Comparative plastid genomics and flow cytometry of five species of the genus Corydalis DC. (Papaveraceae) from the flora of KazakhstanAbstract
Chloroplast genomes of angiosperms exhibit a high degree of structural and sequence conservation, making them valuable resources for phylogenetic and systematic studies. However, the genus Corydalis (Papaveraceae), comprising more than 540 species worldwide, is characterized by a complex infrageneric structure and extensive structural rearrangements within the chloroplast genome, posing significant challenges for taxonomic classification. In the present study, we conducted a comparative analysis of chloroplast DNA sequences from five poorly studied Corydalis species native to the flora of Kazakhstan: Corydalis ledebouriana Kar. & Kir., C. glaucescens Regel, C. sewerzowii Regel, C. schanginii (Pall.) B. Fedtsch., and C. gortschakovii Schrenk. The chloroplast markers trnL intron and atpB–rbcL intergenic spacer were employed to assess both interspecific variation and the conservation of sequences within taxonomic sections. Flow cytometric analysis revealed a broad range of genome sizes and ploidy levels among the investigated species. Corydalis gortschakovii, assigned to the subgenus Capnoides, was the only diploid species, exhibiting the smallest genome size and high measurement stability, consistent with its basal and evolutionarily isolated position within the genus. In contrast, C. schanginii exhibited a tetraploid genome with partial reduction of the monoploid genome size. Species belonging to the sections Pes-gallinaceus and Leonticoides (C. glaucescens, C. ledebouriana, and C. sewerzowii) were characterized by large genomes and high ploidy levels (6×–8×), indicating a close evolutionary relationship and highlighting the important role of polyploidization in their diversification. Phylogenetic analyses based on the trnL–trnF and atpB–rbcL chloroplast regions supported the division of the studied species into the two subgenera, Capnoides and Capnites, and confirmed the monophyly of the sections Leonticoides and Pes-gallinaceus. Corydalis gortschakovii formed a distinct basal lineage, whereas the polyploid species exhibited strong genetic coherence within sections and robust sister-group relationships. Comparison of the two chloroplast markers demonstrated their complementary phylogenetic utility: the atpB–rbcL region provided superior resolution of deeper phylogenetic nodes, whereas the trnL–trnF region was more sensitive in detecting variation at the sectional level. These findings highlight the value of integrating cytogenetic and chloroplast genomic data for resolving phylogenetic relationships within Corydalis, identifying ploidy variation, and improving our understanding of the evolutionary processes shaping the genus, including polyploidization and structural genome rearrangements. The results provide a robust framework for future integrative studies incorporating broader geographic sampling and expanded taxonomic coverage.
Keywords:
Corydalis; chloroplast genome; flow cytometry; Northern Tien Shan
Resumo
Os genomas de cloroplasto das angiospermas apresentam elevado grau de conservação estrutural e de sequência, tornando-se recursos valiosos para estudos filogenéticos e sistemáticos. No entanto, o gênero Corydalis (Papaveraceae), que compreende mais de 540 espécies em todo o mundo, caracteriza-se por estrutura infragenérica complexa e por extensos rearranjos no genoma do cloroplasto, impondo desafios à classificação taxonômica. No presente estudo, foi realizada uma análise comparativa das sequências de DNA do cloroplasto de cinco espécies pouco estudadas de Corydalis nativas da flora do Cazaquistão: Corydalis ledebouriana Kar. & Kir., C. glaucescens Regel, C. sewerzowii Regel, C. schanginii (Pall.) B. Fedtsch. e C. gortschakovii Schrenk. Os marcadores de cloroplasto íntron trnL e espaçador intergênico atpB–rbcL foram utilizados para avaliar variação interespecífica e conservação das sequências dentro das seções taxonômicas. A citometria de fluxo revelou ampla variação no tamanho do genoma e nos níveis de ploidia. Corydalis gortschakovii, pertencente ao subgênero Capnoides, foi a única espécie diploide, apresentando menor tamanho genômico e alta estabilidade das medições, em concordância com sua posição basal e isolamento evolutivo. Em contraste, C. schanginii apresentou genoma tetraploide com redução parcial do tamanho do genoma monoploide. As espécies das seções Pes-gallinaceus e Leonticoides (C. glaucescens, C. ledebouriana e C. sewerzowii) caracterizaram-se por genomas maiores e elevados níveis de ploidia (6×–8×), indicando estreita relação evolutiva e destacando o papel da poliploidização em sua diversificação. As análises filogenéticas baseadas nas regiões do cloroplasto trnL–trnF e atpB–rbcL sustentaram a divisão das espécies nos subgêneros Capnoides e Capnites, confirmando a monofilia das seções Leonticoides e Pes-gallinaceus. Corydalis gortschakovii formou linhagem basal distinta, enquanto as espécies poliploides apresentaram forte coesão genética dentro das seções e relações de grupo-irmão robustas. A comparação entre os dois marcadores de cloroplasto demonstrou sua utilidade filogenética complementar: a região atpB–rbcL proporcionou melhor resolução dos nós filogenéticos mais profundos, enquanto a região trnL–trnF foi mais sensível para detectar variações em nível de seção. Esses resultados destacam a importância da integração de dados citogenéticos e genômicos do cloroplasto para elucidar relações filogenéticas em Corydalis, identificar variações nos níveis de ploidia e ampliar a compreensão dos processos evolutivos que moldaram o gênero, incluindo poliploidização e rearranjos estruturais do genoma. O estudo fornece uma base sólida para futuros estudos integrativos com amostragem geográfica mais ampla e cobertura taxonômica expandida.
Palavras-chave:
Corydalis; genoma do cloroplasto; citometria de fluxo; Tien Shan Setentrional
1. Introduction
In the era of rapidly advancing molecular and bioinformatic approaches in plant systematics, traditional taxonomic concepts are being reassessed and classification boundaries are undergoing substantial revision. This is particularly relevant for large and taxonomically complex genera such as Corydalis DC., which currently comprises 545 accepted species according to the Plants of the World Online (RBG, 2023). The greatest species diversity of Corydalis is concentrated across Eurasia, particularly in the Himalayas and China, where approximately 150 species have been recorded. According to the classical taxonomic studies of Bush (1913), Fedde (1936), the genus possesses two principal centers of species diversity: East Asia and the Mediterranean region. Most sections of Corydalis are considered native to the Sino-Japanese floristic region and subsequently extend westward along the Himalayan mountain system into Central Asia, including the Pamir and Altai Mountains. Species of Corydalis occupy a wide range of ecological habitats, from desert and semi-desert environments to alpine meadows.
The genus Corydalis DC. represents one of the largest and most taxonomically diverse genera within the family Papaveraceae, with a broad distribution throughout the temperate regions of Eurasia, including Kazakhstan and adjacent territories.
According to the Flora of Kazakhstan (Pavlov, 1961) and the taxonomic synopsis by Abdulina (1999), the flora of Kazakhstan comprises 19 species of Corydalis. In contrast, Bajtenov (2001) recognized only 18 species, reflecting differences in the taxonomic circumscription of several taxa. One representative of the genus, Corydalis semenowii Regel, is a rare relict species listed in the Red Data Book of the Republic ofKazakhstan (2014). Within Kazakhstan, species of Corydalis are predominantly distributed in the Northern Tien Shan, where 12 species have been recorded, representing approximately 63% of the national species diversity. These species belong to two subgenera and six sections.
According to the botanical-geographical regionalization of Kazakhstan (Rachkovskaya et al., 2003), the Northern Tien Shan belongs to the Saharo-Gobian Desert Region of the Irano-Turanian Subregion and includes the Trans-Ili, Kungey–Terskey–Ketmen–South Dzungarian, and Kyrgyz subprovinces. Within this territory, the floristic regionalization of Kazakhstan (Pavlov, 1956) recognizes the following floristic districts: 25 — Trans-Ili Alatau; 25a — Ketmen and Terskey Alatau; 26 — Chu-Ili Mountains; and 27 — Kyrgyz Alatau.
According to the monograph Cadastre of the Vegetation of the Almaty Region (Kazakhstan, 2023a, b), seven species of the genus Corydalis have been recorded from this region, confirming its substantial representation and local concentration within the Northern Tien Shan. In recent years, particular attention has been devoted to the taxonomic structure of the genus, as traditional morphological criteria, despite their long-recognized value, often produce conflicting results owing to the high variability of diagnostic characters, the influence of environmental factors, and the complexity of evolutionary processes.
A recent molecular phylogenetic study of Corydalis (Chen et al., 2023a) revealed a deep phylogenetic division of the genus into several well-supported clades corresponding to the subgenera Cremnocapnos, Sophorocapnos, and Corydalis (with Bipapillatae additionally recognized in the expanded classification). Within the subgenus Corydalis, the sectional classification was substantially revised, resulting in the recognition of 39 sections. Nevertheless, the phylogenetic positions of several species from the mountainous regions of Central Asia and Kazakhstan remain insufficiently resolved, highlighting the need for further studies incorporating broader taxonomic sampling and additional molecular markers.
In the present study, chloroplast DNA sequences of the trnL–trnF and atpB–rbcL regions were generated for widespread Corydalis species occurring in the Northern Tien Shan, including C. gortschakovii, C. glaucescens, C. ledebouriana, and C. schanginii. These species were selected as representative components of the regional flora because of their broad distribution ranges and considerable morphological and ecological diversity, making them particularly informative for investigating systematic relationships and evolutionary history within the genus. In addition, the study aimed to determine the genome size and ploidy level of these species within the Northern Tien Shan.
The objective of the present study was to clarify the taxonomic composition of five Corydalis species from the flora of Kazakhstan and to evaluate their genome size and ploidy levels.
2. Materials and Methods
2.1. Geobotanical methods
Classical botanical methods were employed in this study, including route-based field surveys, ecological-systematic, and ecological-geographical approaches (Inelova et al., 2024) (Table 1, Figure 1). Identification of the collected specimens was carried out using the principal taxonomic references, including Flora of Kazakhstan (Pavlov, 1961), Illustrated Guide to the Plants of Kazakhstan (Goloskokov, 1972), as well as publications by specialists on the genusCorydalis. Species names were verified using the Plants of the World Online (POWO) database (RBG, 2023). The authorship of species, genera, and families was cross-checked against the International Plant Names Index (IPNI, 2025) database. Additional reference materials were obtained from the Plantarium online resource (Figure 2). Distribution maps were generated using QGIS version 3.40 (QGIS Development Team, 2025) (Figure 1).
Voucher specimens, geographic coordinates, and administrative districts of the sampled populations.
Species of the genus Corydalis (A) Corydalis sewerzowii Regel; (B) Corydalis ledebouriana Kar. & Kir.; (C) C. glaucescens Regel; (D) C. gortschakovii Schrenk; (E) C. schanginii (Pall.) B.Fedtsch.)
2.2. Flow cytometry
The DNA content was determined by flow cytometry techniques with propidium iodide (PI) staining. Leaves dried with silica gel were used as samples. Samples were chopped with standard using a sharp razor blade in LB01 buffer containing PI (50 µg/ml), RNase (50 µg/ml) (Skaptsov et al., 2024) supplemented with 12 mM sodium thiosulfate and 1% polyvinylpyrrolidone (Doležel et al., 1992). The nuclear suspension was filtered through nylon filter with a pore size 30 μm. Analyses were performed on a Cytoflex (Beckman Coulter, Inc.) cytometer. Peaks with at least 1000 nuclei and a CV of less than 5% were used for analysis. Histograms were visualized and processed using CytExpert software (Beckman Coulter, Inc.). Descriptive statistic was calculated using XLStat (Addinsoft). As an internal standard was used the Petroselinum crispum ‘Moss Curled 2’, 2C= 4.5 pg and Solanum pseudocapsicum, 2C = 2.835 pg (Doležel et al., 1992).
2.3. Phylogenetic analyses methods
Molecular genetic methods were used to analyze samples of five Corydalis DC. species (C. ledebouriana Kar. & Kir., C. glaucescens Regel, C. sewerzowii Regel, C. schanginii(Pall.) B. Fedtsch., and C. gortschakovii Schrenk), which were included in the study for comparative analysis.
The following chloroplast marker sequences were obtained: the trnL intron and the atpB–rbcL intergenic spacer of chloroplast DNA (Sumbembayev et al., 2023).
Total DNA was extracted using the Diamond DNA Plant Kit (Altaybiotech, Russia) according to the manufacturer’s protocol. PCR amplification was performed in a 15 µL reaction mixture based on BioMaster HS-Taq PCR mix (Biolabmix, Russia), with a final primer concentration of 400 nM.
The trnL intron was amplified using the primers TabC (5′-CGAAATCGGTAGACGCTACG-3′) and TabD (5′-GGGGATAGAGGGACTTGAAC-3′) (Taberlet et al., 1991) under the following cycling conditions: 94 °C for 5 min; 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s; with a final extension at 72 °C for 5 min and cooling at 4 °C.
The atpB–rbcL region was amplified using the primers atpBF (5′-AGTAGTAGGATTGATTCTCA-3′) and rbcLR (5′-CAACACTTGCTTTAGTCTCT-3′) (Janssens et al., 2006) under the same cycling profile: 94 °C for 5 min; 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s; followed by a final extension at 72 °C for 5 min and cooling at 4 °C.
Separation and visualization of PCR products were performed using 2% agarose gel electrophoresis in TAE buffer at 80 V. Gels were documented by photography after staining with ethidium bromide (10 µg/mL). A 100 bp DNA Ladder Plus (Thermo Scientific, USA) was used as a molecular weight marker. (An image of the electrophoretic gel may be inserted here if desired.)
PCR products were purified prior to sequencing using MAXLIFE MagnetDNA magnetic beads (MBM-Diagnostic, Russia) according to the manufacturer’s protocol. DNA sequencing was performed on an ABI Prism 3130xl automated sequencer (Applied Biosystems, USA).
For Sanger sequencing, 0.3-0.5 pmol of purified DNA fragments were evaporated to dryness and resuspended in a master mix containing 1-2 µL BigDye Terminator v3.1 (Applied Biosystems, USA), 1× sequencing buffer (Nimagen, USA), and additional reagents for complex regions, in a final volume of 40 µL. The thermal cycling profile consisted of an initial denaturation at 95 °C for 3 min, followed by 50-70 cycles of 95 °C for 25 s, 45-50 °C for 10 s, and 60 °C for 3 min, with a final incubation at 98 °C for 5 min and storage at 4 °C until purification. Sequencing reactions were purified by evaporation, resuspended in 25 µL of Hi-Di formamide, and analyzed on a 3500XL Genetic Analyzer. The obtained sequencing data were processed using Sequence Analysis v.6 software.
Sequence alignment was performed using MEGA 11 (Tamura et al., 2021).
3. Results
3.1. Flow cytometric analysis
As a result of the flow cytometric analysis, quantitative data on nuclear DNA content (2C) were obtained for the investigated representatives of the genus Corydalis. Histogram analysis revealed well-defined nuclear peaks (Figure 3) corresponding to the studied samples and internal standards, indicating high reproducibility of measurements and proper preparation of nuclear suspensions. Only peaks containing at least 1,000 nuclei and characterized by a coefficient of variation below 5% were used for interpretation, ensuring the reliability of DNA content estimates.
Examples of ungated flow cytometric histograms of the Corydalis samples (linear scale). (A) C. gorchakovii; (B) C. sewerzowii; (C) C. glaucescence; (D) C. ledebouriana. Internal standard: P.c. – P. crispum; S.p. – S. pseudocapsicum.
The obtained 2C values varied widely, ranging from 1.136 ± 0.029 pg to 3.119 ± 0.027 pg, reflecting substantial interspecific differentiation in genome size. The lowest values were recorded in C. gorchakovii, whereas the highest values were observed in C. sewerzowii and C. ledebouriana. Conversion into haploid genome size (1C, Gbp) enabled comparison among taxa and suggested differences in ploidy level, ranging from diploid (2x) to putative polyploid states (4x–8x) (Table 2).
Thus, the results of flow cytometry demonstrate pronounced genomic heterogeneity among the studied Corydalis species and confirm the importance of genome size estimation for elucidating cytogenetic structure and interpreting evolutionary divergence within the genus.
3.2. Phylogenetic analysis
To clarify the phylogenetic positions of the investigated taxa and to compare the molecular evidence with the previously obtained flow cytometric data, a plastid phylogenetic tree was reconstructed based on trnL–trnF sequences (Figure 4). Phylogenetic analysis was performed using the TVM+I+G model of nucleotide substitution, selected by JModelTest (–lnL = 3120.74327; AIC = 6983.559934; weight = 0.254675), providing a statistically robust reconstruction of phylogenetic relationships. Node support was assessed using both Bayesian posterior probabilities and bootstrap values.
Plastid phylogenetic tree based on trnL–trnF sequences of Corydalis species. Bayesian posterior probabilities and bootstrap support values are shown together in blue. The specimens analyzed in the present study are highlighted in bold. Sections are indicated by red vertical lines and labeled in red, whereas subgenera are indicated by blue vertical lines. The phylogenetic analysis was performed using the TVM+I+G substitution model selected by JModelTest (–lnL = 3120.74327, AIC = 6983.559934, weight = 0.254675).
The resulting phylogeny revealed a clear hierarchical structure of Corydalis at the subgeneric and sectional levels. The samples analyzed in the present study (shown in bold in Figure 4) formed well-supported clades within their respective sections. Specifically, C. sewerzowii and C. ledebouriana clustered within section Leonticoides of subgenus Capnites with strong statistical support, confirming their close evolutionary relationship and corroborating previous morphological evidence.
Similarly, C. schanginii and C. glaucescens formed a distinct, well-supported clade within section Pes-gallinaceus of subgenus Capnites, demonstrating clear genetic differentiation from representatives of section Leonticoides.
In contrast, C. gortschakovii occupied a separate position within subgenus Capnoides (section Calocapnos), forming an independent lineage clearly separated from the Capnites clade, reflecting a deeper level of evolutionary divergence.
Overall, the reconstructed plastid phylogeny confirms the taxonomic placement of the investigated specimens within their respective subgenera and sections, reveals their evolutionary relationships, and provides a molecular framework for interpreting the genome size variation identified by flow cytometric analysis.
To further validate the inferred phylogenetic relationships and increase the robustness of the phylogenetic reconstruction, an independent plastid phylogenetic analysis was performed using the atpB–rbcL marker (Figure 5). The optimal nucleotide substitution model was selected using JModelTest, with the GTR+G model identified as the best fit to the dataset (–lnL = 4738.25102; AIC = 9948.222605; weight = 0.712823). The use of this model allowed rate heterogeneity among sites to be appropriately accommodated, resulting in a statistically well-supported phylogenetic topology.
Plastid phylogenetic tree based on atpB–rbcL sequences of Corydalis species. Bayesian posterior probabilities and bootstrap support values are shown together in blue. The specimens analyzed in the present study are highlighted in bold. Sections are indicated by red vertical lines and labeled in red, whereas subgenera are indicated by blue vertical lines. The phylogenetic analysis was performed using the GTR+G substitution model selected by JModelTest (–lnL = 4738.25102, AIC = 9948.222605, weight = 0.712823).
The resulting phylogeny of Corydalis clearly resolved the genus into subgenera, indicated by blue vertical lines, and sections, marked by red vertical lines and labels. Combined Bayesian posterior probabilities and bootstrap support values are shown at the nodes in blue, providing an assessment of clade robustness.
The specimens analyzed in the present study (highlighted in bold) formed well-supported clades within subgenus Capnites. Specifically, Corydalis ledebouriana and C. sewerzowii clustered together in a strongly supported clade corresponding to section Leonticoides, confirming their close phylogenetic relationship. Likewise, C. schanginii and C. glaucescens formed a distinct clade within section Pes-gallinaceus, demonstrating clear genetic differentiation from representatives of section Leonticoides.
The upper part of the tree places C. gortschakovii within subgenus Capnoides (section Calocapnos), emphasizing its phylogenetic separation from the Capnites clade. This placement is consistent with its distinct morphological characteristics and the comparatively smaller genome size revealed by flow cytometric analysis.
Overall, the phylogenetic reconstruction based on the atpB–rbcL marker corroborates the subdivision of Corydalis into subgenera and sections, is highly consistent with the topology obtained from the trnL–trnF dataset, and further strengthens the taxonomic interpretation of the investigated specimens.
4. Discussion
4.1. Flow cytometric analysis: genome size and ploidy levels
Flow cytometric analysis enabled the quantitative estimation of nuclear DNA content (2C) and haploid genome size (1C) in the investigated representatives of the genus Corydalis. The obtained data reveal a clear correlation between the phylogenetic position of the taxa and their ploidy levels.
Corydalis gortschakovii, belonging to subgenus Capnoides, possessed the smallest genome among the studied species (2C = 1.136 ± 0.029 pg; 1C = 0.556 Gbp) and exhibited a low coefficient of variation (CV = 2.58%), indicating high measurement precision and genome size stability. This species was the only diploid (2×) taxon among the investigated samples, consistent with its basal position in the phylogenetic trees. These findings support the hypothesis that representatives of subgenus Capnoides have retained the ancestral genomic state. Previous studies by Chen et al. (2023a) and Liu et al. (2024) similarly reported that the basal and phylogenetically isolated position of diploid species is consistent with the evolutionary history of the genus and reflects its early diversification.
Corydalis schanginii exhibited a tetraploid level (4×) with a nuclear DNA content of 2C = 2.806 ± 0.138 pg (1C = 1.372 Gbp). Its genome size was approximately 2.5-fold larger than that of the diploid species, consistent with the expected increase associated with tetraploidy. The relatively higher coefficient of variation (CV = 4.91%) may reflect intraspecific variability. These observations are in agreement with the findings of Jiao et al. (2011), who reported partial genome downsizing following polyploidization, and with the results of Peng et al. (2023a), in which tetraploid representatives of subgenus Capnites exhibited comparable increases in genome size.
Three species belonging to sections Pes-gallinaceus and Leonticoides (C. glaucescens, C. ledebouriana, and C. sewerzowii) possessed relatively large genomes (2C = 2.788-3.119 pg; 1C = 1.363-1.525 Gbp), corresponding to high polyploid levels (6×–8×). Among these taxa, C. sewerzowii had the largest genome (2C = 3.119 pg) and showed excellent measurement reproducibility (CV = 0.85%), whereas C. ledebouriana exhibited the highest coefficient of variation (CV = 6.18%), which may indicate population heterogeneity or variation in ploidy level. The similarity in genome sizes among these species further supports their close evolutionary relationship, consistent with the findings of Xu et al. (2022a), who reported comparable 2C values and confirmed phylogenetic affinity among polyploid representatives of subgenus Capnites.
Overall, the present results provide several important insights into the evolution of Corydalis. A clear relationship exists between subgeneric classification and ploidy level: the diploid subgenus Capnoides contrasts with the predominantly polyploid subgenus Capnites, as reflected by the observed ploidy levels (4×–8×) of the investigated species (Pérez-Gutiérrez et al., 2015b; Peng et al., 2023b). Polyploidization has likely been one of the principal mechanisms driving diversification within subgenus Capnites, while the partial genome downsizing observed in the tetraploid C. schanginii(1C ≈ 1.372 Gbp ≈ 2.47 × 0.556 Gbp) is consistent with well-documented patterns of genome size evolution following whole-genome duplication (Jiao et al., 2011; Xu et al., 2022b).
The statistically similar genome sizes of species belonging to section Leonticoides further support their close evolutionary affinity and are consistent with the plastid phylogenies inferred from the atpB–rbcL and trnL–trnF markers (Liu et al., 2024; Peng et al., 2023a). Taken together, these findings emphasize the importance of cytogenetic data for interpreting phylogenetic relationships, identifying ploidy variation, and improving our understanding of the evolutionary mechanisms underlying diversification within the genus Corydalis (Kluge and Farris, 1969; Doležel et al., 1992; Doležel and Bartos, 2005; Xu et al., 2022b).
4.2. Phylogenetic analysis: structure of the genusCorydalis
Contemporary phylogenetic reconstructions of the genus Corydalis, based on plastid genomes and low-copy nuclear markers (Zhang et al., 2008; Wang, 2006; Xu et al., 2022b; Chen et al., 2023a), have substantially refined the infrageneric structure of the group and confirmed its complex evolutionary history. However, Central Asian taxa have generally been underrepresented in most studies, which has limited the interpretation of their placement within specific sections and subgenera. In this context, our data on Corydalis ledebouriana, C. sewerzowii (sect. Leonticoides), C. schanginii, C. glaucescens (sect. Pes-gallinaceus), andC. gortschakovii (sect. Calocapnos) significantly expand the available molecular sampling and allow refinement of their phylogenetic relationships.
The analysis of two plastid markers (trnL–trnF and rbcL–atpB) consistently supports the division of the investigated taxa into two subgenera, Capnoides and Capnites. The isolated position of C. gortschakovii within section Calocapnos (subgenus Capnoides) is strongly supported by both datasets, in agreement with its morphological recognition as a distinct evolutionary lineage. Unlike several sections reported as polyphyletic in large-scale phylogenomic studies (e.g., Asterostigmata, Pubicaules, Elatae), Calocapnos in our dataset exhibits clear plastid coherence. Nevertheless, given previously reported topological incongruences between plastid and nuclear phylogenies in certain clades (Chen et al., 2023a), the monophyly of this section should be further tested using nuclear markers to exclude potential chloroplast introgression.
Within subgenus Capnites, our results confirm the presence of two well-delimited sections, Leonticoides and Pes-gallinaceus. Section Leonticoides shows high genetic cohesion: C. ledebouriana forms a strongly supported monophyletic group across both markers, confirming its species-level integrity. C. sewerzowii is positioned in close proximity and together they form a well-supported clade. However, the trnL–trnF analysis shows some instability in nodes resolving their exact sister relationships, whereas rbcL–atpB provides higher support for these internal branching patterns. This discrepancy likely reflects differences in phylogenetic signal and resolving power between the markers, emphasizing the importance of combined datasets for more robust inference of sectional relationships.
Section Pes-gallinaceus has been sparsely represented in previous studies, and its placement within major clades has remained insufficiently resolved. Our results confirm the monophyly of C. glaucescens and C. schanginii and their close phylogenetic affinity. At the same time, both species exhibit clear genetic distinctness, indicating completed divergence within a shared evolutionary lineage. The higher nodal support observed in the rbcL–atpB analysis compared to trnL–trnF may reflect differences in evolutionary rates and marker informativeness at this level of divergence.
Overall, our findings are consistent with current views of a heterogeneous and hierarchically structured genus Corydalis, confirming clear subgeneric delimitation and supporting the monophyly of the studied sections within the analyzed sampling framework. At the same time, observed differences in nodal support highlight that further integrative studies with expanded geographic sampling and combined plastid and nuclear genomic data are required to fully resolve intrasectional relationships, particularly within Leonticoides.
4.2.1. Discussion of plastid trnL–trnF data
The phylogenetic analysis of the plastid trnL–trnF region revealed a clear segregation of the investigated Corydalis samples into two major evolutionary lineages corresponding to the subgenera Capnoides and Capnites, which is consistent with previously published phylogenetic studies of the genus (Lidén et al., 1995, 1997; Pérez-Gutiérrez et al., 2015a; Sauquet et al., 2015; Zhang et al., 2008, 2016; Wang, 2006; Xu and Wang, 2022; Chen et al., 2023b).
The inversion in the ndhB–trnR-ACG region, previously reported in 37 Corydalis plastomes (Xu et al., 2022a), was also detected in our samples, confirming its stability and phylogenetic informativeness. The resulting topology is broadly consistent with the current infrageneric classification and allows evaluation of clade robustness based on combined Bayesian posterior probabilities (PP) and bootstrap support (BS), as previously emphasized in related studies (Felsenstein, 1985; Ronquist and Huelsenbeck, 2003; Wang, 2006; Zhang et al., 2016).
According to model selection analysis, the optimal nucleotide substitution model for this dataset was TVM+I+G (–lnL = 3120.74327; AIC = 6983.559934; weight = 0.254675), reflecting both among-site rate heterogeneity and the presence of invariant sites, in agreement with approaches used in earlier phylogenetic reconstructions (Sauquet et al., 2015; Chen et al., 2023b).
Sample R16 (Corydalis gortschakovii) is confidently placed within section Calocapnos of subgenus Capnoides, forming an isolated lineage clearly separated from members of Capnites. Node support is high to moderately high (PP ≈ 0.9; BS > 80), indicating the stability of this placement. Thus, C. gortschakovii is phylogenetically distant from the remaining studied species and confirms the deep divergence between the two subgenera, as previously reported in taxonomic studies of the genus (Lidén et al., 1997; Xu et al., 2022a; Han et al., 2025).
Within subgenus Capnites, two sections are clearly recovered—Leonticoides and Pes-gallinaceus—consistent with current interpretations of infrageneric structure in Corydalis (Wang, 2006; Zhang et al., 2016).
Section Leonticoides includes C. ledebouriana (R1–R4 and an additional sequence) and C. sewerzowii (R9, R12). The monophyly of C. ledebouriana is strongly supported (PP close to 1.0; BS ~80-100), indicating genetic cohesion within the sampled populations, in agreement with previous molecular studies (Pérez-Gutiérrez et al., 2015b; Chen et al., 2023a). C. sewerzowii is positioned closely to the C. ledebouriana clade; however, nodes linking both species show reduced support (e.g., PP ~0.87 / BS ~59; PP ~0.72 / BS ~82). This suggests partial instability of deeper intrasectional relationships, potentially reflecting limited phylogenetic signal, incomplete lineage sorting (ILS), or insufficient population-level sampling, as previously discussed in the literature (Thompson et al., 1997; Zhang et al., 2008; Xu et al., 2022b; Friesen et al., 2026).
Section Pes-gallinaceus includes C. glaucescens (R7, R8 and an additional sequence) andC. schanginii (R13, R14). The monophyly of C. glaucescens is strongly supported (PP = 1.0; BS ≈ 97), whereas C. schanginii forms a distinct subclade with moderate to high support (PP ≈ 0.95; BS ≈ 66 for a key node). Both species cluster together in a single higher-level clade, indicating close phylogenetic affinity while maintaining clear species-level separation, consistent with the findings of Wang (2006) and Zhang et al. (2016).
Overall, the trnL–trnF region supports the subgeneric and sectional delimitation of the studied taxa and demonstrates sufficient resolving power at the interspecific level. However, intrasectional relationships, particularly within Leonticoides, remain partially unresolved, consistent with previous reports highlighting the limited phylogenetic signal of single plastid markers (Sauquet et al., 2015; Chen et al., 2023b; Abdildanov et al., 2025; Friesen et al., 2025; Vesselova et al., 2025; Ussen et al., 2025).
4.2.2. Discussion of plastid rbcL–atpB data
The analysis of the plastid rbcL–atpB region confirmed a clear differentiation of the investigatedCorydalissamples at the subgeneric and sectional levels and demonstrated higher statistical stability for several nodes compared to the trnL–trnF dataset. The optimal nucleotide substitution model for this dataset was GTR+G (AIC weight = 0.71), reflecting pronounced rate heterogeneity across sites in the analyzed region (Xu and Wang, 2020).
Sample R16 (Corydalis gortschakovii), belonging to section Calocapnos of subgenus Capnoides, occupies a basal position within its clade and is completely isolated from representatives of Capnites. High support values (PP = 1.0; BS = 94) confirm its independent phylogenetic position and emphasize the distinctiveness of subgenus Capnoides (Xu and Wang, 2020).
All remaining samples form a monophyletic group corresponding to subgenusCapnites, indicating its phylogenetic coherence. Within this subgenus, two well-defined sections are clearly recovered: Pes-gallinaceus and Leonticoides.
Section Pes-gallinaceus is represented by C. glaucescens R7, R8) and C. schanginii (R13, R14), which form a highly supported clade (PP = 1.0; BS = 99-100). Their sister relationship indicates a recent common ancestor and close evolutionary affinity. The placement of this section relative to the remaining members of Capnites is supported with moderate posterior probability (PP = 0.78), which may reflect a relatively rapid early diversification within the subgenus (Hoot et al., 2015).
Section Leonticoides includes C. ledebouriana (R1–R4) and C. sewerzowii (R9, R12), forming a well-supported clade (PP = 0.92; BS = 98). The basal position of sequence NC_072202 (C. ledebouriana) within this section may indicate proximity to the ancestral lineage of this group. In contrast to the trnL–trnF dataset, sister relationships between C. ledebouriana and C. sewerzowii are more clearly resolved in the rbcL–atpB analysis and are supported by higher statistical values (Wu et al., 1999; Wang, 2006; Hoot et al., 2015).
Overall, the results of the rbcL–atpB analysis confirm the separation of the investigated taxa into three sections within two subgenera and demonstrate the monophyly of Capnites with clear separation from Capnoides. Strong support for the sister pairs C. glaucescens / C. schanginii and C. ledebouriana / C. sewerzowii is consistent with their taxonomic placement and suggests relatively recent diversification within the respective sections (Hoot et al., 1999).
Comparative analysis of the two plastid markers highlights their complementarity: the rbcL–atpB region provides more robust resolution of deeper nodes, whereas trnL–trnF is more sensitive to intrasectional variation and shows partial instability in some clades. Overall, both markers consistently support the current subgeneric and sectional classification of Corydalis and emphasize the informativeness of plastid sequences for reconstructing its phylogenetic structure (Hoot et al., 1999, 2015).
5. Conclusions
The present study provides a detailed characterization of genome size variation and ploidy levels in key representatives of the genus Corydalis, revealing a close association between genomic state and phylogenetic position. The diploid species C. gortschakovii, belonging to subgenus Capnoides, exhibits the smallest haploid genome size and highly stable measurements, supporting its basal and phylogenetically isolated position within the genus. This pattern indicates the retention of an ancestral genomic state and long-term stability of the genomic architecture within this subgenus.
In contrast, polyploid species, including C. schanginii, C. glaucescens, C. ledebourianum, and C. sewerzowii, possess substantially enlarged genomes, reflecting a multi-stage polyploidization process within subgenus Capnites. The observed partial genome downsizing in the tetraploid C. schanginii suggests dynamic post-polyploid diploidization processes, consistent with general evolutionary models of genome expansion followed by stabilization in plants. The variation in ploidy levels and genome sizes within sections Leonticoides and Pes-gallinaceusfurther supports their close evolutionary relationship and reflects sequential divergence events within the subgenus.
Phylogenetic analyses based on two plastid markers allowed refinement of the infrageneric structure of Corydalis and confirmed the monophyly of the investigated sections. Diploid and polyploid lineages are clearly separated, while intrasectional relationships reveal both well-supported and partially unresolved nodes, highlighting the necessity of further integrative approaches incorporating nuclear and plastid datasets. A notable outcome of this study is the apparent stability of major sections with respect to introgression signals, as well as the detection of intraspecific variability in polyploid taxa, which may reflect adaptive processes and local population differentiation.
Overall, the results demonstrate that the integration of cytogenetic and molecular approaches provides a more comprehensive understanding of the evolutionary dynamics of Corydalis, elucidating mechanisms of polyploidization and genome downsizing, and refining intra- and inter-sectional phylogenetic relationships. These findings establish a foundation for future research into genome evolution, adaptation, and speciation within this highly diverse and evolutionarily complex genus.
Data Availability Statement
The entire data set that supports the results of this study was published in the article itself.
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