Taxonomic reevaluation of Daphne pseudomezereum var. koreana (Thymelaeaceae): Evidence for reassignment to D. kamtschatica
Article information
Abstract
Daphne pseudomezereum var. koreana has traditionally been treated as a variety of D. pseudomezereum, a species endemic to the warm-temperate regions of Japan. However, var. koreana occurs primarily in cool-temperate regions of northeastern Asia—including the Baekdudaegan Mountain Range in Korea, southern Manchuria, southern Primorsky Krai, Sakhalin, and Hokkaido—and thus exhibits distinct ecological and biogeographic characteristics from var. pseudomezereum. To reassess its taxonomic position, we conducted molecular phylogenetic analyses using complete chloroplast genome and nuclear ribosomal ITS sequences. Plastome phylogeny supports var. koreana as a sister to D. kamtschatica, whereas nuclear ribosomal internal transcribed spacer phylogeny places it as a sister to var. pseudomezereum. While the phylogenetic discordance complicates its placement, we interpret this discordance as in itself ultimately revealing var. koreana as a lineage bearing an evolutionary identity possibly shaped by ancient hybridization. Evidence from ecology, biogeography, life-history (presence of well-developed rhizomes), and morphology (e.g., growth habit, calyx coloration, anther position) further supports its affinity with D. kamtschatica. Taken together, we propose reclassifying var. koreana as a variety of D. kamtschatica, representing a geographically disjunct southwestern lineage separated from var. kamtschatica by the Sea of Okhotsk. Plants occurring in southern Japan currently considered to be var. koreana may instead represent distinct, undescribed taxa.
INTRODUCTION
The genus Daphne (Thymelaeaceae) is represented by two species on the Korean Peninsula (Yoo et al., 2021; Son et al., 2025). Daphne pseudomezereum A. Gray var. koreana (Nakai) Hamaya is confined to high-elevation habitats along the Baekdudaegan mountain range and extends into cool-temperate regions of northeastern Asia, including Sakhalin and Hokkaido (Fig. 1). Daphne kiusiana Miq. occurs in southern coastal regions, including Jeju Island (Han et al., 2023). These species occupy geographically distinct cool-temperate and warm-temperate regions, respectively. A similar pattern is observed in Japan, where they are mostly separated geographically, with occasional range overlap but differences in ecological habitats.
Distribution map of Daphne pseudomezereum var. koreana, D. pseudomezereum var. pseudomezereum, and D. kamtschatica. Occurrence data were compiled from the Global Biodiversity Information Facility (GBIF, 2015; accessed 3 Aug 2025) database, the floras of Korea and Japan (Murata, 1999; Oh et al., 2016), and published floristic records from the Kamchatka Peninsula and Primorsky Krai (Maximowicz, 1859; Nedoluzhko, 1922; Barkalov et al., 2019; Kozhevnikov et al., 2019).
Daphne pseudomezereum A. Gray var. pseudomezereum is endemic to Japan, occurring in warm-temperate regions (Fig. 1). Interestingly, in Japan, var. pseudomezereum and D. kiusiana frequently occur sympatrically (e.g., Fig. 2). Its habitat markedly contrasts with that of var. koreana. Given their distinct ecological niches, we questioned whether the current taxonomic treatment of var. koreana as an infraspecific taxon of D. pseudomezereum is appropriate. Notably, Murata (1999) also remarked in Flora of Japan that var. koreana occurring on Hokkaido is morphologically more similar to D. kamtschatica than to D. pseudomezereum.
The photograph of Daphne pseudomezereum var. pseudomezereum observed in Inabe City, Mie Prefecture, Japan, occurring in sympatry with D. kiusiana.
Daphne kamtschatica Maxim., occurring in cold-temperate regions, including the Kamchatka Peninsula, southern Sakhalin, and Hokkaido, shares a similar floral morphology and structure with var. koreana, whereas var. pseudomezereum is distinct from both taxa (Table 1). During flowering, var. koreana and var. pseudomezereum differ in their leaf development patterns. In var. koreana, leaves are concentrated toward the distal portions of branches, giving the plant a palm-like growth habit, whereas in var. pseudomezereum, leaves are distributed along the entire stem, resulting in a relatively leafier habit (Fig. 3).
Comparison of morphological characteristics among three Daphne taxa.
Photographs of the three Daphne taxa examined in this study. A. Daphne pseudomezereum var. koreana. B. D. pseudomezereum var. pseudomezereum. C. D. kamtschatica. 1, Flowering individual; 2, Flowers. Photographs of D. kamtschatica were adapted from Nitta and Uchida (2020). Blue arrows indicate anthers included within the calyx tube; the red arrow indicates anthers ca. half-exserted from the calyx tube.
Based on floral morphological and structural characteristics and on the ecological niche of each, we hypothesized that var. koreana is more closely related to D. kamtschatica than to D. pseudomezereum. To test this hypothesis, we conducted molecular phylogenetic analyses using complete chloroplast genome sequences (plastome) and nuclear ribosomal internal transcribed spacer (nrITS) data.
MATERIALS AND METHODS
Plant material sampling and DNA extraction
To clarify the phylogenetic position of D. pseudomezereum var. koreana, we collected leaf samples from three Daphne taxa: D. pseudomezereum var. koreana, D. pseudomezereum var. pseudomezereum, and D. kamtschatica (Table 2). Leaf samples of var. koreana and var. pseudomezereum were collected from their natural habitats in South Korea and Japan, respectively. Voucher specimens of var. koreana (Byeon-Dpk250701) and var. pseudomezereum (Lee-Dpp230303) are deposited in the Department of Biology Education, Chonnam National University (BEC; quercus@jnu.ac.kr). In contrast, the leaf sample of D. kamtschatica used here was obtained from an herbarium specimen collected on the Kamchatka Peninsula, Russian Far East, on 22 July 2003, and is deposited in the Royal Botanic Gardens, Kew (K; Catalogue number: K005971094; Record number: 20879).
Sampling information for the three Daphne taxa used in this study.
Total genomic DNA was extracted from leaf samples of the three taxa using a DNeasy Plant Mini Kit (Qiagen, Seoul, Korea). DNA concentration and quality assessments were conducted with a NanoDrop spectrophotometer (Allsheng Co., Ltd., Hangzhou, China).
Chloroplast genome sequencing, assembly, and annotation
We newly assembled and annotated the plastomes of D. pseudomezereum var. pseudomezereum and D. kamtschatica, while that of D. pseudomezereum var. koreana was obtained from GenBank (GenBank accession No. ON244034) (Yoo et al., 2023). For the newly sequenced taxa, plastome data were generated as follows. The DNA library was constructed with the TruSeq Nano DNA kit (Illumina Inc., San Diego, CA, USA) and sequenced on the Illumina NovaSeqX platform (Macrogen, Seoul, Korea) according to the manufacturers’ instructions. Sequencing generated 150 bp paired-end reads, totaling 75,672,716 for var. pseudomezereum and 84,793,284 for D. kamtschatica. Raw reads were trimmed using the BBDuk plugin in Geneious Prime version 2025.1.2 (Biomatters Ltd., Auckland, New Zealand) and subsequently assembled with NOVOPlasty version 4.1 (Dierckxsens et al., 2017) using the complete plastome of D. laureola as the seed, as this species represents one of the earliest-diverging lineages within the genus Daphne (GenBank accession no. MN201546) (Lee et al., 2022). The assembled sequences were validated for completeness and accuracy by mapping trimmed reads back in Geneious Prime and assessing coverage uniformity, with average depths of 8,748× for var. pseudomezereum and 8,511× for D. kamtschatica. A single consensus genome was determined for each taxon. Each genome was then annotated in Geneious Prime and manually curated for start and stop codons, as well as for intron/exon boundaries. Circular maps were created in OGDRAW 1.3.1 (Greiner et al., 2019). Plastomes of var. pseudomezereum and D. kamtschatica have been deposited in GenBank at the National Center for Biotechnology Information (NCBI) under accession numbers PV752465 and PV752464, respectively.
Assembly of nuclear ribosomal DNA regions
The nrITS region, comprising ITS1, the 5.8S rRNA gene, and ITS2, was amplified from the three Daphne taxa. PCR was performed using primers ITS-p5 (5′-CCT TAT CAY TTA GAG GAA GGA G-3′) (Cheng et al., 2016) and ITS-S3R (5′-GAC CGT TCT CCA GAC TAC AAT-3′) (Chiou et al., 2007). Each reaction was performed in a 20 μL volume containing 10 μL of 2× TopTaq Master Mix (Qiagen), 0.5 μM of each primer, and approximately 15 ng of genomic DNA. The thermal cycling conditions were as follows: initial denaturation at 95°C for 15 min; 35 cycles of 95°C for 30 s, 52°C for 90 s, and 72°C for 60 s; and a final extension at 72°C for 10 min. PCR products were purified using the QIAquick PCR purification kit (Qiagen) and bidirectionally sequenced with the Sanger method on an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA) at Macrogen. The nrITS sequences generated in this study were deposited in GenBank under accession numbers PX056848 (D. pseudomezereum var. koreana), PX056852 (D. pseudomezereum var. pseudomezereum), and PX056858 (D. kamtschatica).
Phylogenetic analysis
To investigate the phylogenetic relationships among the three Daphne taxa—D. pseudomezereum var. koreana, D. pseudomezereum var. pseudomezereum, and D. kamtschatica—we constructed two datasets: a plastome dataset and a nuclear (nrITS) dataset. The plastome dataset included the two newly assembled plastomes of var. pseudomezereum and D. kamtschatica, along with ten Daphne species retrieved from the NCBI database; complete plastid sequences were aligned for a phylogenetic analysis. The nuclear dataset comprised newly generated nrITS sequences of the three target taxa together with nine Daphne species obtained from the NCBI database. After alignment, the commonly amplified region of the nrITS sequences was subjected to a phylogenetic analysis.
Both datasets included three outgroup taxa (Wikstroemia genkwa, Stellera chamaejasme, and Edgeworthia chrysantha, all in Thymelaeaceae), and all sequences were aligned using MAFFT (Katoh et al., 2019). Maximum likelihood (ML) analyses were conducted in IQ-TREE v2.2.0 (Minh et al., 2020) with 10,000 ultrafast bootstrap replicates. The best-fitting substitution models were K3Pu + F + I + G4 for the plastome dataset and GTR + F + I + G4 for the nrITS dataset, as determined by ModelFinder (Kalyaanamoorthy et al., 2017) implemented in IQ-TREE. The resulting phylogenetic trees were visualized and edited in FigTree v1.4.4 (Rambaut, 2018).
RESULTS
The plastomes of D. pseudomezereum var. pseudomezereum and D. kamtschatica were newly assembled, with lengths of 171,019 bp and 171,100 bp, respectively. These are comparable in size to the previously reported plastome of D. pseudomezereum var. koreana (171,152 bp) (Yoo et al., 2023). All three plastomes exhibited an overall GC content of 36.5% and comprised 139 genes, including 93 protein-coding genes, eight rRNAs, and 38 tRNAs. Gene content and order were highly conserved across the three Daphne taxa. A pairwise sequence identity analysis revealed consistently high similarity among the three plastomes, with the highest identity (99.9%) observed between var. koreana and D. kamtschatica, whereas the other two pairs each showed 99.7% identity similarity.
The nrITS sequences obtained from the three taxa were 579 bp in var. koreana, 586 bp in var. pseudomezereum, and 575 bp in D. kamtschatica. Pairwise sequence identity was highest between var. koreana and D. kamtschatica (97.4%), followed by var. koreana and var. pseudomezereum (96.8%).
The ML phylogenetic analyses based on the plastome (179,988 bp) and the nuclear ITS region (599 bp) revealed that the three focal taxa formed a well-supported clade in both trees, while exhibiting conflicting topologies regarding the phylogenetic placement of Daphne pseudomezereum var. koreana. In the plastome phylogeny, var. koreana was placed as a sister to D. kamtschatica (Fig. 4), whereas in the nrITS phylogeny, it was resolved as a sister to var. pseudomezereum (Fig. 5). These results suggest that var. koreana occupies a distinct phylogenetic position depending on the genomic marker analyzed.
Maximum likelihood tree inferred from plastome sequences of Daphne pseudomezereum var. koreana, var. pseudomezereum, and D. kamtschatica, together with nine additional Daphne taxa and three Thymelaeaceae outgroup species retrieved from GenBank. Newly sequenced taxa are indicated by black stars. Bootstrap support values are shown at each node.
Maximum likelihood tree inferred from nuclear ribosomal internal transcribed spacer sequences of Daphne pseudomezereum var. koreana, var. pseudomezereum, and D. kamtschatica, together with nine additional Daphne taxa and three Thymelaeaceae outgroup species from GenBank. Newly sequenced taxa are indicated by black stars. Bootstrap support values are shown at each node.
DISCUSSION
Plastome phylogeny based on complete chloroplast genome sequences shows that D. pseudomezereum var. koreana is most closely related to D. kamtschatica, whereas nrITS phylogeny places var. koreana as a sister to var. pseudomezereum. This phylogenetic discordance may reflect a complex evolutionary history, possibly involving an ancient hybrid origin. To clarify the evolutionary processes underlying this incongruence, further studies incorporating broader sampling and additional nuclear markers will be necessary. However, interestingly, such incongruence itself underscores the unique evolutionary identity of var. koreana within the genus. We believe that the taxonomic implications of this result warrant careful consideration in light of ecological, biogeographical, life-history, and morphological evidence.
Ecologically, var. koreana occurs in cool-temperate regions along the Baekdudaegan mountain range, with a distribution extending into southern Manchuria, southern Primorsky Krai, Sakhalin, and Hokkaido. It is absent from the warm-temperate regions of Honshu and Shikoku (for details, see Taxonomic note). This distribution closely parallels that of D. kamtschatica in both habitat and climate, whereas D. pseudomezereum var. pseudomezereum is restricted to the warm-temperate deciduous forests of Japan (Fig. 1).
Biogeographically, if var. koreana is interpreted as a lineage within D. kamtschatica, its current distribution—spanning the Baekdudaegan mountains, southern Manchuria, southern Primorsky Krai, Sakhalin, and Hokkaido—can be viewed as the southwestern remnant of a lineage formerly widespread across the cool-temperate zone of Northeast Asia. In contrast, D. kamtschatica occupies the northeastern range, occurring on the Kamchatka Peninsula, in southern Sakhalin, and on Hokkaido (recently documented) (Nitta and Uchida, 2020). Taken together, these patterns indicate that vars. koreana and D. kamtschatica represent the southwestern and northeastern segments, respectively, of a lineage that once occupied continuous habitats (Fig. 1). The present disjunction most likely reflects vicariant fragmentation driven by repeated land-bridge dynamics associated with Quaternary climatic oscillations around the Sea of Okhotsk.
Life-history traits further support our interpretation. Both var. koreana and D. kamtschatica develop well-defined rhizomes, indicating a shared evolutionary strategy, whereas their complete absence in var. pseudomezereum highlights a clear divergence in life-history traits. In addition, morphologically, var. koreana shares key features with D. kamtschatica, including a palm-like leaf arrangement during flowering (Fig. 3A), white calyx lobes, anthers included within the calyx tube (Fig. 3B), and a relatively short tube (Table 1).
Taken together, our results indicate that var. koreana represents a genetically and evolutionarily independent lineage. Given its close affinity to D. kamtschatica across ecological, biogeographical, life-history, and morphological traits, we regard it as a variety of that species rather than a separate species. This treatment is further supported by the slight but consistent difference in calyx tube coloration (dark red purple in var. koreana versus entirely white in D. kamtschatica).
TAXONOMIC TREATMENT
Daphne kamtschatica var. koreana (Nakai) E. K. Han & J. H. Lee, comb. nov.
Daphne koreana Nakai, J. Jap. Bot. 13: 880, 1937; Daphne pseudomezereum var. koreana (Nakai) Hamaya, Bull. Tokyo Univ. Forest 55: 72, 1959.—TYPE: KOREA. Gyeongsangbukdo, Yeongyang-gun, Mt. Irwolsan, 27 Jul 1937, T.H. Chung s.n. (holotype TI, [digital photograph!]).
Note: Plants occurring in Honshu (mainly the Kanto Mountain Range) and Shikoku have also been treated as Daphne pseudomezereum var. koreana. These populations, restricted to mountaintops in warm-temperate regions, are characterized by distinctive floral traits—anthers approximately half-exserted from the calyx tube (Nitta and Uchida, 2020), pale green calyx lobes, and a dark red purple calyx tube. Notably, they completely lack rhizomes, making them fundamentally distinct from var. koreana in terms of their life-history strategy (Nitta and Uchida, 2020). These distinctions suggest they may represent separate taxonomic entities more closely allied with D. pseudomezereum than with var. koreana. Considering that the type locality of D. pseudomezereum var. koreana is situated in Korea, it is taxonomically more appropriate not to apply this name to populations occurring in Honshu and Shikoku. Consequently, we conclude that D. kamtschatica var. koreana (Nakai) E. K. Han & J. H. Lee does not occur in the warm-temperate regions of Japan.
Notes
ACKNOWLEDGMENTS
We sincerely appreciate the Herbarium, Royal Botanic Gardens, Kew (K), for granting access to Daphne kamtschatica specimens and for providing the leaf material used in this study. We are also grateful to Dr. Noritoshi Nitta (Hokkaido Research Organization, Japan) for granting permission to use the photographs of D. kamtschatica. Our sincere thanks go to the anonymous reviewers for their efforts to improve the manuscript. This work was funded by the National Research Foundation of Korea (No. RS-2022-NR074927) and by the Korea National Arboretum (KNA 1-1-30-25-1).
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.