1. Introduction
The chloroplast genome serves as a powerful tool for evolutionary studies, offering advantages through its maternal inheritance, structural conservation, and moderate mutation rate [1,2]. Most angiosperm chloroplast genomes display a quadripartite structure, comprising a large single copy (LSC), small single copy (SSC), and two inverted repeat (IR) regions [3,4]. However, exceptions have been reported, such as the loss of inverted repeats (IRs), which may influence genome stability and evolutionary dynamics [5]. The advent of high-throughput sequencing technologies have enabled comprehensive chloroplast genome analyses, facilitating phylogenetic reconstruction, hybrid identification, and molecular marker developments [6,7,8].
Rhododendron L. (Ericaceae), one of the largest angiosperm genera with over 1000 species globally, holds significant horticultural value. China represents its primary diversity center, comprising approximately 571 species (409 endemic) [9,10]. This genus includes economically important species prized for ornamental and medicinal applications [11,12] while also playing crucial roles in ecosystem stability and biodiversity conservation [13,14]. However, taxonomic uncertainties persist due to extensive hybridization, recent adaptive radiation, morphological plasticity, and limited genomic data [15,16,17,18]. Traditional morphology-based classifications often prove inadequate, necessitating molecular approaches for robust phylogenetic resolution [19,20].
Rhododendron purdomii Rehder & E. H. Wilson, an evergreen shrub or small tree species primarily distributed in the Qinling-Daba Mountains of Central China, is threatened by climate change and anthropogenic activities [21,22]. As a medicinal plant, the aerial parts of Rh. purdomii contain terpenoids, fatty acids, brassins, and phenolic acids, which have anti-inflammatory and antitussive effects [23]. Listed as “Vulnerable” in China’s Higher Plant Red List [24], this ecologically and horticulturally significant species remains genomically understudied, with prior research limited to morphology, microsatellite marker development, and species distribution modeling [21,22,25,26]. As a member of the taxonomically complex subgenus Hymenanthes (>300 species), Rh. purdomii exhibits unresolved subsection-level classification, variably assigned to Taliensia, Selensia, or Pontica within Rh. subg. Hymenanthes by different authorities [9,16,17,27,28]. The absence of complete chloroplast genome data impedes phylogenetic placement and evolutionary studies within this challenging group.
This study presents the sequencing and characterization of the entire chloroplast genome of Rh. purdomii to (1) analyze its structural features, gene composition, and evolutionary patterns; (2) compare it with other Rhododendron species to identify genomic variations; and (3) reconstruct phylogenetic relationships to resolve the taxonomic placement of Rh. purdomii. Our findings provide critical genomic resources for Rh. purdomii and elucidate the understanding of chloroplast genome evolution within the genus Rhododendron.
2. Materials and Methods
2.1. Plant Material, DNA Extraction, and Sequencing
Fresh leaves of Rh. purdomii were sampled from Xi’an Zhuque National Forest Park, Shaanxi Province, China (33°49′18.11″ N, 108°36′34.08″ E, 2425 m; Figure 1). Voucher specimens were deposited at the Zhengzhou University Herbarium (ZZU, established in 2006, Zhengzhou, China; accession number: LY2021052815). Genomic DNA was isolated via a cetyltrimethylammonium bromide (CTAB) method [29]. The extracted genomic DNA was subjected to the following quality control tests: 1. Visual inspection of extracted DNA samples to check for the presence of particulate matter or impurities; 2. Agarose gel electrophoresis to assess DNA degradation; 3. Nanodrop spectrophotometry to evaluate DNA purity (A260/A280: 1.8–2.0, A260/A230: >2.0); 4. Qubit fluorometric quantification to ensure a total DNA quantity ≥ 1 μg. High-quality DNA was utilized for library preparation and sequencing. Paired-end sequencing (150 bp) was performed on the Illumina NovaSeq platform (Illumina, San Diego, CA, USA), while long-read sequencing was conducted on the Oxford Nanopore PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK). All sequencing workflows were executed by Wuhan Benagen Technology Co., Ltd. (Wuhan, China).
2.2. Assembly and Annotation of Chloroplast Genome
After quality control of sequencing data, the chloroplast genome was assembled using Flye v2.8.3 [30] with default parameters. Gaps in the assembly were resolved using GapCloser v1.12 [31]. Genome annotation was performed using CPGAVAS2 (
2.3. Data Analysis
2.3.1. Repeat Sequence Analysis
Genomic repeats were identified using REPuter (
2.3.2. Comparative Analysis of the Chloroplast Genome
For comparative genomics analyses, we used ten complete chloroplast genomes from the Rhododendron subgenus Hymenanthes: Rh. purdomii (NC_086505.1), Rh. henanense subsp. Lingbaoense Fang (MT239363), Rh. williamsianum Rehder & E. H. Wilson (PP690644), Rh. griersonianum Balf. f. & Forrest (MT533181), Rh. calophytum Franch (OM373082), Rh. platypodum Diels (MT985162), Rh. oreodoxa var. fargesii (Franch.) D. F. Chamb (OL639014), Rh. przewalskii Maxim (OL871190), Rh. delavayi Franch (MN413198), and Rh. shanii Fang (MW374796) (Supplementary Table S1). Multiple sequence alignment was generated using MAFFT within the PhyloSuite v1.2.2 platform [39]. Nucleotide diversity (Pi) was estimated via sliding-window analysis (window length: 600 bp; step size: 200 bp) in DnaSP v6.12 [40]. Genomic sequence divergence was visualized using mVISTA (
2.3.3. Phylogenetic Analysis
To resolve the phylogenetic position of Rh. purdomii within Ericaceae, we constructed a phylogenetic tree using the chloroplast genomes of newly annotated Rh. purdomii and the other 26 Rhododendron species retrieved from the National Center for Biotechnology Information (NCBI,
3. Results
3.1. Chloroplast Genome Structure
We obtained 221,648 reads with an average length of 9385.48 bp for long-read sequencing and 34,235,862 reads for paired-end sequencing (150 bp). The complete chloroplast genome of Rh. purdomii (208,062 bp) exhibited a canonical quadripartite structure, featuring a LSC region of 110,618 bp, an SSC region of 2606 bp, and two IR regions of 47,419 bp each (Figure 2 and Table 1). The average coverage depth of the final assembly was 178.62×.
The overall GC content was 35.81%, with regional values of 35.27% (LSC), 30.05% (SSC), and 36.59% (IR) (Table 1). The genome contained 146 functional genes, consisting of 96 protein-coding, 42 tRNA, and 8 rRNA genes (Table 1). Of these, 113 genes were unique. Thirteen genes contained single introns, while two genes (rps12 and ycf3) contained two introns (Table 2). Notably, rps12 exhibited trans-splicing with three exons. Additionally, two putative pseudogenes (petA and ndhH) were identified.
The Rh. purdomii chloroplast genome contained 67,354 codons, with UUU being the most frequent codon and CGC the least frequent (Figure 3). The most abundant amino acid was leucine (Leu), while tryptophan (Trp) was the least abundant one. Among 30 codons with an RSCU value ≥ 1, AGA exhibited the highest RSCU, whereas CGC showed the lowest preference (Figure 3).
3.2. Repeat Sequences
A total of 96 long repeats were identified in the Rh. purdomii cp genome, comprising 46 tandem repeats (48%), 32 forward repeats (33%), and 18 palindromic repeats (19%) (Supplementary Figure S1). Tandem repeats were generally shorter than 60 bp, whereas forward and palindromic repeats typically exceeded 89 bp in length. A total of 171 SSRs were detected, comprising 69 mononucleotides, 18 dinucleotides, 66 trinucleotides, 16 tetranucleotides, one pentanucleotide, and one hexanucleotide (Supplementary Figure S2a). Among these, A (mononucleotide) and TA (dinucleotide) repeats were the dominant repeat types. In addition, ten (A) repeats and four (GAA) repeats were the most abundant in mononucleotides and trinucleotides (Supplementary Figure S2a). SSR distribution analysis revealed that 144 SSRs (84%) were located in intergenic regions, 16 (9%) in protein-coding regions, and 11 (7%) in intronic regions (Supplementary Figure S2b,c).
3.3. Genome Comparison and Nucleotide Diversity
Among the ten Rhododendron species analyzed, chloroplast genomes were largely conserved, with sequence divergence highest in the LSC region and then the IR regions (Figure 4). Comparative analyses identified hypervariable coding regions (accD, rps9) and non-coding regions (trnK-UUU-ycf3, trnI-CAU-rpoB, trnT-GGU-accD, rpoA-psbL, rpl20-trnC-GCA, trnI-CAU-rrn16, and trnI-CAU-rps16), which constitute candidate molecular markers for species identification within Rhododendron.
Comparative analysis of IR boundaries revealed species-specific junction patterns (Figure 5). The LSC/IRb boundary in Rh. shanii, Rh. Griersonianum, and Rh. purdomii was positioned between ycf15 and trnR. The SSC/IRb boundary consistently retained the complete ndhF gene within the SSC region, occupying identical positions in Rh. williamsianum, Rh. shanii, Rh. przewalskii, Rh. griersonianum, Rh. henanense subsp. lingbaoense, Rh. delavayi, and Rh. purdomii. The SSC/IRa junction exhibited expansion into the IRa region by 37−316 bp beyond ndhF. Most species displayed the LSC/IRa junction 128 bp downstream of trnH, with exceptions observed in Rh. przewalskii, Rh. henanense subsp. lingbaoense, and Rh. delavayi.
Nucleotide diversity (Pi) across the chloroplast genomes exhibited substantial regional variation (range: 0 to 0.23452), with a mean value of 0.00690 (Figure 6). Three regions with high nucleotide diversity (Pi value higher than 0.045) were identified in the LSC region of trnI-CAU-rpoB, the IR region of trnM-CAU-trnI-CAU, and the SSC region of ndhF. Notably, ndhF exhibited the highest nucleotide diversity, highlighting its potential as a molecular marker for population genetic studies in Rhododendron.
3.4. Phylogenetic Relationship
Phylogenetic reconstruction confirmed the monophyly of Rhododendron, with all 27 examined species forming a highly supported clade (Figure 7). However, the topological structure within Rhododendron did not fully align with traditional taxonomic classifications at the subgenus level [9]. While subgenera Hymenanthes and Azaleastrum were supported as monophyletic, subgenera Tsutsusi and Rhododendron were not. The ten species within subgenus Hymenanthes formed a well-supported monophyletic group and were resolved as sister clades to other subgenera (Azaleastrum, Tsutsusi, Choniastrum, and Pentanthera). Within Hymenanthes, the three species of subsection Fortunea (Rh. calophytum, Rh. platypodum, and Rh. oreodoxa var. fargesii) were clustered into a single clade (Supplementary Table S1). In contrast, subsection Taliensia (Hymenanthes) was not monophyletic. Notably, Rh. purdomii formed a sister clade with Rh. henanense subsp. lingbaoense. Among subgenus Rhododendron, the three species in subsection Lapponica (Rh. capitatum, Rh. nivale, and Rh. thymifolium) were clustered into one clade, while the two species of subsection Triflora (Rh. ambiguum and Rh. concinnum) formed a distinct clade (Supplementary Table S1 and Figure 7).
4. Discussion
In the present study, the chloroplast genome of Rh. purdomii was determined to be 208,062 bp in size, similar to other Rhododendron species, including Rh. henanense subsp. lingbaoense (208,015 bp) [45], Rh. griersonianum (206,467 bp) [46], Rh. delavayi (202,169 bp) [47], and Rh. oreodoxa var. fargesii (200,787 bp) [48]. Furthermore, most Rhododendron chloroplast genomes exhibit conserved quadripartite structures, demonstrating conservation in genome size, gene number, and GC content. Introns, which play an important role in gene expression and regulation [49], were identified in the Rh. purdomii chloroplast genome. Specifically, ycf3 and rps12 genes each contain two introns. This intron configuration aligns with observations in the chloroplast genome of Rh. oreodoxa var. fargesii [48], indicating evolutionary structural conservation in intron organization among these species.
Codon usage bias, a critical evolutionary feature of chloroplast genomes, arises from the interplay of natural selection and mutation [50]. In the Rh. purdomii chloroplast genome, codons encoding leucine were the most frequent, while tryptophan codons were the least frequent. Notably, 27 of the 30 codons with an RSCU index ≥ 1 terminated with A or U, mirroring patterns observed in other Rhododendron species [51,52]. This underscores a pronounced preference for A/U-ending codons, likely shaped by evolutionary pressures such as selection, mutation, and gene regulation. Such codon usage patterns provide insights into gene expression mechanisms and molecular evolution [53].
The chloroplast genome of Rh. purdomii displayed the highest abundance of tandem repeats, with most repeats measuring less than 60 bp in length. These repetitive sequences are known to facilitate genome recombination and structural rearrangements in chloroplast genomes [54]. SSRs represent valuable molecular markers for both basic and applied botanical research [55]. Our analysis identified 171 SSRs in the Rh. purdomii chloroplast genome, predominantly distributed in intergenic regions. This count is comparatively lower than those reported for congeneric Rhododendron species Rh. farrerae (270 SSRs) [56] and Rh. × pulchrum (221 SSRs) [51], highlighting the genetic complexity within this plant family. Chloroplast-derived SSRs (cpSSRs) have proven effective as polymorphic markers for interspecific and intraspecific differentiation at population scales [57]. The SSRs and repetitive sequences identified in this study establish a foundation for future studies on population genetics and evolutionary patterns within Rhododendron species.
The evolutionary dynamics of IR regions, particularly their expansion and contraction, are fundamental to chloroplast genome evolution, directly influencing genome size and gene organization [58,59]. These boundary regions (LSC/IRa/SSC/IRb) generally exhibit high conservation of gene content within genera, with only minor shifts in IR borders reported across closely related species [60,61]. Boundary comparisons across Rhododendron chloroplast genomes revealed distinct positional variations, including differential base positions and copy numbers for key genes (ycf15, trnR, ndhF, and trnH) at the IR-LSC and IR-SSC junctions.
Sequence comparisons using the mVISTA method demonstrated high similarity in coding regions among the nine examined chloroplast genomes, with most structural divergence localized to non-coding sequences in the LSC region. This pattern aligns with broader trends observed in angiosperm chloroplast genomes [62,63]. Nucleotide diversity (Pi) analysis across ten Rhododendron species identified three highly variable regions, including the ndhF region in the SSC compartment, which displayed the highest polymorphism. Similar variability hotspots have been documented in Chenopodium album [64] and Utricularia amethystine [65], suggesting their potential utility as molecular markers for phylogeographic research and population genetic analyses.
As a species-rich genus with persistent taxonomic challenges, Rhododendron exhibits complex and contentious classification schemes and phylogenetic relationships [17]. Previous phylogenetic investigations have employed molecular markers including nuclear internal transcribed spacers (ITSs), chloroplast DNA (cpDNA) fragments, and SSRs [66,67], yet these approaches yielded insufficient resolution for robust phylogenetic reconstruction. Recent advances in chloroplast genome sequencing have demonstrated superior utility in resolving taxonomic uncertainties among closely related taxa [6,7]. Our chloroplast genome-based phylogeny aligns closely with traditional taxonomic frameworks for Rhododendron [11,28] (Supplementary Table S1). Despite limited taxon sampling, two of the six recognized subgenera of Rhododendron were supported as monophyletic. The ten species within subgenus Hymenanthes formed a strongly supported clade, consistent with prior phylogenetic analyses [16,17]. Our findings validate the taxonomic elevation of former sections Azaleatrum and Choniastrtum (previously classified under subgenus Azaleastrum) to the subgeneric rank [20,28,68]. Rh. × pulchrum is considered as a horticultural cultivar of “Omurasaki” and Hirado azalea cultivars with its putative parents being Rh. scabrum, Rh. ripense, Rh. × mucronatum, and other related cultivars [69,70]. This species’ hybrid origin may account for the non-monophyly of subg. Rhododendron in this study. At the subsection level, monophyly was confirmed for subsection Fortunea (subgenus Hymenanthes) and subsections Lapponica and Triflora (subgenus Rhododendron). These results underscore the efficacy of the chloroplast genome for elucidating phylogenetic relationships within this large genus. Notably, subgenus Tsutsusi was not supported as monophyletic, and subsection Taliensia (subgenus Hymenanthes) was also not monophyletic. These inconsistencies align with prior reports [20,71], emphasizing the need for expanded sampling and multilocus approaches to refine lower-level taxonomy. Interestingly, Rh. purdomii formed a sister relationship with Rh. henanense subsp. lingbaoense, warranting denser taxon sampling to clarify its phylogenetic position within subgenus Hymenanthes. Given the very limited number of species we used, our study only provides a preliminary exploration of the phylogenetic relationships within the genus Rhododendron. Meanwhile, it should be noted that molecular techniques alone are not sufficient, and further studies should be conducted in combination with the use of morphological, phenological, and phytochemical markers.
5. Conclusions
This study presents the first complete chloroplast genome assembly of the threatened horticultural and medicinal species Rh. purdomii. The circular genome spans 208,062 bp, exhibiting a typical quadripartite structure and encoding 146 functional genes: 96 protein-coding genes, 42 tRNA genes, and 8 rRNA genes. Structural characterization identified 96 long repetitive sequences and 171 SSRs. Comparative analysis revealed two highly variable coding regions (accD, rps9) and seven polymorphic intergenic spacers (trnK-UUU-ycf3, trnI-CAU-rpoB, trnT-GGU-accD, rpoA-psbL, rpl20-trnC-GCA, trnI-CAU-rrn16, and trnI-CAU-rps16), which represent promising molecular markers for species authentication and population studies. Phylogenetic reconstruction using whole chloroplast genomes strongly supports the utility of these data for resolving complex relationships within Rhododendron. Notably, Rh. purdomii forms a sister clade with Rh. henanense subsp. lingbaoense, underscoring their close evolutionary affinity. In conclusion, this study provides critical genomic resources that advance systematic studies and clarify some taxonomic uncertainties in the genus Rhododendron. The identified molecular markers and phylogenetic insights will inform conservation strategies and sustainable utilization of Rh. purdomii and its congeners.
Conceptualization, N.Z. and Y.L.; methodology, L.Y.; validation, N.Z., Y.L. and S.Z.; formal analysis, L.Y.; investigation, Y.L.; resources, Y.L.; writing—original draft preparation, L.Y.; writing—review and editing, Y.L. and N.Z.; supervision, S.Z.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.
Data are contained within the article and
We wish to thank Peiliang Liu, Shuai Li, and Hao Dong for their help during the field survey. This work was partly supported by the Supercomputing Center in Zhengzhou University (Zhengzhou).
The authors declare no conflicts of interest.
Footnotes
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Figure 1 The morphology of Rhododendron purdomii Rehder & E. H. Wilson. (a,b) Plant and habitat, (c) inflorescence, (d) branch, (e) leaf, (f) capsular fruit, (g) seedling, (h,i) flower bud.
Figure 2 Circular visualization of Rh. purdomii Rehder & E. H. Wilson chloroplast genome. Genes are color-coded by functional category, with arrows indicating transcriptional direction. GC content is represented by darker gray, while lighter gray reflects AT content.
Figure 3 Codon content and codon usage of 20 amino acids and stop codons of all protein-coding genes of Rh. purdomii Rehder & E. H. Wilson.
Figure 4 Comparative complete chloroplast genome architectures of ten Rhododendron species. The VISTA-based identity plots were visualized through mVISTA in Shuffle-LAGAN.
Figure 5 Analysis of expansion and contraction of inverted repeats in the ten Rhododendron chloroplast genomes.
Figure 6 Nucleotide diversity analysis across ten Rhododendron chloroplast genomes. Sliding window analysis with genomic position on the X-axis and Pi values on the Y-axis.
Figure 7 Maximum likelihood (ML) phylogenetic tree of Ericaceae based on 29 complete chloroplast genomes. Numbers at the nodes represent bootstrap support values.
Features of the Rh. purdomii Rehder & E. H. Wilson complete chloroplast genome.
| Genome Features | Rh. purdomii |
|---|---|
| Genome size (bp)/GC content (%) | 208,062/35.81 |
| LSC size (bp)/GC content (%) | 110,618/35.27 |
| SSC size (bp)/GC content (%) | 2606/30.05 |
| IR size (bp)/GC content (%) | 47,419/36.59 |
| Total gene number | 146 |
| Protein-coding genes | 96 |
| tRNAs | 42 |
| rRNAs | 8 |
LSC, large single copy region; SSC, small single copy region; IR, inverted repeat.
Genes present in the Rh. purdomii Rehder & E. H. Wilson chloroplast genome.
| Category of Genes | Group of Genes | Name of Genes | ||||
|---|---|---|---|---|---|---|
| Photosynthesis-related genes | photosystem I | psaA | psaB | psaC | psaI | psaJ |
| photosystem II | psbA | psbB | psbC | psbD | psbE | |
| psbF | psbI | psbJ | psbK | psbL | ||
| psbM | psbN | psbT | psbZ | psbH | ||
| NADH dehydrogenase | ndhA * | ndhB * | ndhC | ndhD | ndhE | |
| ndhF | ndhG | ndhI | ndhJ | ndhK | ||
| ndhH | ||||||
| ATP synthase | atpA | atpB | atpE | atpF * | atpH | |
| rubisco | rbcL | |||||
| cytochrome b/f complex | petB * | petD * | petG | petL | petN | |
| petA | ||||||
| cytochrome c synthesis | ccsA | |||||
| Transcription- and translation-related genes | ribosome proteins | rpl2 | rpl14 | rpl16 * | rpl20 | rpl22 |
| transcription | rpoA | rpoB | rpoC1 | rpoC2 | ||
| translational initiation factor | infA | |||||
| RNA genes | ribosomal RNA | rrn4.5 | rrn5 | rrn16 | rrn23 | |
| transfer RNA | trnA-UGC * | trnC-GCA | trnD-GUC | trnE-UUC | trnF-GAA | |
| trnfM-CAU | trnG-GCC | trnG-UCC * | trnH-GUG | trnI-CAU | ||
| trnI-GAU * | trnK-UUU * | trnL-CAA | trnL-UAA * | trnL-UAG | ||
| trnM-CAU | trnN-GUU | trnP-UGG | trnQ-UUG | trnR-ACG | ||
| trnR-UCU | trnS-GCU | trnS-GGA | trnS-UGA | trnT-GGU | ||
| trnT-UGU | trnV-GAC | trnV-UAC * | trnW-CCA | trnY-GUA | ||
| Other genes | fatty acid synthesis | accD | ||||
| carbon metabolism | cemA | |||||
| RNA processing | matK | |||||
| Unknown | conserved reading frames | ycf1 | ycf3 ** | ycf4 | ycf15 | ycf68 |
* Gene containing a single intron, ** Gene containing two introns.
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1 School of Agricultural Sciences, Zhengzhou University, No. 100 Science Avenue, Zhengzhou 450001, China; [email protected]
2 School of Life Sciences, Zhengzhou University, No. 100 Science Avenue, Zhengzhou 450001, China; [email protected] (N.Z.); [email protected] (S.Z.), Henan Funiu Mountain Biological and Ecological Environment Observatory, Zhengzhou University, No. 100 Science Avenue, Zhengzhou 450001, China