Genetic insights into species diversity and taxonomy of Ulva (Ulvales, Chlorophyta) in coastal Korea

Sampling and specimen processing

Our comprehensive dataset included 130 Ulva specimens examined both genetically and morphologically (Fig. 1, Supplementary Table S2), including 51 specimens collected in 2023 from the coastal regions of Jeju Island, Geoje, and Tongyeong, in the south coast of Korean peninsula, and 79 Ulva herbarium specimens housed at the Jeju National University Herbarium (JNUB) collected during expeditions spanning 2017 to 2023. The JNUB specimens were prepared as vouchers for other studies, including investigations on bloom-forming species (2017–2018) and bioactivity evaluations of Ulva species (2021–2023). The former study focuses on Jeju Island, while the latter study covers Jeju Island (Hallim-eup, Seongsan, Udo, Biyangdo), Namildae (Sacheon-si, Gyeongsangnam-do), Yeongdeok, Uljin (Gyeongsangbuk-do), Yangyang (Gangwon-do), Ulleungdo, and Taean (Chungcheongnam-do), representing the western, southern, and eastern coasts of Korea and its offshore islands. None of the sequences of the specimens in these two studies has been previously submitted to NCBI or published. Fresh specimens were stored in a cool bag and transported to the laboratory immediately after collection. For each fresh specimen, a small fragment was thoroughly washed in clean seawater to remove epiphytes, followed by careful drying and preservation in silica gel for subsequent DNA extraction. The remaining portion was mounted and dried as herbarium sheets and deposited at JNUB.

Fig. 1

Sampling map of Ulva specimens across Korea, including both newly collected and herbarium samples, used in this study.

Molecular data acquisition

Approximately 1 cm² of fresh thallus or 0.5–1 cm² of dried thallus, depending on specimen availability, was used for each DNA extraction. Total genomic DNA was extracted using the MagPurix Plant DNA Extraction Kit v.1.3 (P02014; Zinexts Life Science Corporation, New Taipei City, Taiwan). AccuPower PCR Premix (Bioneer, Daejeon, Korea) was used according to the manufacturer’s protocol for all PCR reactions. Three genetic markers were amplified employing the forward primer of Hayden et al. (2003) and the reverse primer and PCR conditions described in Blomster et al. (1998) for ITS, and Saunders and Kucera (2010) for rbcL and tufA. Primer sequences and annealing temperatures for all three markers are listed in Supplementary Table S3. A total of 89 ITS sequences, 92 tufA sequences, and 92 rbcL sequences were generated (Supplementary Table S2).

Phylogenetic analyses

Only unique Korean haplotypes (30 ITS, 28 tufA, and 49 rbcL), identified using CD-HIT (threshold 0.999) (Huang et al. 2010) on the public Galaxy platform (Mareuil et al. 2017), were included in further analyses. Public sequences from GenBank, including the closest BLAST hits of the Korean sequences, were added to the datasets of the three markers. Species from Umbraulva E. H. Bae & I. K. Lee, Percursaria Bory de Saint-Vincent, and Ulvaria Ruprecht were used as outgroup taxa. Alignments were constructed using the MAFFT online server using default settings (Katoh and Standley 2013) and subsequently inspected and trimmed manually in MEGA X (Kumar et al. 2018). The final alignment lengths were 620 bp (316 variable sites, 256 parsimony-informative sites) for ITS, 607 bp (206 variable sites, 159 parsimony-informative sites) for tufA, and 972 bp (224 variable sites, 134 parsimony-informative sites) for rbcL. Single gene maximum likelihood (ML) analyses with ultrafast bootstrapping (1,000 replicates) were conducted using the IQ-TREE v. 1.6.12 on the web server (Trifinopoulos et al. 2016), with the best-fit partitioning schemes and models of molecular evolution selected using the Bayesian information criterion (BIC) criterion using ModelFinder integrated in IQ-TREE (Kalyaanamoorthy et al. 2017).

Representatives of each species clade, chosen based on the closest related sequences identified in the single gene phylogenies and curated names in the literature and/or GenBank, were included in the concatenated sequences (tufA + rbcL + ITS) (Supplementary Table S4). Since only a short fragment of rbcL (110 bp) is available for the U. conglobata lectotype, and this fragment has been shown to fall within the same clade as Ulva sp. 1 (Matsumoto and Shimada 2015, Hughey et al. 2021), we used Ulva sp. 1 and specimen JNU0871 as representatives for this lineage. This approach avoids the instability caused by extremely short sequences while still preserving the phylogenetic placement of the lectotype. For the ML phylogenetic analysis, the best-fit partitioning schemes by gene and models of molecular evolution determined using the BIC criterion in ModelFinder integrated in IQ-TREE were TPM3u + F + I + G4 for tufA, TPM3u + F + I + G4 for rbcL, and TIM + F + I + G4 for ITS.

Morphological observations

Both freshly collected and herbarium specimens underwent meticulous examination, with morphological features recorded, encompassing external morphology, including size, shape of the thallus, branches, and holdfast, as well as cell characteristics such as size, shape, arrangement in different parts of the thallus, number of pyrenoids per cell, and chloroplast positions. Herbarium specimens were rehydrated following in-house protocols using warm water with a small amount of detergent or, for some samples, a mild buffer solution. Microphotographs were taken using a microscope (BX43; Olympus, Tokyo, Japan) equipped with an EOS 600D digital camera (Canon, Tokyo, Japan) at magnifications ranging from 10× to 100×. Cell dimensions were measured directly from calibrated microphotographs, using the smallest and largest values observed across all specimens examined to represent the morphological range for each species.

Single gene trees

The tufA phylogenetic tree resolved the Korean sequences into 12 well-supported species-level clades (Supplementary Fig. S1). Of these, seven were blade-like Ulva species, identified as U. adhaerens Kaoru Matsumoto & S. Shimada, U. arasakii, U. australis, U. californica, U. lacinulata, U. ohnoi, and U. pseudo-ohnoi, among which U. australis emerged as the most prevalent species in our dataset. The remaining five species were identified as tubular species, namely U. aragoënsis (Bliding) Maggs, U. compressa, U. linza, U. torta/clathratioides, and an unidentified species.

The rbcL phylogenetic tree delineated 13 species, exhibiting similarity with the tufA gene-based phylogenetic tree, albeit with lower support values (Fig. 2). Notable differences with the tufA results included the identification of a specimen from the Udo channel at a depth of 15–20 m as U. iliohaha H. L. Spalding & A. R. Sherwood. Because no tufA sequence was obtained for this specimen, this identification could not be verified in the tufA-based analysis. Additionally, the unidentified tubular species in the tufA phylogenetic tree clustered with sequences of U. partita K. Ichihara described from Japan (pairwise intraspecific divergence 0.11–0.13%). A Korean specimen (U52_GD_SM_L_1), forming a clade with sequences designated U. californica in the tufA tree, did not cluster with other U. californica rbcL sequences but stood as a distinct clade. The rbcL sequence of the U. pseudo-ohnoi holotype and the specimen JNU0871 clustered within the same clade as the sequence of Ulva sp. 1 from Japan (representative for the U. conglobata type specimen) (pairwise intraspecific divergence 0%), affirming their conspecificity based on the rbcL gene.

Fig. 2

Maximum likelihood tree based on the rbcL alignment. Numbers at the nodes indicate UF bootstrap values for 1,000 replicates. Outgroup taxa were pruned out of the tree. The representative Korean sequences generated in this study are highlighted in bold.

The phylogenetic tree reconstructed using ITS sequences delineated 13 genetic groups (Supplementary Fig. S2), largely concordant with the results of the tufA and rbcL phylogenetic trees. The Korean specimen JNU0871, identified as U. pseudo-ohnoi in the tufA-based phylogenetic tree, instead clustered in the ITS tree with the Japanese Ulva sp. 1 and specimens identified as U. ohnoi, U. reticulata Forsskål, and U. spinulosa Okamura & Segawa (pairwise intraspecific divergence 0.002–0.22%).

Concatenated phylogenetic tree

Although there are discrepancies in the single gene trees (Supplementary Fig. S3), the concatenated phylogenetic tree of tufA, rbcL, and ITS confirmed the presence of 15 genetically distinct Ulva species in Korea, with all nodes well supported (UF bootstrap values mostly > 98) (Table 1, Fig. 3). The blade-like species included U. adhaerens, U. arasakii, U. australis, U. californica, U. iliohaha, U. lacinulata, U. lactuca, U. ohnoi, U. pseudo-ohnoi, and U. tanneri H. S. Hayden & Waaland. The tubular species comprised U. aragoënsis, U. compressa, U. linza, U. partita, and U. torta/clathratioides. Among these, four species, namely U. aragoënsis, U. iliohaha, U. lacinulata, and U. partita, are newly recorded in Korea.

Table 1

Current understanding of Ulva species diversity in Korea based on molecular and/or morphological evidence

Fig. 3

Maximum likelihood tree based on the concatenated (rbcL, tufA, and ITS) alignment. Numbers at the nodes indicate UF bootstrap values for 1,000 replicates. Outgroup taxa were pruned out of the tree. The representative Korean sequences generated in this study are highlighted in bold.

In the concatenated phylogenetic tree, the sequences of the U. pseudo-ohnoi holotype (tufA + rbcL), the specimen JNU0871 (tufA + rbcL + ITS), clustered with the Japanese Ulva sp. 1 (rbcL + ITS). It provides further evidence that U. pseudo-ohnoi and Ulva sp. 1 are the same species.

Morphological observations

A summary of the morphology of specimens within the species complex Ulva conglobata, “U. pseudo-ohnoi” holotype, U. conglobata lectotype, Ulva sp. 1, and the newly collected U. conglobata JNU0871, is provided in Table 2. The “U. pseudo-ohnoi” holotype (blade-like morphology) from Jeju Island, Korea exhibit similarities in cell shape, thallus thickness, and chloroplast position with the U. conglobata lectotype, Ulva sp. 1 from Japan, and U. conglobata JNU0871. The primary differences are observed in the presence or absence of rosette-like gross morphology and denticulate margins.

Table 2

Summary of morphological features of Ulva conglobata lectotype, Ulva sp. 1, U. pseudo-ohnoi holotype, and U. pseudo-ohnoi JNU0871

The featured morphology (gross morphology, surface view, and cross-sections of the middle part of the thallus) of five tubular and 10 blade-like Ulva in Korea is summarized in Table 3 and Figs 4 & 5. Additional morphological details, including morphological variation, surface view and cross-sections of the basal part of the 15 Ulva species from Korea are presented in Supplementary Text S1 and Figs S4–S18. Based on these results and morpho-anatomical observations of Ulva specimens from Lee et al. (2019), we developed a preliminary identification key for Ulva species in Korea. While the key offers a practical field reference for ecologists and the general public, it should be used with caution. Due to the high morphological variation within Ulva species, it is best complemented with DNA analysis for accurate species identification. As further variations are documented, the key should be updated accordingly. Traits marked “(in part)” indicate morphologically overlapping species complexes.

Table 3

Summary of morphology of Korean Ulva species

Fig. 4

Summary of morphology of five tubular Ulva in Korea. The featured morphology includes the whole thallus/thalli (A, D, G, J & M), the surface view (B, E, H, K & N), and the cross-section (C, F, I, L & O) of the middle thallus. (A–C) Ulva aragoënsis. (D–F) Ulva compressa. (G–I) Ulva linza. (J–L) Ulva partita. (M–O) Ulva torta/clathratioides. Scale bars represent: A, D, J & M, 1 cm; B, F, K, L, N & O, 50 μm; C, E, H & I, 100 μm; G, 2 cm.

Fig. 5

Summary of morphology of 10 blade-like Ulva in Korea. The featured morphology includes the whole thallus/thalli (A, E, I, M, Q, U, Y, CC, GG & KK), the surface views (B, C, F, G, J, K, N, O, R, S, V, W, Z, AA, DD, EE, HH, II, LL & MM), and the cross-section (D, H, L, P, T, X, BB, FF, JJ & NN) of the middle thallus. (A–D) Ulva adhaerens. (E–H) Ulva arasakii. (I–L) Ulva australis. (M–P) Ulva californica. (Q–T) Ulva conglobata. (U–X) Ulva iliohaha. (Y–BB) Ulva lacinulata. (CC–FF) Ulva lactuca. (GG–JJ) Ulva ohnoi. (KK–NN) Ulva tanneri. Scale bars represent: A, E, U, Y & GG, 2 cm; B, 200 μm; C, D, G, K, L, N–P, R, AA, BB, DD, II, JJ & NN, 50 μm; F, H, J, Z & HH, 100 μm; I, M, Q, CC & KK, 1 cm; S, T, V-X, EE, FF, LL & MM, 20 μm.

Key to the species of Ulva in South Korea

Diversity and distribution

Based on our sampling, we observed a slightly higher diversity of blade-like species in terms of both species richness and abundance compared to tubular species. Specifically, our study, along with previous studies, recorded 10 blade-like species in Korea, whereas only five tubular species were identified (Table 1). Among the blade-like species, U. australis (44) had the highest number of specimens and the widest distribution, being found from Ulleungdo, across the East and South coasts, to Jeju Island. Ulva lacinulata (5) was the second most widely distributed species, occurring along the East and South coasts and on Jeju Island. Ulva californica (3) was present along the East coast and Jeju. Seven other blade-like species, U. adhaerens (4), U. arasakii (7), U. lactuca (Lee et al. 2019), U. ohnoi (2), U. conglobata (previously known as U. pseudo-ohnoi) (2), and U. tanneri (3) were found in various locations around Jeju Island, except for U. iliohaha (1), which has so far only been observed in the Udo Channel, specifically in sandy bottom and rhodolith beds at depths of 5 to 15 m in turbid waters. It was recorded during our 2023 sampling period, and additional field observations in 2024 showed the same seasonal and habitat pattern. Among the tubular Ulva, U. linza (21) was found to be the most abundant and widely distributed species, ranging from Ulleungdo to the East and South coasts, as well as Jeju Island. Ulva partita (11) was observed in Ulleungdo, the South coast, and Jeju Island. Ulva compressa (7) was found in Ulleungdo, the East and West coasts, and Jeju Island. Ulva aragoënsis (5) was recorded on the South coast and Jeju Island. Finally, U. torta (2) was exclusively found on Jeju Island.

Expanding the knowledge of distribution and morphological variation of Ulva conglobata

A previous study of Hughey et al. (2021) noted genetic similarities between the rbcL sequences of the U. conglobata lectotype (Yokohama, Japan) and Ulva sp. 1 from a nearby locality (Yokosuka). Subsequently, Carneiro et al. (2023) formally synonymized U. pseudo-ohnoi with U. conglobata. Our phylogenetic analysis confirms that rbcL sequences of the U. conglobata lectotype formed a clade with U. pseudo-ohnoi holotype, Ulva sp. 1, and specimen JNU0871. Ulva sp. 1, U. conglobata, U. pseudo-ohnoi, and JNU0871 are also morphologically highly similar, providing additional evidence for their conspecificity (Table 3).

Our DNA-based findings extend the known distribution range of U. conglobata, previously reported only from Japan (Kjellman 1897, Hughey et al. 2021), to Korea. Based on morpho-anatomical analysis, Hughey et al. (2021) documented the distribution of U. conglobata across multiple localities in the western Pacific and Indian Oceans, and called for DNA analyses of rosette forms of Ulva outside of Japan to verify this range of U. conglobata. However, this rosette morphology is not unique for U. conglobata. A recent study in Vietnam further highlights the discrepancy between morpho-anatomical and DNA-based species identification, revealing that a specimen with rosette-like morphology was genetically identified as U. lactuca (Tran et al. 2023). Additionally, rosette-like morphology has been observed in other Ulva species, including U. tanneri, U. pertusa Kjellman (synonym of U. australis), and U. fasciata Delile (synonym of U. lactuca) (Matsumoto and Shimada 2015, this study). These findings demonstrate significant morphological overlap among species, and reinforce the necessity of molecular data for accurate species identification, particularly for taxa exhibiting high morphological plasticity. Existing records of Ulva with rosette-like morphology currently assigned to U. conglobata require reassessment using DNA-based methods.

The distinct morphological forms observed in U. conglobata, a large-bladed “U. pseudo-ohnoi” morphotype restricted to the upper subtidal zone (Lee et al. 2019), and a rosette-like morphotype confined to the upper intertidal, may reflect phenotypic plasticity in response to contrasting environmental conditions. This variation in thallus form, aligned with specific ecological niches, highlights the potential for environmentally induced morphological flexibility in U. conglobata. Although subtle genetic differences exist between these morphotypes, their biological significance remains unclear. These patterns raise the possibility that ecological factors could contribute to early divergence, but this requires further investigation. Differences in desiccation pressure, light regimes, or predation between intertidal (rosette form) and subtidal (blade-like form) habitats could influence both morphology and genetic structure. Further research, including genomic analyses and common garden experiments, are needed to determine the extent of genetic divergence and validate the hypothesis of incipient speciation within this intriguing species complex.

Integrative assessment of species diversity and distribution of Ulva in Korea

This study provides a comprehensive assessment of Ulva diversity along the Korean coast, integrating molecular and morphological approaches. Our findings confirm the limitations of morphology-based identification, given the high variability observed in Ulva species, and reinforce the necessity of molecular tools for accurate species delimitation and taxonomic classification. By analyzing three genetic markers (tufA, rbcL, and ITS) alongside morpho-anatomy features, we identified 15 genetically distinct Ulva species and provided detailed morphological descriptions for each species. Among these, there are 10 blade-like species: U. adhaerens, U. australis, U. arasakii, U. californica, U. conglobata, U. iliohaha, U. lactuca, U. ohnoi, U. tanneri, and U. lacinulata, as well as five tubular species: U. aragoënsis, U. compressa, U. linza, U. torta, and U. partita.

Species identification was based on DNA data from type specimens, obtained either directly or indirectly through validated reference sequences representing the type concept. This included the types of U. adhaerens, U. australis, U. californica, U. conglobata, U. iliohaha, U. ohnoi, U. lacinulata, U. lactuca, U. tanneri, and U. partita, as well as one recently combined species (U. aragoënsis), for which the sequence from the publication of establishing the new combination serves as a molecular reference of the current species concept (Krupnik et al. 2018). Because only short DNA fragments were recovered from the holotype of U. lactuca and the type specimen of U. australis, species identification was based on verified reference sequences. Ulva lactuca was identified using DNA from the lectotype of U. lobata (Kützing) Harvey, which has been demonstrated to be conspecific with U. lactuca (Hughey et al. 2019). Likewise, U. australis was identified using sequences of Australian material (Kraft et al. 2010) shown to be identical to the type specimen (Hanyuda et al. 2018). Although this study did not directly include sequences of type specimens of U. californica and U. tanneri because they were not available at the time of analysis, the identification of the analyzed lineages was anchored to publicly available reference sequences whose identities have been subsequently confirmed through comparison with type material (Hughey et al. 2024).

For the remaining five species, identification relied on integrative comparisons combining morphology, ecology, type locality, and DNA similarity to well-characterized reference collections (Shimada et al. 2003, Kraft et al. 2010, Steinhagen et al. 2019). The absence of DNA sequences from many type specimens, particularly tubular Ulva species, remains a major obstacle to taxonomic resolution. Although the number of sequenced type specimens has increased in recent years, some historical types will likely remain unsuitable for molecular analysis (Tran et al. 2022). While comparisons with modern reference material cannot provide absolute taxonomic certainty, they represent a pragmatic and reproducible framework for aligning molecular lineages with traditional species names and for facilitating future comparisons when type-derived DNA data become available (Hughey et al. 2024).

Our results largely confirm previous molecular and morphological records of Ulva in Korean waters while refining and correcting several species boundaries. Eleven previously reported species, U. arasakii, U. californica, U. adhaerens, U. australis, U. compressa, U. conglobata, U. lactuca, U. ohnoi, U. linza, U. torta, and U. tanneri, were genetically verified, confirming their taxonomic validity. Notably, U. torta shares a similar molecular species concept with U. clathratioides L. G. Kraft, Kraft & R. F. Waller, but the absence of type sequences for U. torta precludes confirmation of conspecificity. In this study, we adopt the name U. torta for this molecular species concept, as it has nomenclatural priority over U. clathratioides. Furthermore, we report four new Ulva records for Korea, including two blade-like species (U. iliohaha and U. lacinulata) and two tubular species (U. aragoënsis and U. partita). In contrast, our data indicate that U. pseudo-ohnoi is conspecific with U. conglobata.

Eight taxa previously recorded in Korea based on morphology, including U. flexuosa, U. clathrata, U. intestinalis, U. polyclada Kraft, U. prolifera, U. reticulata, U. rigida C. Agardh, and U. sublittoralis Segawa, were not genetically confirmed in our study. For example, U. flexuosa specimens identified by Kang et al. (2019) based on tufA and ITS sequences cluster with U. aragoënsis in our prior analysis (personal note), suggesting misidentification. The absence of other taxa in our data may represent true absences due to habitat-restricted or rare species not encountered during our sampling, or unresolved cases requiring molecular re-evaluation. These findings collectively update the checklist of Korean Ulva and provide a revised molecular framework for future taxonomic and ecological studies.

The known ranges of U. australis, U. aragoënsis, U. compressa, U. californica, U. lacinulata, U. linza, and U. partita are genetically confirmed to extend from Jeju Island to mainland coasts. Other species remain restricted to Jeju, with U. iliohaha recorded only from the Udo channel. This pattern may reflect denser sampling around Jeju, or genuinely higher diversity of Ulva in this marine diversity hotspot.

An interesting trend observed in our data is that a greater number of blade-like species were identified than tubular species, which contrast with patterns observed in Vietnam (Tran et al. 2023), subtropic-warm temperature regions of Japan (Ogawa et al. 2013), Australia (Kraft et al. 2010), and New Caledonia (Lagourgue et al. 2022). These regions situated in subtropical to tropical climates, where tubular Ulva diversity is often greater. Given that our sampling primarily targeted marine environments, whereas tubular species are more prevalent in brackish habitats, the low diversity of tubular taxa in Korea may reflect a sampling bias rather than a true biogeographical pattern. Broader surveys in estuarine and brackish systems will be necessary to evaluate this observation more rigorously and to enable meaningful regional comparisons.

This study represents the most comprehensive molecular assessment of Ulva diversity in Korea to date, identifying 15 distinct species and refining taxonomic classifications. Our findings underscore the importance of integrative taxonomy in resolving species boundaries within Ulva, particularly given the genus’s high morphological plasticity. These results have broader implications for ecological monitoring, aquaculture, and the management of green tides in Korea. Future research should focus on expanding sampling to brackish environments and conducting further molecular analyses to clarify the taxonomic status of morphologically identified species.