Introduction
Parasitic weeds represent one of the most serious biological constraints to global agriculture, causing extensive yield losses and threatening food security across diverse cropping systems (Erdogan, 2022). Unlike ordinary weeds that merely compete for soil and light resources, parasitic plants directly attach to their hosts and extract water, nutrients, and assimilates, often leading to severe physiological stress and yield decline (Twyford, 2018). Among these, the genus Cuscuta (dodder) of the family Convolvulaceae stands out as one of the most destructive. Comprising about 170–200 species with nearly cosmopolitan distribution (Costea et al., 2015), Cuscuta species parasitize a wide range of dicotyledonous crops (Bernal Galeano et al., 2022). Their rapid spread and ability to form dense haustorial networks result in substantial economic and ecological impacts (Ngare et al., 2021). Although Cuscuta occurs globally, regional variations in host range and species composition exist (Kaiser et al., 2015), necessitating accurate and context-specific identification, particularly in agricultural regions such as Korea. In Korea, the genus is majorly represented by C. pentagona, C. japonica, C. chinensis and C. australis (Park et al., 2019). These species parasitize several economically important crops such as soybean (Glycine max) and balloon flower (Platycodon grandflorum) as well as native vegetation.
Accurate species identification in Cuscuta remains a major taxonomic challenge. These holoparasites exhibit extreme morphological reduction, lacking leaves, and bearing highly simplified thread-like stems (Stefanovic et al., 2007). Consequently, diagnostic features are often restricted to floral structures, but which are often subtle, variable, and sometimes unavailable (Wright et al., 2011). Moreover, convergent evolution and phenotypic plasticity frequently produce cryptic similarities among species, making morphological identification unreliable (McNeal et al., 2007). As a result, misidentification has been a recurrent problem in both research and management programs. To overcome these limitations, DNA barcoding has become a powerful and standardized tool for species discrimination and phylogenetic analysis (Park et al., 2020). Chloroplast regions together with nuclear ribosomal regions are particularly valued for their universality, high amplification success, and balanced discriminatory power in closely related plant groups (Yang et al., 2012). A combination of markers including rbcL, matK, and trnL–trnF (Revill et al., 2005) together with ITS (Ibiapino et al., 2022) have consistently demonstrated their effectiveness in resolving species boundaries and reconstructing phylogenetic relationships within Cuscuta lineages, making them a strong framework for molecular identification.
Despite this progress, the taxonomy and distribution of Cuscuta in Korea remain poorly resolved, with current records based largely on morphological traits (Hwang et al., 2013). This uncertainty complicates the management efforts against these parasitic weeds. Therefore, using DNA barcoding is essential for clarifying species boundaries and establishing a precise reference framework. The present study aimed to identify and characterize Cuscuta accessions collected from different regions of Korea using multiple DNA barcode markers (rbcL, matK, trnL–trnF, and ITS). By integrating these loci, this study seeks to resolve taxonomic ambiguities and provide a molecular foundation for accurate species identification and distribution assessment of Cuscuta in Korea.
Materials and Methods
Sample collection and preparation of the Cuscuta seeds for germination
Thirty Cuscuta accessions were collected from different key agricultural zones across Korea (Table I). Fifty seeds from each of the thirty accessions were scarified in concentrated sulfuric acid (96%; Janssen Pharmaceuticalaan, Geel, Belgium) for 10 min. The seeds were then thoroughly rinsed under running tap water for 2 h. Following scarification, seeds were transferred into micro tubes f illed with distilled water. All tubes were wrapped in aluminium foil to prevent light exposure and stored at 4°C for 3 days to allow adequate water absorption.
Preparation of growth medium and planting of seeds.
Murashige and Skoog (MS) basal media (Murashige & Skoog, 1962), solidified with 0.4% (w/v) plant agar, and adjusted to pH 5.7 was autoclaved and dispensed into magenta boxes to cool and solidify. Thirty seeds from each sample tube were then aseptically arranged on the surface of the media. Cultures were incubated in a growth chamber at 25/15°C (day/night) under a 14/10 h photoperiod. Germination and seedling growth were monitored for 15 days (Fig. 1).
Genomic DNA extraction, PCR amplification, and purification
Young Cuscuta stems were used as the source material for DNA extraction. After 15 days of growth, when most seeds had germinated and developed sufficient tissue mass, shoots from each magenta box were harvested. Genomic DNA was extracted using the DNeasy® Plant Mini Kit (QIAGEN Inc., Germany) following the manufacturer’s protocol. DNA concentration and purity were quantified using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, USA).
PCR was performed using primer pairs targeting the rbcL, trnL-trnF, matK, and ITS regions. Each 25 μL reaction contained 1 μL of template DNA, 12.5 μL of 4× PCR Master Mix (containing Taq DNA polymerase, reaction buffer, dNTPs, and MgCl2; Thermo Scientific), 1 μL of each primer (100 pmol), and 9.5 μL of nuclease-free water. Annealing conditions were optimized for each primer pair using gradient PCR with temperatures ranging from 54–65°C (Table 2). Amplifications were carried out on a T100™ Thermal Cycler (Bio-Rad, USA) with the following cycling profile: Initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 94°C for 30 s, annealing at the optimized temperature for 1 min, and extension at 72°C for 1 min; followed by a final extension at 72°C for 10 min. PCR products were verified by capillary electrophoresis using the QIAxcel Advanced System (QIAGEN Inc., Germany) using the QIAxcel DNA High-Resolution Kit (QIAGEN Inc., Germany). Amplicons were then purified with the QIAquick® PCR Purification Kit (QIAGEN Inc., Germany) and high-quality products were sequenced.
Sequence retrieval and phylogenetic analysis
Nucleotide sequences obtained from this study were complemented with homologous sequences retrieved from the National Center for Biotechnology Information (NCBI) nucleotide database (https://www.ncbi.nlm.nih.gov/). All sequences were aligned using ClustalW in MEGA6 software (Tamura et al., 2013) to ensure positional homology. Phylogenetic analyses were performed using MEGA6 software under the maximum likelihood criterion. The tree was inferred using the Nearest-Neighbor Interchange (NNI) heuristic search method. To root the phylogeny, Arabidopsis thaliana was used as the outgroup and branch support was assessed with 1,000 bootstrap replicates to evaluate the robustness of the inferred relationships. Bootstrap values above 80% were considered to indicate strong support.
Intraspecific and interspecific variation among the identified species
To assess genetic variation within and between the identified Cuscuta species, pairwise evolutionary divergences were estimated based on trnL-trnF sequences. For intraspecific variation, the average number of nucleotide substitutions per site was computed by averaging over all sequence pairs within each species group. Interspecific variation was similarly calculated as the mean evolutionary divergence between species groups. In both analyses, the Tamura–Nei substitution model was applied, and variance estimates were obtained through 1,000 bootstrap replicates to ensure reliability of the mean distances (Tamura and Nei, 1993).
Results
Amplification patterns of the barcode regions
Three barcode regions (rbcL, trnL-trnF, and ITS) were successfully amplified across the 30 accessions (Fig. 2). For rbcL and ITS, fragment sizes were relatively consistent among accessions, ranging from approximately 600–700 bp for rbcL and 600–750 bp for ITS. Conversely, variation was observed in the trnL-trnF region; while most samples produced fragments of ~500 bp, two accessions, S12 and S22, consistently yielded longer fragments of ~950 bp. A similar distinction was observed with the matK marker, where amplification succeeded only for these two accessions. These results demonstrate overall marker reliability across accessions, while highlighting the distinct amplification patterns in S12 and S22 that set them apart from the rest.
Phylogenetic clustering of Cuscuta accessions
Phylogenetic analysis placed all accessions within the expected Cuscuta clade, with Arabidopsis thaliana serving as an outgroup. Based on rbcL sequences, the 30 accessions were differentiated into four well-supported clades (Fig. 3A). The majority clustered with reference sequences of C. pentagona (Clade I), while three accessions grouped distinctly with C. australis (Clade II), confirming its separation from C. pentagona. Five accessions clustered with C. campestris and C. chinensis (Clade III), indicating that these two species were not fully resolved by rbcL. Notably, two accessions, S12 and S22, were distantly separated from the other clades and grouped with C. japonica (Clade IV). Meanwhile, analysis of the trnL-trnF region reinforced these findings but provided greater resolution. Accessions were again divided into four major clusters consistent with rbcL, but this marker effectively separated C. campestris from C. chinensis, confirming that none of the sampled accessions belonged to C. chinensis (Fig. 3B). The ITS region exhibited limited variability. Except for S12 and S22, which consistently clustered with C. japonica, all other accessions produced nearly identical sequences, with the accession from Jeju Island showing only a slight divergence. Similarly, matK amplification was successful only in S12 and S22, also confirming their identity as C. japonica. Therefore, ITS and matK were informative only for distinguishing C. japonica.
Overall, a combination of rbcL and trnL-trnF markers proved most effective for resolving the Cuscuta accessions, enabling the identification of four species: C. pentagona, C. australis, C. campestris, and C. japonica. Among these, C. pentagona was the most widely distributed, present in 67% of the sampled locations, while other species showed narrower distribution ranges. Otherwise, ITS and matK did not contribute to species-level resolution beyond confirming C. japonica.
Patterns of intraspecific and interspecific variation among identified species
Results demonstrated a generally low intraspecific variation, with mean substitution values ranging from 0.000 to 0.004 (Table 3). The highest intraspecific divergence was observed within C. australis, primarily driven by the sequence difference of the accession collected from Jeju Island (Table 4). In contrast, interspecific divergence values were substantially higher, ranging from 0.012 to 0.897. Among the species, C. japonica exhibited the greatest genetic differentiation, showing markedly higher pairwise distances (0.824–0.879) from all other taxa. This distinct divergence pattern indicates a strong level of molecular differentiation of C. japonica from the other resolved Cuscuta species.
Table 4
Pairwise interspecific genetic distances (%) among Cuscuta species estimated using the Neighbor-Joining method. The matrix reflects evolutionary divergence and species differentiation based on trnL-trnF sequences
Distribution of Cuscuta species in Korea
To visualize the regional patterns of occurrence, the identified Cuscuta species were mapped across Korea (Fig. 4).
Discussion
This study aimed to resolve Cuscuta species in Korea using a multi-locus DNA barcoding approach. The analysis identified four distinct species, C. pentagona, C. australis, C. campestris, and C. japonica, validating the utility of molecular markers in delineating morphologically cryptic taxa (Letsiou et al., 2024). Despite earlier reports of C. chinensis in Korea (Kim et al., 2007), our study resolved all the putative samples as C. campestris suggesting a possibility of prior morphological misidentification. Among the tested loci, the combination of rbcL and the trnL-trnF spacer emerged as the best informative strategy, providing the most consistent specieslevel resolution. The strong performance of rbcL reflects primer universality and amplification reliability (CBOL Plant Working Group, 2009), whereas trnL-trnF contributed increased discriminatory power through indel-rich variation in the noncoding region (Drábková et al., 2004), together offering a pragmatic, and cost-effective identification toolkit. Consistent with previous studies (Pleines et al., 2009), noncoding plastid regions such as trnL-trnF tend to offer superior discriminatory power compared to coding loci, thanks to their higher substitution rates and reduced functional constraints. In contrast, matK amplified only in two accessions (S12 and S22), pointing to gene loss or severe degradation of this locus in most sampled lineages, an expected consequence of plastome reduction in parasitic taxa (Banerjee & Stefanović, 2023). Similarly, while ITS amplified across all accessions, it showed meaningful variation only for the same accessions, reinforcing their strong genetic separation from the remaining accessions.
Phylogenetic analysis revealed clear patterns of diversity and distribution among the studied accessions. Cuscuta pentagona emerged as the most geographically widespread species, occurring across diverse environments. Its broad distribution has been noted previously (Kim et al., 2007), and may be attributed to its strong adaptability to persist and flourish even under sub-optimal growing conditions (Jang and Kuk, 2020). Interestingly, C. japonica exhibited strong phylogenetic distinctness, consistently forming an isolated lineage across all markers. The successful amplification of matK in this species, unlike in others, suggests a relatively conserved or partially functional plastome. While most Cuscuta lineages, particularly in the subgenus Grammica, have extensively lost photosynthesis-related genes (Banerjee and Stefanović, 2023), species such as C. japonica in the subgenus Monogynella have retained several of these genes (Claude et al., 2025). Given that plastome reduction is a gradual process associated with increasing parasitic dependence (Chen et al., 2024), C. japonica may represent an intermediate stage in the evolutionary transition from hemi-parasitism to full holoparasitism. Meanwhile, the narrower distribution of C. campestris and C. australis may suggest more specialized ecological associations. Otherwise, the four resolved species offer a clear snapshot of the current Cuscuta diversity across the major agricultural zones in Korea
Despite the global richness of the genus Cuscuta (Costea et al., 2015), our study revealed a remarkably narrow genetic diversity in Korea. Such a limited variation may be linked to a combination of ecological filtering and introduction history. Cuscuta pentagona and C. campestris are cosmopolitan species that have spread widely through seed contamination and agricultural exchange (Barath, 2021), whereas C. australis (Wu et al., 2019) and C. japonica (Zagorchev et al., 2025) represent indigenous East Asian lineages. Thus, the limited diversity here may explain a history of few introduction events coupled with the adaptive success of some lineages that successfully established across diverse habitats. Overall, the findings provide an updated taxonomic baseline of Cuscuta in Korea and offer essential information for designing targeted management strategies in agricultural systems.
Conclusions
This study provided an updated molecular perspective of the diversity and distribution of Cuscuta species in Korea. Four species were confirmed; C. pentagona, C. campestris, C. australis and C. japonica, with C. pentagona being the most widely spread. This relatively broader distribution of C. pentagona suggests its particular management importance. Importantly, C. chinensis, previously reported in Korea, was not detected in any of the sampled regions; instead, all putative accessions were resolved as C. campestris, thereby correcting earlier distribution records. The intraspecific variation was generally low, indicating the stability of the identified species. Meanwhile, the combined use of rbcL and trnL-trnF proved most effective for accurate species delimitation, offering a practical and cost-effective tool for Cuscuta systematics. Going forward, expanding sampling and testing additional barcodes will strengthen the findings and establish a standardized molecular framework for the reliable identification of Cuscuta species across Korea.
Acknowledgements
This study was supported by the Rural Development Administration (RDA) through the Research Project (Project No. PJ017669) and the 2025 On-the-Job Training for Young Scientists Research Program of the Korea-Africa Food and Agriculture Cooperation Initiative (KAFACI), Rural Development Administration (RDA), Republic of Korea.
