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. 2026 May 27;16(6):e73728. doi: 10.1002/ece3.73728

Comparative Analysis of Complete Chloroplast Genomes of Caltha scaposa for Identification and Phylogenetic Analysis

Le Chen 1,2, Yongming Fan 3, Qihang Chen 4, Xiaonan Yu 2,✉, Tai Gao 1,2,✉
PMCID: PMC13239713  PMID: 42256135

ABSTRACT

The complete chloroplast genome of Caltha scaposa, a high‐altitude wetland species native to the Himalayas and Hengduan Mountains, was assembled and characterized to clarify its phylogenetic position and explore plastome structural variation within the tribe Anemoneae. The circular genome is 160,219,155,293 bp in length and exhibits a typical quadripartite structure, including a LSC (large single copy) region of 79,994 bp, an SSC (small single copy) region of 17,075 bp, and a pair of IRs (inverted repeats) of 26,410 bp each. The genome encodes 132 genes, comprising 87 protein‐coding genes, 37 tRNA genes, and four rRNA genes. A total of 54 SSRs (simple sequence repeats) were identified, predominantly mononucleotide A/T repeats. Comparative analysis with nine related species from the genera Anemone and Pulsatilla revealed significant variation in IR boundaries. Caltha scaposa possesses the shortest IR regions, while all examined Pulsatilla species exhibit expanded IRs, resulting in structural rearrangements. Nucleotide diversity analysis indicated that intergenic spacers, especially ndhE‐ndhG (Pi = 0.1195), are highly variable, whereas tRNA genes are extremely conserved. Codon usage analysis revealed a bias toward A/T‐ending codons. Phylogenomic reconstruction based on 40 complete plastomes using maximum likelihood and Bayesian methods robustly placed C. scaposa as a sister lineage to the clade comprising Anemone and Pulsatilla within tribe Anemoneae. These findings clarify the systematic position of C. scaposa , highlight dynamic IR evolution in Anemoneae, and provide valuable genomic resources for future evolutionary and population genetic studies in Ranunculaceae.

This study assembled and characterized the complete chloroplast genome of Caltha scaposa, revealing it has the shortest inverted repeat regions among examined Anemoneae taxa and identifying highly variable markers for future phylogenetic work. Phylogenomic analysis of 40 plastomes robustly resolves C. scaposa as the sister lineage to the Anemone–Pulsatilla clade, clarifying its systematic position within Ranunculaceae. These findings provide critical genomic resources for understanding plastome evolution and species diversification in high‐altitude wetland plants.

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1. Introduction

The plants chloroplast genome is a highly conserved circular DNA molecule, typically ranging from 120 to 160 kb in size, and generally exhibits a quadripartite structure comprising two inverted repeats (IRs) that separate the large and small single‐copy regions (Shaw et al. 2007). This structural conservation, together with moderate sequence variation and maternal inheritance in most angiosperms, makes the plastome a valuable tool for phylogenetic reconstruction, species identification, and evolutionary investigations across various taxonomic scales (Wicke et al. 2011). In recent years, rapid advancement of high‐throughput sequencing has greatly facilitated plastome assembly, allowing for comparative genomic studies across diverse plant lineages and revealing both conserved features and lineage‐specific structural rearrangements (Daniell et al. 2021).

Caltha is a small but widely distributed genus within the family Ranunculaceae (Wang et al. 2001). Caltha scapose (Figure 1), a species native to the high‐altitude wetlands of the Himalayas and the Hengduan Mountains, represents an ecologically distinctive member adapted to cold and moist environments (Zhang, Meng, et al. 2017). Although nuclear and morphological data have provided some insights into the systematics of Caltha (Zhang et al. 2017), its precise phylogenetic placement within Ranunculaceae, particularly its relationship with other early‐diverging genera such as Anemone and Pulsatilla, remains incompletely resolved due to limited molecular evidence.

FIGURE 1.

FIGURE 1

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Caltha scapose (The photos are sourced from iPlant).

Recent phylogenomic studies have increasingly incorporated complete plastomes to resolve both deep and shallow nodes within Ranunculaceae (Ji et al. 2023). Notably, members of the tribe Anemoneae, including Anemone, Pulsatilla, and allied genera, have been shown to exhibit dynamic plastome evolution, characterized by IR boundary shifts, gene losses, and inversions (Liu et al. 2018). These structural changes often coincide with speciation events and may reflect adaptive responses to environmental pressures. Comparative analyses employing tools such as mVISTA and IR boundary mapping have proven effective in identifying both conserved coding regions and hypervariable non‐coding spacers, which serve as potential phylogenetic markers or indicators of genomic instability.

In this context, the plastome of C. scaposa offers a critical opportunity to expand our understanding of plastid genome evolution in basal eudicots by assembling and characterizing its complete chloroplast genome and conducting comparative analyses with closely related taxa, including eight Pulsatilla species.

2. Materials and Methods

2.1. Plant Materials and DNA Extraction

Caltha scaposa was collected from Deqin County, Diqing Tibetan Autonomous Prefecture, Yunnan Province, China. Upon collection, the leaves were immediately stored at 4°C. No specific permissions were required for the collection of plant samples from this location. Voucher specimens are deposited at the Aquatic Plant Application and Germplasm Enhancement Laboratory of the Yellow River Basin, North China University of Water Resources and Electric Power, NCWU. Genomic DNA was isolated from fresh leaf material employing the CretMag Multi Sample DNA Kit (Suzhou Cretaceous Biotechnology Co. Ltd., Suzhou, China), which utilizes a magnetic bead‐based extraction method as described by Zhou et al. (2022). The extracted DNA was evaluated for quality and quantity: the A260/A280 ratio was 1.78 as assessed by NanoDrop spectrophotometry, the concentration was measured at 83.2 ng/μL via Qubit fluorometry, and agarose gel electrophoresis revealed distinct bands with no evidence of degradation.

2.2. Sequencing, Complete Genomes Assembly, and Annotation

Sequencing was performed on the Illumina HiSeq 4000 platform (Shanghai OE Biotech. Co. Ltd., Shanghai, China) with 150 bp paired‐end reads. FastQC v0.12.0 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed: December 1, 2025) was used for quality control of the raw data (Chen et al. 2018). The complete chloroplast genome was assembled using GetOrganelle v1.7.7.1 (https://github.com/Kinggerm/GetOrganelle, accessed December 21, 2025) (Jin et al. 2020), with the cp genome of Caltha palustris (NCBI accession: MG581742) serving as a reference. The results showed that the assembled chloroplast genome achieved an average coverage depth of 150X. Annotation of the C. scaposa chloroplast genome was performed using the online platform CPGAVAS2 (http://www.herbalgenomics.org/cpgavas/) (Shi et al. 2019), followed by manual curation using Geneious R11 (Biomatters Ltd., Auckland, New Zealand). The circular genome map was generated using OGDRAW v1.2 (http://ogdraw.mpimp‐golm.mpg.de/, accessed January 24, 2026) (Lohse et al. 2007).

2.3. Identification of Simple Sequence Repeats

Simple sequence repeats (SSRs) were detected using MISA (https://webblast.ipk‐gatersleben.de/misa/, accessed December 22, 2025) (Beier et al. 2017). The minimum number of repeats for SSR motifs of lengths 1 to 6 was set to 10, 6, 5, 5, 5, and 5, respectively.

2.4. Comparative Analysis of cp Genomes

Comparative analyses of chloroplast genomes, with Caltha scaposa as the reference against eight closely related species, were performed using mVISTA in Shuffle‐LAGAN mode (https://genome.lbl.gov/vista/mvista/submit.shtml, accessed December 23, 2025) (Frazer et al. 2004). The boundaries of the inverted repeat (IR) regions were visualized using IRscope (https://irscope.shinyapps.io/irapp/, accessed: December 24, 2025) (Amiryousefi et al. 2018), comparing C. scaposa with nine related taxa. Using CPStools v2.5, nucleotide diversity (Pi) was calculated (Huang et al. 2024). Based on previous phylogenetic studies and taxonomic considerations, nine species were selected for comparative analyses. Their chloroplast genome sequences were retrieved from the NCBI database, with the following accession numbers: Anemone taipaiensis (NC_050873.1), Anemone tomentosa (NC_039451.1), Pulsatilla campanella (NC_061038.1), Pulsatilla cernua (MK860687.1), Pulsatilla chinensis (MK569491.1), Pulsatilla dahurica (MK860685.1), Pulsatilla saxatilis (NC_070435.1), and Pulsatilla tongkangensis (NC_061398.1).

2.5. Codon Usage Analysis

Codon usage bias was assessed using CodonW v1.4.2 (https://sourceforge.net/projects/codonw/files/codonw/Win32‐Executables‐1.4.2/, accessed: December 252025), from which relative synonymous codon usage (RSCU) values were calculated (Sharp and Li 1987). Following standard criteria, an RSCU value of 1.0 indicates no bias in codon usage, whereas values exceeding 1.0 reflect a preference for particular codons.

2.6. Phylogenetic Analysis

Phylogenetic analysis was performed on 40 complete chloroplast genomes, which included the newly sequenced C. scaposa genome from this study and 39 cp genome sequences downloaded from the NCBI database. Paeonia obovata (accession number JQ952561.1) was designated as the outgroup. For the analysis, protein‐coding sequences (CDS) were extracted using PhyloSuite v1.2.3, then aligned with MAFFT v7.505 (Katoh et al. 2019). Poorly aligned regions and divergent sites were removed with trimAl v1.2 (Capella‐Gutiérrez et al. 2009), and protein‐coding genes were refined using MACSE v2.0.3 (Ranwez et al. 2018). A phylogenetic tree was reconstructed employing both maximum likelihood (ML) and Bayesian inference methods, with ML bootstrap support evaluated using IQ‐TREE based on 5000 replicates (Katoh et al. 2019; Nguyen et al. 2015).

3. Results

3.1. Cp Genome Features of C. scaposa

The chloroplast genome of C. scaposa contains a total of 132 genes (by copy number), including 87 protein‐coding genes, 37 tRNA genes, and 4 rRNA genes (Figure 2). These genes are classified into three functional categories: photosynthesis‐related, self‐replication‐related, and others (Table 1). Due to the quadripartite structure, 17 genes are duplicated in the inverted repeat (IRs) regions. These include several protein‐coding genes including rpl2, rpl23, rps7, rps19, ycf2, 8 rRNA gene copies, and multiple tRNA genes (trnI‐CAU, trnA‐UGC). Owing to copy‐dependent repair via gene conversion between the two IR regions, the sequences of these duplicated genes are maintained with perfect identity. A total of 18 intron‐containing genes were identified in the C. scaposa chloroplast genome, including 15 genes (ndhB, ndhA, petB, petD, atpF, rpl16, rpl2, rps16, rpoC1, trnK‐UUU, trnG‐UCC, trnL‐UAA, trnV‐UAC, trnI‐GAU, and trnA‐UGC) with a single intron, and 3 genes (rps12, clpP, and ycf3) with two introns each, accounting for a total of 21 introns. Notably, the rps12 gene undergoes trans‐splicing: its 5′ exon is located in the large single‐copy (LSC) region, whereas the 3′ exon resides in the IR region (Figure 2). The complete cp genome sequence of C. scaposa has been deposited in GenBank under accession number PZ091414.

FIGURE 2.

FIGURE 2

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The circular complete cp genome map of Caltha scaposa . Genes shown outside the outer circle are transcribed clockwise, whereas those shown inside are transcribed counterclockwise.

TABLE 1.

The basic cp. genomes information of Caltha scaposa .

Category Gene group Gene name
Photosynthesis Subunits of photosystem I psaB, psaA, psaI, psaJ, psaC
Subunits of photosystem II psbA, psbK, psbI, psbM, psbD, psbC, psbZ, psbJ, psbL, psbF, psbE, psbB, psbT, psbN, psbH
Subunits of NADH dehydrogenase ndhJ, ndhK, ndhC, ndhB (2) #, ndhF, ndhD, ndhE, ndhG, ndhI, ndhA#, ndhH
Subunits of cytochrome b/f complex petN, petA, petL, petG, petB#, petD#
Large subunit of rubisco rbcL
Subunits of ATP synthase atpA, atpF, atpH, atpI, atpE, atpB
Self‐replication Proteins of large ribosomal subunit rpl33, rpl20, rpl36, rpl14, rpl16#, rpl22, rpl2(2) #, rpl23(2), rpl32
Proteins of small ribosomal subunit rps12(2) ##, rps16#, rps2, rps14, rps4, rps18, rps11, rps8, rps3, rps19(2), rps7(2), rps15
Subunits of RNA polymerase rpoC2, rpoC1#, rpoB, rpoA
Ribosomal RNAs rrn16S (2), rrn23S (2), rrn4.5S (2), rrn5S (2)
Transfer RNAs trnH‐GUG, trnK‐UUU#, trnQ‐UUG, trnS‐GCU, trnG‐UCC#, trnR‐UCU, trnC‐GCA, trnD‐GUC, trnY‐GUA, trnE‐UUC, trnT‐GGU, trnS‐UGA, trnG‐GCC, trnfM‐CAU, trnS‐GGA, trnT‐UGU, trnL‐UAA#, trnF‐GAA, trnV‐UAC#, trnM‐CAU, trnW‐CCA, trnP‐UGG, trnI‐CAU(2), trnL‐CAA(2), trnV‐GAC(2), trnI‐GAU(2)#, trnA‐UGC(2)#, trnR‐ACG(2), trnN‐GUU(2), trnL‐UAG
Other genes Maturase matK
Protease clpP##
Envelope membrane protein cemA
Acetyl‐CoA carboxylase accD
c‐type cytochrome synthesis gene ccsA
Conserved open reading frames ycf3##, ycf4, ycf2(2), ycf1(3)
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Note: #, intron number; (n), gene copy number.

3.2. Analysis of SSRs

A total of 54 SSR loci were identified in the chloroplast genome of C. scaposa . Among them, 48 were mononucleotide repeats (A/T or C/G) and 6 were dinucleotide repeats (AT/AT). No tri‐ or higher‐order nucleotide repeats were detected. Within the mononucleotide repeats, A/T repeats were predominant, accounting for 47 loci, while only one C/G repeat was identified. The predominance of A/T repeats is consistent with the general characteristic of plant chloroplast genomes being A/T‐rich. The detailed distribution of all SSRs is shown in Table 2.

TABLE 2.

Number of Caltha scaposa SSRs.

Repeats 5 6 7 8 9 10 11 12 13 14 Total
A/T 0 0 0 0 0 26 15 2 1 3 47
C/G 0 0 0 0 0 1 1
AT/AT 0 5 1 6
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3.3. Comparative Analysis of Genome Structure in C. scaposa and Its Related Species

Caltha scaposa has the shortest IR regions, measuring 26,410 bp, whereas the two Anemone species have IR lengths of approximately 28,500 bp (Figure 2). In contrast, all Pulsatilla species possess significantly longer IRs, ranging from 31,117 to 31,293 bp. In both Caltha and Anemone, the rps19 gene is entirely located within the LSC region. However, in Pulsatilla species, the IRa region has expanded into the SSC region, resulting in truncation of the ndhF gene. The genes located at these boundaries include rps4, rps19, rpl36, ndhF, ycf1, and trnH genes. The position of the trnH gene also varies with the contraction or expansion of IRa, showing inconsistent distances from the IRa boundary across different species. The shifts in IR boundaries have directly altered the gene content in these junction regions. Notably, rps19 and ycf1 are the primary genes affected by these boundary movements. The ycf1 gene spans the SSC/IRb junction and, in all examined species, appears as an incomplete pseudogene within IRb, while its intact copy resides in the SSC region. The length of the intact ycf1 in the SSC varies slightly among species, reflecting the precise positions of the boundary shifts. The ndhF gene is located at the IRb/SSC boundary, with the larger part located in the SSC region. Due to asymmetric IR expansions, truncated pseudogene copies of ndhF and rps19 have formed in certain species (Figure 3).

FIGURE 3.

FIGURE 3

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Comparison of the LSC, SSC, and IR regions among 9 selected cp genomes in the family Caltha, Anemone, and Pulsatilla. The gaps between the genes and the boundaries are indicated by the base lengths. Genes are denoted by colored boxes. JLB, junction between LSC and IRb; JSB, junction between IRb and SSC; JLA, junction between IRa and LSC; JSA, junction between SSC and IRa.

The mVISTA genome alignment plot illustrates the sequence conservation and variable regions among the chloroplast genomes of C. scaposa and its close relatives, using C. scaposa as the reference (Figure 4). The complete chloroplast genomes of nine Ranunculaceae species, including C. scaposa , two Anemone species, and seven Pulsatilla species—exhibit overall high sequence conservation, yet display several distinct non‐conserved regions concentrated in specific genomic intervals, accompanied by evident structural variations and elevated sequence divergence in intergenic spacers. Notably, in the genomic region spanning approximately 130–140 kb, which encompasses genes such as ycf1, trnH‐GUG, and rps12, all Pulsatilla species show markedly reduced sequence similarity compared to the reference, while Caltha and Anemone retain high conservation in this interval. This region corresponds precisely to the SSC, IRb junction, and its low conservation aligns closely with the previously observed IR expansion and boundary shifts, indicating that it is a hotspot for genomic rearrangements and sequence diversification. Additionally, in the intervals around 80–90 kb and 110–120 kb, Pulsatilla species exhibit multiple localized insertions, deletions, or repetitive segments, whereas Caltha and Anemone display continuous high conservation across the same regions. Furthermore, intergenic spacers generally show lower sequence conservation, with the trnH‐psbA and trnL‐trnF regions being particularly divergent. These non‐coding regions thus represent key sources of sequence variation among the studied taxa.

FIGURE 4.

FIGURE 4

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Sequence alignment of 9 Anemone, Caltha, Pulsatilla cp. genomes using the mVISTA program with C. scaposa as a reference. x‐axis: The coordinates in the cp genome, y‐axis: Percent identity within 50%–100%. The transcriptional direction of genes indicated by gray arrows. Regions where the identity curve falls to the baseline (~50%) indicate sequences that are highly divergent or absent in the query genome. The gene names are indicated directly above the dark gray arrows.

3.4. Codon Usage Patterns in C. scaposa and Its Close Relatives Cp Genomes

Codon usage bias analysis of the C. scaposa chloroplast genome reveals significant differences in codon usage, indicating a pronounced codon preference (Figure 5). AAA (Lys) has the highest frequency at 41.234%, corresponding to 2171 occurrences. In contrast, AAG (Lys) is used much less frequently, at only 15.536%, suggesting that AAA is the predominant preferred codon for lysine. ATT (Ile) has a frequency of 41.139%, making it the most commonly used codon for isoleucine. GAA (Glu) shows a frequency of 38.024%, serving as the primary codon for glutamic acid. For arginine, AGA (17.322%) is the most frequently used, while CGC (3.305%) is the least used. For serine, TCT (20.664%) is the most frequent, whereas AGC (4.805%) is the least frequent. Most high‐frequency codons end with A or T, consistent with the general characteristic of plant chloroplast genomes favoring A/T‐ending codons. High‐frequency codons such as AAA, ATT, GAA, TTT, and AAT may represent optimal codons, potentially related to tRNA abundance or translation efficiency.

FIGURE 5.

FIGURE 5

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Codon usage in percentages (the right column) and RSCU values (the left column) of 20 amino acids. Each codon for an amino acid is shown with different colors.

3.5. Nucleotide Diversity Analysis

The Pi value range of the C. scaposa chloroplast genome is from 0.00000 to 0.119509(Figure 6). The region with the highest value is the intergenic spacer ndhE‐ndhG, with a value of 0.119509. A large number of regions show extremely low polymorphism, such as the tRNA genes trnE‐UUC, trnV‐GAC, and trnY‐GUA. Among the analyzed protein‐coding and RNA genes, matK (0.03554), accD (0.03089), and ndhF (0.045987) have relatively high Pi values, while rpl23 (0.00138) and rps12 (0.00179) exhibit extremely low Pi values. Almost all tRNA genes have a Pi value of 0.00000, indicating extreme sequence conservation. In general, intergenic regions show polymorphism values that are higher than or significantly higher than those in gene‐coding regions. Regions of high polymorphism are mainly concentrated in the intergenic spacers.

FIGURE 6.

FIGURE 6

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Sliding window analysis of cp genomes from 9 species. Nucleotide diversity (Pi) of intergenic regions in coding regions.

3.6. Phylogenetic Tree Construction and Analysis of Species Clusters

Based on the CDS region of the chloroplast genome and using Paeonia obovata as the outgroup, phylogenetic trees constructed by maximum likelihood and Bayesian inference methods clearly reconstruct the classical phylogenetic relationships within the core subfamilies of Ranunculaceae (Figure 7). All analyses strongly support (bootstrap/posterior probability 100/100) the formation of a monophyletic clade within Ranunculaceae, consisting of the tribes Aquilegieae and Thalictreae as sister groups. Notably, the genus Thalictrum exhibits multiple well‐supported species‐level branches, reflecting a potentially rapid history of species diversification within this genus. This clade, in turn, forms a sister group to another monophyletic clade comprising the tribes Trollieae and Adonideae. The relationships among species within these tribes are clearly resolved, with generally high branch support values. This topology receives nearly complete support in both trees (maximum likelihood bootstrap 100, Bayesian posterior probability 100), which is fully consistent with recent studies based on genomic data and resolves long‐standing controversies regarding the relationships among these tribes.

FIGURE 7.

FIGURE 7

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ML phylogenetic (left) and Bayesian tree (right) of 40 complete cp genomes. Bootstrap values and branch lengths are shown near each node. Different colors represent the differences in the clustering results.

Caltha scaposa is firmly nested within the tribe Anemoneae. It is not directly related to Thalictreae or Trollieae but is more closely related to the clade containing Pulsatilla and Anemone. Caltha scaposa is the sister group to the entire Pulsatilla and Anemone clade. This relationship receives 92% bootstrap support in the maximum likelihood tree and an even higher 93% posterior probability in the Bayesian tree. This strong supporting evidence indicates that after divergence from a common ancestor, one lineage evolved into the modern Pulsatilla and Anemone, while the other evolved into the Caltha lineage represented by C. scaposa (Odago et al. 2022; Raman et al. 2020). The divergence between C. scaposa and the most recent common ancestor of Pulsatilla and Anemone occurred earlier than the divergence between Pulsatilla and Anemone themselves. Paeonia obovata , used as the outgroup in the analysis, is consistently placed outside all core lineages of Ranunculaceae. It is extremely long branch length confirms the distant phylogenetic relationship and prolonged independent evolutionary history between Paeonia and the core Ranunculaceae lineages, while also highlighting the relatively closer relationships among the various groups within Ranunculaceae (Rui et al. 2025). The highly supported phylogenetic tree provides compelling molecular systematic evidence for the taxonomic delineation within Ranunculaceae, particularly among genera in the tribe Anemoneae. C. scaposa , as a clearly defined independent evolutionary branch, is positioned as a distinct lineage within the tribe Anemoneae of Ranunculaceae, forming a sister group relationship with the clade comprising Pulsatilla and Anemone. Its status at the genus level is fully confirmed. With the phylogenetic position of C. scaposa clarified, subsequent research can more precisely use this as a basis to conduct comparative genomics analyses, investigate species formation mechanisms, and estimate the divergence time between Caltha and the Pulsatilla‐Anemone lineage by integrating fossil evidence.

4. Discussion

As reported by Daniell et al. (2016), plant chloroplast genomes typically display a highly conserved quadripartite structure, comprising large single‐copy (LSC) and small single‐copy (SSC) regions separated by two inverted repeat (IR) regions. The complete chloroplast genome of C. scaposa provides crucial molecular evidence for resolving the phylogenetic position of the genus Caltha within the early‐diverging eudicot family Ranunculaceae. Our phylogenomic analyses, based on 40 complete plastomes, robustly place C. scaposa as the sister group to the clade comprising Anemone and Pulsatilla, with high bootstrap (92%) and posterior probability (93%) support. This finding strongly corroborates the monophyly of the tribe Anemoneae and clarifies that Caltha represents an independent evolutionary lineage that diverged prior to the split between Anemone and Pulsatilla. Although Liu et al. (2018) first proposed the sister relationship between Caltha and the (Anemone and Pulsatilla) clade, our study substantially advances this finding by employing 40 complete plastomes. Our results confirm that the Caltha lineage diverged prior to the split between Anemone and Pulsatilla.

Comparative structural analysis revealed significant variation in the inverted repeat (IR) boundaries among Caltha, Anemone, and Pulsatilla. Notably, C. scaposa possesses the shortest IR regions (26,410 bp), while all examined Pulsatilla species exhibit markedly expanded IRs (31,117 ~ 31,293 bp). This expansion in Pulsatilla has led to the truncation of the ndhF gene at the IRb/SSC junction, a structural rearrangement not observed in Caltha or Anemone. These lineage‐specific IR dynamics are consistent with a growing body of evidence in angiosperms showing that IR expansion/contraction is a major driver of plastome evolution (Hu et al. 2024). Specifically, our results go beyond Liu et al. (2018) by pinpointing IR expansion as the cause of ndhF truncation exclusively in Pulsatilla, which was not examined in their study. Furthermore, a recent study on the plastomes of the tribe Anemoneae (Hu et al. 2024) reported similar IR‐boundary shifts correlating with ndhF integrity, supporting our conclusion. Song et al. (2024) also found structural variation of the ndhF gene at IR boundaries in Delphinium and identified ndhF‐trnL and ycf1 as hypervariable molecular markers for phylogenetic studies. These cross‐generic comparative analyses suggest that IR boundary dynamics may be a common mechanism of plastome evolution in Ranunculaceae and even basal eudicots. The mVISTA alignment further highlighted that the genomic region around 130–140 Kb (encompassing ycf1, trnH, and rps12) is a hotspot for sequence divergence, particularly in Pulsatilla, which directly correlates with these dynamic IR boundary shifts. These findings underscore that IR boundary dynamics are a major driver of structural and sequence evolution within the Anemoneae tribe.

Nucleotide diversity (Pi) analysis identified the intergenic spacer ndhE‐ndhG as the most variable region (Pi = 0.1195), while tRNA genes were found to be extremely conserved (Pi≈0.0000). This pattern, where non‐coding regions harbor higher polymorphism than coding regions, is typical of plant plastomes (Bian et al. 2024) and highlights the utility of these hypervariable spacers as potential molecular markers for population genetics and species identification within Caltha and its relatives. Compared to Liu et al. (2018), who did not perform nucleotide diversity scanning, our Pi analysis provides the first quantitative assessment of variable hotspots in the Caltha plastome, directly enabling marker development for this genus. Furthermore, the codon usage bias in C. scaposa , characterized by a strong preference for A/T‐ending codons, aligns with the overall A/T‐rich nature of its plastome and may reflect optimization for translational efficiency (Ji et al. 2023). To place this finding in a broader evolutionary context, we compared codon usage patterns between Caltha and the genera Aconitum and Delphinium using recently published Ranunculaceae plastome data. The A/T bias in C. scaposa is highly consistent with that of the congeneric species C. palustris , indicating that A/T preference is a general feature of Ranunculaceae plastomes, a conclusion further supported by previous studies on the tribe Anemoneae (Hu et al. 2024). Additionally, a study on Ranunculus muricatus (Wan et al. 2025) also reported a similar A/T bias pattern, further confirming the conservation of A/T preference in Ranunculaceae.

In conclusion, the plastome of C. scaposa not only resolves its long‐debated systematic position but also reveals lineage‐specific genomic features that contribute to our understanding of plastid genome evolution in basal eudicots. The clear sister relationship between Caltha and the (Anemone and Pulsatilla) clade (Szczecińska and Sawicki 2015), coupled with distinct IR structures and sequence divergence patterns, provides a solid foundation for future studies on the tempo and mode of diversification in the Anemoneae tribe.

Author Contributions

Le Chen: conceptualization (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal). Yongming Fan: conceptualization (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal). Qihang Chen: conceptualization (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal). Xiaonan Yu: conceptualization (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal). Tai Gao: conceptualization (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal).

Funding

This research was funded by the China National Botanical Garden (Northern Garden), grant number T000002812929.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

We acknowledge Associate Research Professor Juntong Chen (Kunming Institute of Botany, Chinese Academy of Sciences) for his contribution in identifying the Caltha scaposa specimens.

Contributor Information

Xiaonan Yu, Email: yuxiaonan626@126.com.

Tai Gao, Email: gaotai36@outlook.com.

Data Availability Statement

The chloroplast genome sequence of Caltha scaposa has been submitted to NCBI (accession number: PZ091414).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The chloroplast genome sequence of Caltha scaposa has been submitted to NCBI (accession number: PZ091414).

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