein-coding genes among the CP genomes of Murraya. EasyCodeML v1.41[31] was used to calculate nonsynonymous (dN) and synonymous (dS) substitution rates, as well as their ratio (ω). Each protein-coding gene was aligned separately using ClustalW (codons) in MEGA11[32] after removal of stop codons. Based on these alignments, a maximum likelihood (ML) tree was constructed using IQ-TREE v2.0.5.[33] The preset mode and site models (M0, M3, M1a, M2a, M7, M8, M8a) in EasyCodeML were applied to calculate dN, dS, ω, and likelihood ratio test values. The Bayesian empirical Bayes method was used to estimate posterior probabilities (PP) and detect positive selection in the genes. PPs greater than 0.95 and 0.99 indicate sites under positive selection and strong positive selection, respectively.
2.6.　 Phylogenetic analysis
Phylogenetic trees were constructed using ML and Bayesian inference (BI) methods. Three datasets were analyzed as follows: (1) complete CP genomes, (2) concatenated coding sequences (CDSs) of 78 shared protein-coding genes (including only 1 IR region), and (3) 109 shared noncoding sequences (NCS) of intergenic regions (including only 1 IR region). Sequence alignments were performed using MAFFT v7.453,[34] and aligned sequences were trimmed with TrimAl v1.4[35] using a gap threshold of 0.9 and a consistency score (cons) of 60. ML analysis was conducted in IQ-TREE with 1000 ultrafast bootstrap replicates, and the best-fitting substitution model was selected using the ModelFinder plugin. BI analysis was performed in MrBayes v3.2.6[36] via PhyloSuite v1.2.2[37] for 10,000,000 generations, with the optimal substitution model also determined using ModelFinder[38] in PhyloSuite. Phylogenetic trees were visualized and edited using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Zanthoxylum armatum (NC_050250.1) was used as the outgroup to root the trees.
2.7.　 Divergence time estimation
Divergence times of Murraya species were estimated using MCMCtree in PAML v4.9[39] based on the complete CP genomes. The ML tree constructed by IQ-TREE was used as the input topology. Three fossil calibrations were applied: the node of Citrus species was set at 8 million years ago (Mya)[40]; the node of the branch containing Clausena excavata was set at 27.23 Mya[41]; and the node of the outgroup (Z. armatum) was set at 35-40 Mya.[42] The Markov Chain Monte Carlo process was run for 20,000 generations with a sampling frequency of every 100 iterations, discarding the first 20,000 samples as burn-in. Divergence times were estimated using the independent rates clock model and the HKY85 substitution model, with 95% highest posterior density (HPD) intervals. The resulting divergence times were then mapped onto the ML tree.
2.8.　 Molecular marker development
Gene and intergenic regions among Murraya species were analyzed using mVISTA and DnaSP. Primers for molecular markers were designed with Primer Premier 5.0,[43] and their accuracy was verified through polymerase chain reaction (PCR) amplification. The 25 μL PCR reaction contained 12.5 μL 2× M5 Super FastTaq PCR MasterMix (Mei5 Biotechnology Co., Ltd., Beijing, China), 0.5 μL forward primer (10 μmol/L), 0.5 μL reverse primer (10 μmol/L), 0.5 μL DNA template, and 11.0 μL dH2O. The PCR amplification was performed on a Veriti 96 PCR system (Applied Biosystems, Waltham, Massachusetts, USA) under the following conditions: initial denaturation at 95 °C for 3 minutes; 35 cycles of 94 °C for 10 seconds, 58 °C for 15 seconds, and 72 °C for 10 seconds; and a final extension at 72 °C for 5 minutes. PCR products were evaluated by 1.5% agarose gel electrophoresis, followed by purification and sequencing.
3.　Results
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3.1.　 Features of the Murraya CP genomes
All 33 Murraya CP genomes exhibited a typical quadripartite structure, consisting of 2 IR regions (IRa and IRb), a large single-copy region (LSC), and a small single-copy region (SSC). The total lengths of the CP genomes ranged from 158,573 bp (M. kwangsiensis var. macrophylla) to 160,817bp (M. exotica) (Fig. 2). Specifically, the CP genome lengths of M. exotica ranged from 160,178 to 160,817 bp, while those of M. paniculata ranged from 160,261 to 160,369bp. The lengths of the LSC, SSC, and IR regions varied from 86,813 bp (M. microphylla) to 87,812 bp (M. kwangsiensis), 17,983 bp (M. euchrestifolia) to 18,620 bp (M. paniculata), and 26,328 bp (M. kwangsiensis var. macrophylla) to 27,577 bp (M. exotica), respectively. The total GC content ranged from 38.54% to 38.65%, with average GC contents of 37.04%, 33.46%, and 42.94% for the LSC, SSC, and IR regions, respectively.
These CP genomes contained 112 unique genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes (Supplemental Table S2, https://links.lww.com/STCM/A68). The ndhD gene was annotated as a pseudogene in M. exotica, one sample of M. paniculata, M. euchrestifolia, M. kwangsiensis, and M. kwangsiensis var. macrophylla due to the absence of a valid start codon. Seven protein-coding genes (ndhB, rpl2, rpl23, rps12, rps19, rps7, and ycf2), 7 tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC), and 4 rRNA genes (rrn4.5, rrn5, rrn16, and rrn23) were duplicated in the IR regions. Interestingly, compared with other Murraya species, trnH-GUG was duplicated in the LSC region of M. kwangsiensis, whereas 1 copy of rps19 and ycf2 was lost in M. kwangsiensis var. macrophylla. Furthermore, 3 protein-coding genes contained 2 introns, while 6 proteincoding genes and 6 tRNA genes contained 1 intron across all Murraya samples (Supplemental Fig. S1, https://links.lww.com/STCM/A68). An intron of the ndhF gene was observed in 4 samples of M. exotica.
Nucleotide diversity (π) values were calculated for 112 genes and 109 intergenic regions in Murraya CP genomes (Fig. 3). The top 5 genes with the highest π values, in ascending order, were matK (0.0100), ycf1 (0.0098), ccsA (0.0097), ndhF (0.0096), and rps15 (0.0084). The top 5 intergenic regions with the highest π values, in ascending order, were ndhI-ndhA (0.0351), trnH-GUG-psbA (0.0303), rpl23-rpl2 (0.0288), rpl2-rpl23 (0.0288), and trnG-UCC-trnR-UCU (0.0260). Overall, the average π value of genes was lower than that of intergenic regions.
3.2.　 Repeat analysis
Four types of long repeats (forward, reverse, complement, and palindromic) were identified in the CP genomes of 33 Murraya samples. A total of 1233 long repeats were detected, including 407 forward repeats, 68 reverse repeats, 42 complement repeats, and 716 palindromic repeats (Supplemental Fig. S2, https://links.lww.com/STCM/A68). The number of long repeats per species ranged from 31 (M. exotica) to 49 (M. paniculata). Most repeats (70.9%) were 30-39 bp in length, 18.7% were 40-49 bp, and 10.4% exceeded 50 bp (Supplemental Table S3, https://links.lww.com/STCM/A68). The longest repeat was a palindromic repeat of 398 bp, which was the only repeat longer than 70 bp.
A total of 7924 SSRs were identified in the CP genomes of 33 Murraya samples, including 5738 mononucleotide SSRs, 1544 dinucleotide SSRs, 292 trinucleotide SSRs, 284 tetranucleotide SSRs, 25 pentanucleotide SSRs, and 41 hexanucleotide SSRs. The number of SSRs varied among the 8 species, ranging from 236 (M. microphylla) to 254 (M. kwangsiensis). Notably, no pentanucleotide SSRs were detected in the CP genomes of M. exotica and M. alata. Most SSRs were located in the LSC regions, followed by the SSC and IR regions. Among all SSR types, A/T mononucleotide SSRs were the most abundant, accounting for 66.48%, followed by AT/AT dinucleotide SSRs, accounting for 13.24% (Supplemental Fig. S3, https://links.lww.com/STCM/A68).
3.3.　 Comparative genomes analysis
The boundaries of the LSC/IR and SSC/IR regions were compared among the 8 Murraya species (Fig. 4). Four M. exotica samples, each approximately 160,816 bp in length, exhibited IR region expansion compared with other samples. In contrast, M. kwangsiensis var. macrophylla, which had the shortest CP genome, showed IR region contraction. Genes located near the LSC/IR junction included rps3, rpl22, rps19, rpl2, and trnH. The rpl22 gene was entirely located in the IRb region in M. kwangsiensis, but in the LSC region of M. kwangsiensis var. macrophylla, whereas in the other 6 species, it crossed the LSC/IRb junction. The trnH gene was positioned in the LSC region in all 8 species, with M. kwangsiensis containing 2 copies in the LSC, an unusual feature. Due to IR contraction in M. kwangsiensis var. macrophylla, the rps19 gene shifted into the LSC region, losing 1 copy, while the 2 rpl2 genes in the IR regions extended to the LSC/IR boundary, and the trnH gene was located farther from the LSC/IRa boundary. The ndhF gene contracted inward by 148 bp from the SSC/IRb junction in M. kwangsiensis var. macrophylla, whereas it expanded in other species. IR expansion in some M. exotica individuals resulted in an additional intron in the ndhF gene. The ycf1 gene was located near the SSC/IRa junction, differing only in its distance from the junction among species.
The 8 Murraya CP genomes exhibited highly similar sequences. As observed in other angiosperms, the IR regions were more conserved than the LSC and SSC regions, and coding regions were more conserved than noncoding regions. Highly divergent regions were mainly located in intergenic spacers and introns, including trnH-GUG-psbA, petN-psbM, rpl32-trnL-UAG, and ccsA-ndhD. Additionally, several genes contained variable regions, such as matK, rps3, ndhF, ccsA, and ycf1 (Fig. 5). Collinearity analysis of the CP genomes, using M. exotica as the reference, revealed no obvious rearrangements, indicating that the Murraya CP genomes are relatively conserved (Supplemental Fig. S4, https://links.lww.com/STCM/A68).
3.4.　 Selection pressure analysis
The ratio (ω) of dN to dS substitutions (dN/dS) was calculated for all 78 shared protein-coding genes across 33 complete Murraya CP genomes. Using the M8 (β and ω > 1) model, 10 protein-coding genes were identified as being under positive selection, with PP values greater than 0.95 (Table 1). Among these genes, rbcL showed the highest number of positively selected amino acid sites (4), followed by psaJ (3), ndhD (2),ndhF (2), rpl2 (2), rpl20 (2), ycf1 (2), accD (1), ccsA (1), and rpl32 (1) (Table 1).
3.5.　 Phylogenetic analysis
Six phylogenetic trees were constructed using ML and BI methods based on complete CP genomes, 78 shared protein-coding genes, and 109 shared NCSs. The topologies of these trees were highly similar, with strong bootstrap support and PP values (Fig. 6; Fig. S5-S7, https://links.lww.com/STCM/A68). The 33 Murraya samples were divided into 2 main groups. Three species from sect. Murraya (M. exotica, M. paniculata, and M. alata) clustered together, whereas 5 species from sect. Bergera (M. euchrestifolia, M. kwangsiensis, M. kwangsiensis var. macrophylla, M. microphylla, and M. koenigii) formed a separate group. Within the sect. Murraya group, different populations of M. exotica and M. paniculata each formed monophyletic clades, with M. exotica being the sister lineage to M. alata. However, the genus Murraya was found to be nonmonophyletic, which is inconsistent with traditional taxonomy. The sect. Murraya group showed a closer relationship to M. caloxylon, C. maxima, and C. medica, while the sect. Bergera group was more closely related to C. excavata.
3.6.　 Divergence time estimation
The divergence between Aurantieae and Clauseneae was estimated at approximately 35.48 Mya (95% HPD: 30.17-39.82 Mya). The split between Bergera and Clausena occurred around 31.40 Mya (95% HPD: 25.55-36.67 Mya), while species within sect. Bergera diverged approximately 21.92 Mya (95% HPD: 15.57-28.15 Mya). Diversification of sect. Murraya species and M. caloxylon was estimated at 16.13 Mya (95% HPD: 9.55-23.43 Mya). The common ancestor of sect. Murraya species diverged around 9.11 Mya (95% HPD: 4.90-13.87 Mya), with the most recent divergence occurring between M. exotica and M. alata at approximately 5.85 Mya (95% HPD: 2.88-9.47 Mya) (Supplemental Fig. S8, https://links.lww.com/STCM/A68).
3.7.　 Molecular marker development
To develop a high-resolution molecular marker for distinguishing M. exotica and M. paniculata, the ycf1 region was selected, and specific primers were designed targeting conserved sequences (forward primer: 5'-TTTTCTATCTACCCTTACTATTCCA-3'; reverse primer: 5'-GTGGTACTAATCTAGCCCATTTA-3'). To verify the marker's effectiveness at the species level, the primers were used to amplify the ycf1 region in 6 taxa, including M. exotica, M. paniculata, M. alata, M. euchrestifolia, M. kwangsiensis var. macrophylla, and M. microphylla. The PCR products were approximately 450 bp and shared ≥90% sequence identity with their original sequences (Fig. 7; Supplemental Fig. S9, https://links.lww.com/STCM/A68). One species-specific single-nucleotide polymorphism (SNP) was identified in M. exotica, and 2 SNPs were identified in M. paniculata. These SNPs provide a reliable molecular basis for the identification of M. exotica and M. paniculata.
4.　Discussion
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4.1.　 Variations among the Murraya species
In this study, we assembled and analyzed the CP genomes of Murraya species. As in most angiosperms, all Murraya CP genomes exhibited a typical quadripartite circular structure, consisting of 2 SC regions and 2 IR regions.[44] Comparison of the 33 CP genomes revealed that their structures were relatively conserved, with only minor differences in genome size, composition, and gene order. The previously reported total length of M. koenigii was 159,402 bp,[45] while the CP genomes assembled in this study ranged from 158,573 to 160,817 bp, consistent with that of M. koenigii. Gene composition and arrangement are often related to evolutionary processes. All Murraya CP genomes contained 112 unique genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Similar to other angiosperms, chlB, chlL, chlN, and trnP-GGG were absent in the CP genomes of Murraya.[46] Additionally, the infA gene, a translation initiation factor, was lost in Murraya, as reported in some other Rutaceae species such as C. sinensis[47] and Z. bungeanum,[48] although it remains present in Camellia japonica.[49] This suggests that the loss of infA may have occurred independently within Rutaceae. Although the loss of ndh genes is widely reported in terrestrial plant CP genomes,[50,51] the Murraya CP genomes retained 10 complete ndh genes, except for a pseudogenized ndhD in some samples. Further investigation into the mechanisms and functional consequences of gene loss, pseudogenization, or transfer could provide valuable insights into the evolution of Murraya and the Rutaceae family.
Long repeat sequences and SSRs are widely distributed in CP genomes and can contribute to genome variation and rearrangement, significantly influencing species evolution.[52,53] These features have been extensively applied in population genetics and phylogenetic studies.[54,55] In the Murraya CP genomes, 4 types of long repeats were detected, including forward, reverse, complement, and palindromic repeats. Among these, palindromic repeats were the most abundant, and the majority of repeats were 30-39 bp in length, consistent with reports from other land plant CP genomes.[56,57] The types and distribution of SSRs were similar across Murraya species. A/T mononucleotide repeats were the most common, and other SSR types also exhibited high A/T content, reflecting the generally high A/T composition of CP genomes in most plants.[58] The abundance of SSRs varied among genomic regions, but, as in other Rutaceae species,[49] most were located in the LSC region. These long repeats and SSRs provide valuable information for assessing genetic diversity, evolutionary patterns, and phylogenetic relationships in Murraya, particularly for M. exotica and M. paniculata.
4.2.　 Expansion and contraction of IR regions in the Murraya species
Contraction and expansion of the IR regions in angiosperm CP genomes are common evolutionary events.[59,60] Even closely related species often exhibit slight differences in IR boundaries, and such contraction or expansion is an important factor affecting CP genome size.[61] In this study, the IR/SC boundaries of 8 Murraya species were analyzed. The results showed that, except for M. kwangsiensis var. macrophylla, the other 7 Murraya species had highly similar gene distributions at the IR/SC boundaries, resembling those of C. maxima. Significant differences were observed at the LSC/IR boundary of M. kwangsiensis var. macrophylla, where rps19 and rpl22 were entirely located in the LSC, and one copy of rps19 was lost, a pattern similar to that reported in Duabanga grandiflora.[62] Additionally, the ycf1 gene extended further into the IR region. In most monocots (except Alismatales and a few Acorales), the trnH-rps19 cluster is partially or completely located in the IR regions, whereas in dicots it is typically situated in the LSC region.[63] Interestingly, in Elaeagnaceae (eg, Elaeagnus and Shephedia) and Rhamnaceae (Rhamnus), duplication of the trnH gene has been reported.[64] In contrast to other Murraya species, M. kwangsiensis contains 2 copies of the trnH gene in the LSC region, positioned 5-6 bp away from the IR region. Further studies are warranted to elucidate the evolutionary significance of IR region contraction and expansion in Murraya.
4.3.　 DNA markers from the comparative analysis
Comparative analysis of the CP genomes showed that the Murraya species had highly similar genomes with conserved structures and no obvious structural rearrangements. As in most angiosperms, the IR regions were more conserved than the SC regions,[65] while the SSC regions exhibited the highest sequence divergence among the Murraya species. Moreover, coding regions were more conserved than noncoding regions.[66]Sequence divergence analysis indicated that the 8 Murraya species could be divided into 2 groups, consistent with the traditional classification. M. paniculata and M. alata showed higher sequence similarity to M. exotica, whereas M. euchrestifolia, M. kwangsiensis, M. kwangsiensis var. macrophylla, M. microphylla, and M. koenigii exhibited higher divergence relative to M. exotica. Several highly divergent regions were identified within the Murraya CP genomes. In particular, the intergenic region trnH-psbA showed a high degree of divergence, consistent with its reported utility as a candidate DNA barcode in grasses and legumes from temperate sub-alpine grasslands.[67] Additionally, the coding region ycf1 exhibited substantial variation. For land plants, ycf1 has been recognized as one of the most promising plastid DNA barcodes, generally outperforming matK, rbcL, and trnH-psbA at the species level.[68] In this study, ycf1 was demonstrated to be effective for the accurate identification of M. exotica and M. paniculata, both of which are used for anti-inflammatory purposes. Several specific SNPs were also observed in other Murraya species, though additional samples are required for further validation. The molecular marker developed here will facilitate the identification of M. exotica and M. paniculata, supporting the efficient utilization and conservation of wild resources. Future research on other highly variable regions may yield additional DNA markers for Murraya identification and phylogenetic studies.
4.4.　 Adaptive evolution of Murraya species
The ratio (ω = dN/dS) is widely used to assess selective pressure,[69] where ω > 1, ω = 1, and ω < 1 indicate positive, neutral, and purifying selection, respectively. In this study, most protein-coding genes of Murraya had ω values below 1, indicating purifying selection. However, 10 genes were identified as undergoing positive selection. Similar findings have been reported in other angiosperms,[70-72] suggesting that genes under positive selection play crucial roles in CP biogenesis and function. The accD gene encodes the β-carboxyl transferase subunit of acetyl-CoA carboxylase, which is involved in maintaining plastid compartments.[73] The ccsA gene encodes a c-type cytochrome synthesis protein, functioning in the delivery of heme from the stroma to the lumen and catalyzing heme ligation reactions.[74]NdhD and ndhF encode subunits of NADH-plastoquinone oxidoreductase, which mediate cyclic electron transport in photosystem I by forming a supercomplex with the photosystem I.[75] The psaJ gene encodes a photo-system I subunit necessary for the stability and optimal excitation of photosystem I.[76] The rbcL gene encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, which is essential for CO2 fixation in plants.[77] Notably, rbcL contained the highest number of positively selected amino acid sites (4) among the Murraya species, indicating a potential role in their adaptive evolution. The rpl2, rpl20, and rpl32 genes encode ribosomal proteins, which are essential for maintaining ribosome integrity.[78] Finally, the ycf1 gene encodes Tic214, a protein crucial for CP protein import in green tissues.[79] Given that Murraya species are predominantly distributed in warm, high-light environments, positive selection on these genes may contribute to their adaptation to such conditions.
4.5.　 Phylogenetic inference and divergence time of the Murraya species
CP genomes exhibit highly conserved structure and gene composition, with variable nucleotide substitution rates across different regions, making them highly suitable for phylogenetic analysis at multiple taxonomic levels and providing significant advantages in plant phylogenetic studies.[80] In contrast to traditional classifications of Murraya, the monophyletic groups of sect. Murraya and sect. Bergera did not cluster into a single clade. Consistent with previous molecular studies, sect. Murraya was monophyletic and sister to M. caloxylon, whereas sect. Bergera was monophyletic and sister to C. excavata.[18-20,81] Phylogenetic analysis based on the pangenome also supported the close relationship between sect. Bergera and Clausena.[82] This taxonomic arrangement is further supported by chemotaxonomic evidence.[83,84] Sect. Murraya and sect. Bergera produce distinct alkaloids, namely, yuehchukene in one group and girinimbine in the other, and differ in volatile oil composition. Notably, yuehchukene has been isolated from the stem and root barks of M. caloxylon, reinforcing the close relationship between M. caloxylon and sect. Murraya.[85] Collectively, evidence from morphotaxonomy, chemotaxonomy, and molecular phylogeny supports a generic-level division within Murraya rather than a sectional classification.[86] Accordingly, Merrillia and sect. Murraya should be placed in the tribe Aurantieae, while sect. Bergera should be assigned to the tribe Clauseneae. Species including M. euchrestifolia, M. kwangsiensis, M. kwangsiensis var. macrophylla, M. microphylla, and M. koenigii should be transferred to Bergera. Furthermore, the phylogenetic trees confirmed that M. exotica and M. paniculata are distinct taxa,[21] with M. exotica showing a closer relationship to M. alata than to M. paniculata. These results provide robust phylogenetic evidence supporting the recognition of M. exotica and M. paniculata as independent species and contribute to resolving longstanding taxonomic confusion within the genus. However, the CP genome and nuclear genome data for Murraya and other Rutaceae species remain limited. Additional genomic data will be necessary to further elucidate the evolution and phylogenetic relationships within Murraya and the broader Rutaceae family.
Based on the complete CP genome, we estimated the divergence times of Murraya species. The most recent common ancestor of sect. Murraya and Bergera was estimated to have existed approximately 35.48 Mya (95% HPD: 30.17-39.82 Mya), which is earlier than the 22.76 Mya estimated by Shi et al.[87] In contrast, Nguyen et al., using ITS sequences of Rutaceae, estimated the divergence of sect. Murraya and Bergera at 66.9 Mya.[19] These discrepancies likely reflect differences in molecular data selection, taxon sampling, fossil calibrations, and analytical methods. Yang et al., using 4995 SC gene families, estimated the diversification of M. paniculata and other Aurantieae species to be approximately 25 Mya,[88] which is close to our estimate of 28.01 Mya (95% HPD: 21.00-34.70 Mya). Our estimate for the divergence of M. exotica and M. paniculata (9.11 Mya, 95% HPD: 4.90-13.87 Mya) was also consistent with the previous estimate of 5.86 Mya.[87] These divergence time estimations based on complete CP genomes provide valuable insights into the evolutionary history of Murraya. Nevertheless, further validation using whole-genome data is warranted.
5.　Conclusion
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In this study, we analyzed the complete CP genomes of 33 samples representing 8 Murraya species, 5 of which were reported for the first time. The CP genomes are highly conserved, exhibiting the typical quadripartite structure with similar genome size, gene content, and gene order. We identified long repeat sequences and SSRs, and observed that contraction and expansion of the IR regions were associated with genome size, gene duplication, and intron occurrence. Comparative analyses revealed highly variable regions, including ndhI-ndhA, trnH-GUG-psbA, rpl32-trnL, matK, and ycf1. Ten protein-coding genes (rbcL, psaJ, ndhD, ndhF, rpl2, rpl20, ycf1, accD, ccsA, and rpl32) showed evidence of positive selection. Phylogenetic analyses and divergence time estimations based on CP genomes supported the classification of M. exotica and M. paniculata as 2 distinct species. Additionally, a molecular marker based on ycf1 was developed for the accurate identification of these 2 species.
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doi: 10.1097/st9.0000000000000091
  • Receive Date：2025-06-13
  • Online Date：2026-06-25
  • Published：2026-03-25
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  • Received：2025-06-13
  • Accepted：2025-09-08
Affiliations
    aState Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
    bExperimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China

Corresponding:

* Yuan Yuan and, China Academy of Chinese Medical Sciences, No 16 Nanxiao street, Dongzhimen, Dongcheng District, Beijing 100700, China. E-mail address: (Y. Yuan),
Chao Jiang, China Academy of Chinese Medical Sciences, No 16 Nanxiao street, Dongzhimen, Dongcheng District, Beijing 100700, China. (C. Jiang).
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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