A New Record of Antithamnion hubbsii (Ceramiales, Rhodophyta) from the Korean Coast: Invasive Species Interactions with Native and Non-Native Communities

1. Introduction

Marine ecosystems serve as habitats for various algal species, which play crucial roles as primary producers. Red algae (Rhodophyta) have adapted to diverse environments and are widely distributed throughout the world’s oceans (Rueness et al., 2007). Among them, the genus Antithamnion has received continuous attention from marine biologists due to its morphological diversity and ecological significance. For instance, Antithamnion nipponicum has been introduced to new regions, such as the Norwegian coast, highlighting the impact of globalization on marine ecosystems (Rueness, 2007). Additionally, Antithamnion sparsum has been recently recorded in the Northwest Atlantic, showcasing the ongoing spread of non-indigenous species in marine environments (Brooks and Krumhansl, 2023).

The study by Cho et al. (2007) made significant contributions to resolving taxonomic confusion within the genus Antithamnion. Their research clarified the relationship between A. hubbsii and A. nipponicum while providing molecular evidence that A. nipponicum is distinct from A. pectinatum. However, later finds of a similar alga from the Azores (Athanasiadis & Tittley, 1994) were classified as A. pectinatum (Montagne) Brauner, with A. nipponicum reduced to synonymy (with a question mark), further illustrating the historical ambiguity surrounding these taxa. According to their findings, A. hubbsii and A. nipponicum have nearly identical rbcL gene sequences and share morphological characteristics, leading to the proposal that they may represent the same species. They confirmed the presence of A. nipponicum along the Pacific and Atlantic coasts of North America, suggesting it was a recent introduction from Japan.

However, misidentifications within the genus Antithamnion remain common due to the high degree of morphological similarity among species (Cho et al. 2007). In particular, A. nipponicum and A. hubbsii possess very similar morphological features, making them difficult to distinguish using traditional methods alone. Cho et al. emphasized that the key identifying features of A. hubbsii include indeterminate lateral axes of pinnae, oppositely arranged pinnules, and gland cells located on the adaxial surface of pinnules.

Until now, only A. nipponicum has been reported from Korean waters, with no confirmed presence of A. hubbsii. However, through detailed morphological observations and molecular phylogenetic studies of specimens collected from the eastern coast of Gangneung, it was revealed that some specimens previously identified as A. nipponicum are actually A. hubbsii. This represents the first report of A. hubbsii in Korean waters, deepening our understanding of red algal biodiversity in Korea.

The introduction pathway of marine invasive species is often linked to international maritime activities. In the case of A. hubbsii in Korean waters, two potential introduction vectors are worthy of investigation. First, given that A. hubbsii has been well-documented along the Pacific coast of North America (Cho et al., 2007), increasing cruise ship traffic between North American ports and Korean destinations could serve as a potential vector. Cruise ships carry large volumes of ballast water and provide hull surface area for biofouling organisms, both of which are recognized as major pathways for marine species introductions (Carlton & Geller, 1993). Second, considering that Japan is geographically proximate to Korea and that A. nipponicum is native to Japanese waters, natural dispersal or smaller-scale maritime activities between Japan and Korea could have facilitated the introduction of A. hubbsii, especially if it was previously misidentified in Japan as well.

Our research employs both traditional morphological and modern molecular approaches to accurately identify and characterize A. hubbsii from Korean waters. This integrated approach is essential for distinguishing between morphologically similar species and establishing the phylogenetic relationships between Korean specimens and those from other regions. By documenting this new record and investigating its introduction pathway and ecological context, this study contributes to our understanding of marine biodiversity in Korea and provides valuable information for monitoring and managing non-native species in Korean coastal ecosystems.

2. Materials and Methods

2.1. Sampling, Culture and Microscopy

The samples were collected in the rocky intertidal of Sungeut beach (37°49′2.6″N, 128°53′40.5″E), Gangneung-si, Gangwon-do, Korea, on January 6, 2024. Culture conditions consisted of sterilized seawater supplemented with IMR (Institute of Marine Resources) medium as described by Klochkova et al. (2006). Samples were maintained at 20°C under cool-white fluorescent lights providing >20 µmol photons m⁻² s⁻¹, with a 16 h light ⁄ 8 h dark cycle. Culture maintenance involved transfer to new IMR medium every 7-14 days. Samples including the type specimen were deposited at the National Marine Biodiversity Institute of Korea (MABIK: AL00100***).

Live imaging was performed using an Olympus BX54 research microscope featuring differential interference contrast (DIC) capabilities and integrated with a Samsung iPolis imaging system (Samsung, Suwon, Korea). Nuclear visualization was performed following cell fixation in culture media using microwave irradiation for a few seconds. Subsequently, cells were stained with Hoeschst 33342 (1 μl/mL in the IMR medium) for 15 minutes under dark conditions prior to microscopic examination. Light and fluorescence microscopy images were merged using Adobe Photoshop CS6 software. The images were then analyzed using ImageJ software (NIH, http://imagej.nih.gov/ij/) to quantify the length of the cells and the diameter of the nuclei.

2.2. Molecular Analysis

Total DNA was extracted from fresh or dried samples using the Chelex method (Zuccarello et al. 1999). The partial sequence of the plastid-encoded ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) was amplified using the primer pairs F321 and R1150 (Kang et al. 2020). PCR conditions followed a touchdown PCR as follows: initial denaturation at 94°C for 4 min, followed by 10 cycles of 94°C for 1 min, 55°C for 30sec, and a decrease in annealing temperature by 1°C per cycle, and 72°C for 1 min; followed by 25 cycles of 94°C for 1min, 45°C for 30sec, and 72°C for 1 min and a final step at 72°C for 5 min. Additionally, the partial sequence of the plastid-encoded photosystem I P700 chlorophyll a apoprotein A1 gene (psaA) was amplified using the primer pairs psaA130F (5’-AACWACWACTTGGATTCGAA-3’) and psaA1760R (5’-CCTCTWCCWGGWCATCWCAWGG-3’) (Yoon et al. 2002; Saunders & Moore 2013). The PCR conditions for psaA followed a touchdown protocol: initial denaturation at 94°C for 2 min, followed by 5 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min; then 35 cycles of 94°C for 30 s, 46.5°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 7 min. PCR products were purified using Zymoclean^TM Gel DNA recovery Kit (Zymo Research, USA). PCR products were sent for commercial Sanger sequencing using both sets of PCR primers (Cosmogene Tech, Daejeon, Korea). Sequences were edited and assembled in Geneious^® Prime 2025.1.2 (https://www.geneious.com).

For phylogenetic analyses, the newly determined rbcL and psaA sequences were analyzed alongside sequences from various Antithamnion and Anthithamnionella species, plus related genera downloaded from GenBank. Details of all specimens used in this study, including collection information, voucher numbers, and GenBank accession numbers, are presented in Table 1. The data set was aligned using MAFFT in Geneious^® Prime 2025.1.2 (https://www.geneious.com) together with our new sequences of Antithamnion hubbsii collected from Korea (PV453999). Phylogenetic analyses were performed using the maximum likelihood (ML) method with IQ-TREE 2.3.4 (Minh et al. 2020). Codon positions were partitioned and the best-fit model was determined using ModelFinder (Kalyaanamoorthy et al. 2017) and partition model (Chernomor et al. 2016). The rbcL DNA substitution models were selected for each codon (i.e., first codon: TN+F+I; second codon: F81+F+I; third codon: HKY+F+G4) were selected. The psaA DNA substitution models were selected for each codon (i.e., first codon: TIM2+F+G4; second codon: TIM+F+I; third codon: TPM3u+F+I) were selected. Support for each internal branch was determined by non-parametric bootstrap (500 replicates) (Felsenstein 1985). Bayesian inference analysis was conducted with MrBayes v. 3.2 (Ronquist et al. 2012) with partitioned codons using variable rates and six rate categories. Two parallel runs of Markov chain Monte Carlo were performed for 5,000,000 generations, sampling every 1,000 generations. Estimated sample sizes, split frequencies, and stationarity were checked after each run. After analysis, 10% of generations were removed as burn-in and the posterior probabilities were visualized in Figtree v1.1.4 (Rambaut 2009). Two DNA-based species delimitation methods were used to determine species status. For the Assemble Species by Automatic Partitioning (ASAP) method, the Kimura (K80) Ts/Tv 2.0 model was selected and analyzed from the ASAP online platform (https://bioinfo.mnhn.fr/abi/public/asap/). A tree-based method (Poisson-Tree processes, PTP; Zhang et al. 2013) was used online using the ML tree topology. Additionally, the Automatic Barcode Gap Discovery (ABGD) method was applied using the online ABGD platform (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html) with default parameters, including a relative gap width (X) of 1.5 and prior intraspecific divergence (P) values ranging from 0.001 to 0.1 (Puillandre et al. 2012). The final tree was then edited using Adobe Illustrator 2024 (Adobe, San Jose, CA, USA).

3. Results

3.1. Morphological Observations

Genus: Antithamnion

Species: Antithamnion hubbsii

Type locality: Intertidal, Pacific Grove, California, USA; (Dawson 1925)

Collection site in Korea: Antithamnion hubbsii collected from Sungeut beach, Gangneung-si, Gangwon-do (37°49′2.6″N, 128°53′40.5″E); collected 6 January 2024.

Habitat: Intertidal, epiphytic on other macroalgae.

Holotype: Dawson E.Y. 1925. Specimens collected from intertidal zone, Pacific Grove, California, USA; deposited in the University of California Herbarium, Berkeley (UC), accession number UC 261983.

Paratype: KNU000620-KNU000622 (Sungeut beach (37°49′2.6″N, 128°53′40.5″E), Gangneung-si, Korea, 30 March 2013, Eunyoung Shim and Soo Yeon Kim); deposited in the National Marine Biodiversity Institute of Korea (MABIK).

Specimens examined: KNU000620- KNU000622 (Sungeut beach (37°49′2.6″N, 128°53′40.5″E), Gangneung-si, Korea, 30 March 2013, Eunyoung Shim and Soo Yeon Kim).

GenBank accession number: PV453999 (rbcL), PV454032 (psaA)

Description : Antithamnion hubbsii is a delicate red alga with filamentous thallus organization displaying distinct morphological features. Thalli are generally small, reaching approximately 1-5 cm in height, with a characteristic pinnate branching pattern (Figure 1B). Antithamnion hubbsii is characterized by a thallus composed of both prostrate and erect axes. The erect axis comprises main and indeterminate lateral axes of unlimited growth bearing opposite pinnae of determinate growth. Indeterminate lateral axes are irregularly produced from the basal cell of pinnae in erect axes (Figure 1D).

Vegetative features: The main axis consists of prominent axial cells (Ax) that are cylindrical and elongated (Figure 1A). Branching is primarily distichous (arranged in two rows), with distinct pinnae (Pn) and pinnules (Pl) forming a regular, feather-like appearance (Figure 1A). Apical cells (AC) are prominent at the tips of growing branches and are responsible for indeterminate growth (Figure 1A). Rhizoids (rh) develop from the lower portions of the thallus and serve as attachment structures (Figure 1A). Each branch originates from a basal cell (BC) at the nodes of the main axis (Figure 1C). A distinctive feature is the presence of spherical gland cells (GC) situated adjacent to basal cells of lateral branches (Figure 1C). These gland cells are typically colorless to pale and conspicuous under light microscopy, borne on pinnae, adaxial, and covering 2 cells as shown in Table 1.

Table 1. Distinguishing characteristics of local and morphologically similar Antithamnion spp. as well as Antithamnionella.

Species	Gland cells	Indeterminae laterals	Pinnae	Branching plane	Tetrasporangia
New collection, Antithamnion hubsii Shim & Kim, 2024	Borne on pinnae, adaxial, covering 2 cells	Paired	Pinnate (diploid) Adaxially pectinate (haploid)	Distichous	1-cell pedicel
Antithamnion hubsii Dawson, 1963	Borne on pinnae, adaxial, covering 2 cells	Paired	Pinnate	Distichous	NA
Antithamnion nipponicum Yamada et Inagaki	Borne on pinnae, adaxial, covering 2 cells	Paired	Pinnate	Distichous	1-cell pedicel
Antithamnion cruciatum Nägeli, 1847	On pinnae, covering 2 cells	Paired w/ determinate lateral	Branched decussate	Decussate	1-cell pedicel or sessile, tetrahedral
Antithamnion defectum Kylin, 1925	On pinnae, covering 2 cells	Unpaired	Branched, pectinate	Distichous	Pedicellate, undefined pedicel length
Antithamnion densum Howe, 1914	On pinnae, covering 2 cells	Paired w/ determinate lateral	Adaxially pectinate	Distichous	1-cell pedicel
Antithamnion sparsum Tokida, 1932	On pinnae, covering 2 cells	Unpaired	Adaxially pectinate	Distichous	1-cell pedicel
Antithamnionella spirographidis Wollaston, 1968	On primary branches, partly covering 1 cell	Unpaired	Simple	Distichous	Sessile

Table 1. Distinguishing characteristics of local and morphologically similar Antithamnion spp. as well as Antithamnionella.

Species	Gland cells	Indeterminae laterals	Pinnae	Branching plane	Tetrasporangia
New collection, Antithamnion hubsii Shim & Kim, 2024	Borne on pinnae, adaxial, covering 2 cells	Paired	Pinnate (diploid) Adaxially pectinate (haploid)	Distichous	1-cell pedicel
Antithamnion hubsii Dawson, 1963	Borne on pinnae, adaxial, covering 2 cells	Paired	Pinnate	Distichous	NA
Antithamnion nipponicum Yamada et Inagaki	Borne on pinnae, adaxial, covering 2 cells	Paired	Pinnate	Distichous	1-cell pedicel
Antithamnion cruciatum Nägeli, 1847	On pinnae, covering 2 cells	Paired w/ determinate lateral	Branched decussate	Decussate	1-cell pedicel or sessile, tetrahedral
Antithamnion defectum Kylin, 1925	On pinnae, covering 2 cells	Unpaired	Branched, pectinate	Distichous	Pedicellate, undefined pedicel length
Antithamnion densum Howe, 1914	On pinnae, covering 2 cells	Paired w/ determinate lateral	Adaxially pectinate	Distichous	1-cell pedicel
Antithamnion sparsum Tokida, 1932	On pinnae, covering 2 cells	Unpaired	Adaxially pectinate	Distichous	1-cell pedicel
Antithamnionella spirographidis Wollaston, 1968	On primary branches, partly covering 1 cell	Unpaired	Simple	Distichous	Sessile

Figure 1. Morphological features of Antithamnion hubbsii. tetrasporophyte. Tetrasporophyte of A. hubbsii growing attached to intertidal rocks with distinctive bilateral branching pattern. (A) Light microscopy image showing pinnate branching pattern. AC: apical cell; IB: indeterminate branch; Pn: pinnule; PI: pinna; rh: rhizoid. Scale bar = 100 μm. (B) Higher magnification of central axis showing cellular organization. Ax: axial cell; BC: basal cell; GC: gland cell. Scale bar = 100 μm. (C) Herbarium specimen of A. hubbsii tetrasporophyte showing overall thallus morphology and branching pattern. Scale bar = 1 cm. (D) Close-up view of lateral branches showing the insertion of indeterminate branches (IB) and the presence of gland cells (GC) at the adaxial surface of pinnules. Note the repeated bilateral branching and presence of reproductive structures (R). Ax: axial cell; BC: basal cell. Scale bar = 100 μm.

Figure 1. Morphological features of Antithamnion hubbsii. tetrasporophyte. Tetrasporophyte of A. hubbsii growing attached to intertidal rocks with distinctive bilateral branching pattern. (A) Light microscopy image showing pinnate branching pattern. AC: apical cell; IB: indeterminate branch; Pn: pinnule; PI: pinna; rh: rhizoid. Scale bar = 100 μm. (B) Higher magnification of central axis showing cellular organization. Ax: axial cell; BC: basal cell; GC: gland cell. Scale bar = 100 μm. (C) Herbarium specimen of A. hubbsii tetrasporophyte showing overall thallus morphology and branching pattern. Scale bar = 1 cm. (D) Close-up view of lateral branches showing the insertion of indeterminate branches (IB) and the presence of gland cells (GC) at the adaxial surface of pinnules. Note the repeated bilateral branching and presence of reproductive structures (R). Ax: axial cell; BC: basal cell. Scale bar = 100 μm.

Reproductive structures: The tetrasporophyte bears spherical tetrasporangia (T) that develop on specialized short branches (Figure 2A). Tetrasporangia are typically borne singly or in pairs on the adaxial side of pinnae, adjacent to gland cells (Figure 2B). Each tetrasporangium is characterized by a distinctive 1-cell pedicel structure that differentiates it from other species in the genus (Table 1). Nuclear staining reveals the presence of a single large nucleus (N) in ordinary vegetative cells (Figure 2C; white arrow). Within each tetrasporangium, nuclear staining confirmed the presence of four small nuclei (n) corresponding to the four daughter cells formed during meiotic division (Figure 2C; red arrows), confirming the typical pattern of tetrasporogenesis seen in other red algae.

Sexual dimorphism: Male and female gametophytes show distinct morphological differences (Figure 3). These reproductive structures were observed after culturing tetrasporangia for 3-6 months under laboratory conditions. Male gametophytes display dense clusters of spermatangia (arrowheads) borne on spermatangial parent cells on special branches on the adaxial side of pinnules, giving a more tufted appearance (Figure 3A,A’). Female gametophytes maintain a more regular branching pattern with procarps (arrows) developing on the basal cells of pinnae (Figure 3B). Detailed examination of the female reproductive structures reveals the development of carpogonial branch cells (CB1-3) arranged in a characteristic pattern, with the terminal carpogonium extending a trichogyne (tr) for spermatial reception (Figure 3B’). The carpogonial branch is supported by a distinct supporting cell (Su) connected to the axial cell (Ax). The branching in female gametophytes appears more orderly and pinnate compared to the clustered organization in male plants.

Carposporophyte development: The development of carposporophytes was not observed in our cultured specimens during the study period. Therefore, further investigations under controlled conditions will be required to characterize the post-fertilization stages in A. hubbsii.

Distinguishing characteristics: As shown in Table 1, A. hubbsii can be distinguished from morphologically similar species by a combination of features. It has paired indeterminate laterals, pinnate arrangement in diploid phase and adaxially pectinate in haploid phase, and distichous branching plane. These characteristics, together with the distinctive gland cell arrangement and 1-cell pedicel of tetrasporangia, clearly differentiate A. hubbsii from other related species such as A. nipponicum, A. cruciatum, and A. defectum. While A. hubbsii shares some features with A. nipponicum, such as paired indeterminate laterals and distichous branching, our molecular analyses demonstrate that they represent distinct species.

This combination of morphological features, particularly the arrangement of pinnae, location of gland cells, and pattern of tetrasporangia development, supports the identification of this specimen as Antithamnion hubbsii, consistent with its phylogenetic placement shown in the molecular analyses.

3.2. Phylogenetic Analyses

The 1467 base pairs (bp) of rbcL and 1523 bp of psaA were sequenced from samples collected in Korea. The phylogenetic trees were constructed by aligning the newly generated sequences with those downloaded from GenBank (Table 2, Figure 4).

Molecular phylogenetic analyses based on both rbcL (Figure 4A) and psaA (Figure 4B) sequences revealed that specimens collected from Sungeut Beach in Gangneung, South Korea formed a well-supported clade with previously reported Antithamnion hubbsii. In the rbcL phylogeny, our Korean A. hubbsii (PV453999) formed a strongly supported clade with A. hubbsii from California (AY591930) with bootstrap values of 98/99 for ML/Bayesian analyses. This clade was sister to A. nipponicum sequences with minimal genetic distance (0.25-0.29%). The psaA gene analysis similarly grouped our Korean A. hubbsii specimen (PV454032) with previously reported A. hubbsii (MK814610) with high statistical support (99.4/100), forming a sister relationship to A. nipponicum (AY295136) with only 0.34% genetic distance.

Both genes consistently placed the newly reported Korean specimens within the A. hubbsii lineage, distinct from but closely related to A. nipponicum. The ASAP and PTP analyses confirmed the species delimitation, specifically highlighting the distinct status of A. hubbsii from Sungeut Beach in Gangwon-do, South Korea. This represents the first report of A. hubbsii in Korean waters, as previous records of this species were primarily from the United States.

Figure 4. Maximum Likelihood (ML) phylogenetic trees of Antithamnion species based on (A) rbcL and (B) psaA gene sequences. Numbers at nodes indicate bootstrap support values (%) for ML analysis. The trees were rooted using Cystoclonium purpureum and Ceramium piluliferoides as outgroups for rbcL and psaA, respectively. Newly sequenced A. hubbsii specimens from Korea are indicated in bold. The matrices to the right of each tree summarize species delimitation results based on three methods: ASAP (Assemble Species by Automatic Partitioning), PTP (Poisson Tree Processes), and ABGD (Automatic Barcode Gap Discovery). Black squares indicate groups recognized as separate species by each method. Conflicting results between ASAP and PTP analyses highlight the taxonomic uncertainty between A. hubbsii and A. nipponicum.

Figure 4. Maximum Likelihood (ML) phylogenetic trees of Antithamnion species based on (A) rbcL and (B) psaA gene sequences. Numbers at nodes indicate bootstrap support values (%) for ML analysis. The trees were rooted using Cystoclonium purpureum and Ceramium piluliferoides as outgroups for rbcL and psaA, respectively. Newly sequenced A. hubbsii specimens from Korea are indicated in bold. The matrices to the right of each tree summarize species delimitation results based on three methods: ASAP (Assemble Species by Automatic Partitioning), PTP (Poisson Tree Processes), and ABGD (Automatic Barcode Gap Discovery). Black squares indicate groups recognized as separate species by each method. Conflicting results between ASAP and PTP analyses highlight the taxonomic uncertainty between A. hubbsii and A. nipponicum.

4. Discussion

The present study documents the first confirmed occurrence of Antithamnion hubbsii in Korean waters, based on both detailed morphological characteristics and molecular phylogenetic evidence. The species has likely been misidentified as A. nipponicum in previous surveys due to their striking morphological similarities. This finding highlights the taxonomic complexity within the genus Antithamnion and emphasizes the importance of integrating molecular tools with traditional morphological approaches for accurate species identification in red algae (Saunders, 2005; Freshwater et al., 2010).

The taxonomic history of Antithamnion species has been marked by numerous revisions and reclassifications over the past century. The genus was first established by Nägeli (1847) and has since undergone significant taxonomic refinement. Our findings contribute to this ongoing taxonomic discourse by providing molecular evidence for the presence of A. hubbsii in Korean waters. The extremely close genetic relationship between A. hubbsii and A. nipponicum revealed in our analyses (0.25-0.29% divergence in rbcL and 0.34% in psaA) reinforces the taxonomic challenges within this genus. While Cho et al. (2007) previously suggested these taxa might represent the same species based on minimal genetic differences, our ASAP analysis results support their distinction as separate species despite these minimal differences. This conclusion contradicts our PTP analysis results, which grouped them as a single species, reflecting the ongoing methodological challenges in delimiting species boundaries within closely related red algal taxa (Payo et al., 2013; Leliaert et al., 2014).

Although the origin of A. hubbsii in Korean waters remains uncertain, its presence calls for a re-assessment of regional red algal biodiversity, particularly in taxa with cryptic or morphologically similar species. Previous studies have shown that minimal genetic divergence can mask true taxonomic identity, highlighting the necessity for refined field sampling and the application of multi-locus molecular approaches (Steen & Scrosati, 2004; Freshwater et al., 2010).

At present, it remains unclear whether A. hubbsii is native to Korean waters or introduced. Regardless of its origin, its presence calls for a re-evaluation of red algal biodiversity in the region, particularly within genera that include cryptic or morphologically similar species. Previous studies have emphasized that even minimal genetic differences can obscure true taxonomic boundaries in red algae (Saunders, 2005; Payo et al., 2013). Future studies should aim to resolve these uncertainties through expanded field surveys and more refined molecular analyses (Steen & Scrosati, 2004; Freshwater et al., 2010).

These taxonomic challenges are not only of academic interest but also have important implications for biodiversity monitoring and non-native species management. Accurate identification of A. hubbsii contributes to our understanding of species diversity in Korean coastal ecosystems and may aid in improving detection of potential introductions. Given the morphological and genetic ambiguity between A. hubbsii and A. nipponicum, clarifying their taxonomic relationship is crucial (Leliaert et al., 2014; Olenin et al., 2011). A systematic re-evaluation of the genus Antithamnion will be essential to establish robust species boundaries, which in turn can inform ecological research and conservation strategies (Williams & Smith, 2007).