1. INTRODUCTION

The genus Roseolithon L.M.Coutinho & Barros-Barreto was delineated from Lithothamnion Heydrich through combined molecular and morphological investigations (Coutinho et al. 2021). This taxonomic change was initiated following the sequencing of the type species Lithothamnion muelleri Lenormand ex Rosanoff by Jeong et al. (2021). Roseolithon is distinguished by the set of key morphological traits: (1) a monomerous thallus structure with a plumose, non-coaxial hypothallus; (2) flared epithallial cells; (3) subepithallial cells that vary in length and may be shorter than, similar to, or longer than the cells beneath them; (4) cell fusions connecting adjacent filaments; (5) multiporate tetra/bisporangial conceptacle chambers; and (6) pore canals surrounded by rosette cells located in surface depressions, resulting in a distinctly pitted appearance (Coutinho et al. 2021).

Roseolithon is presently comprised of sixteen recognized species. As its establishment, Coutinho et al. (2021) described seven new species: R. goytacae L.M.Coutinho, F.P.Gomes & Barros-Barreto; R. karaiborum L.M.Coutinho & Barros-Barreto; R. potiguarae L.M.Coutinho & Barros-Barreto; R. purii L.M.Coutinho & Barros-Barreto; R. tamoioi L.M.Coutinho & Barros- Barreto; R. tremembei L.M.Coutinho & Barros- Barreto; R. tupii L.M.Coutinho & Barros-Barreto. Subsequently, Richards et al. (2022) added three species: R. louisianense J.L.Richards & Fredericq; R. occidentaleatlanticum J.L.Richards & Fredericq; R. rhodolapidosum J.L.Richards & Fredericq. In 2023, Gabrielson et al. transferred three species from Lithothamnion: R. crispatum (Hauck) P.W.Gabrielson, Maneveldt, Hughey & V.Peña; R. indicum (Foslie) P.W.Gabrielson, Maneveldt, Hughey & V.Peña; R. superpositum (Foslie) P.W.Gabrielson, Maneveldt, Hughey & V.Peña. In 2024, Min- Khant-Kyaw et al. introduced two new species: R. littorale Min-Khant-Kyaw, A.Kato & M.Baba; R. sabulosum Min-Khant-Kyaw, A.Kato & M.Baba from Japan. In the same year, Gabrielson et al. described R. mirabile (Foslie) P.W.Gabrielson, J.J.Lamb & Hughey, which was later validated by Gabrielson in 2025.

The species of Roseolithon display a wide and diverse range of distribution patterns (Guiry and Guiry 2025). Roseolithon crispatum, for instance, is widely distributed across Africa, Asia, Australia and New Zealand, the Caribbean, Europe, the Middle East, North and South America, and the western Atlantic. In contrast, six species (R. goytacae, R. karaiborum, R. potiguarae, R. purii, R. tamoioi, and R. tremembei) are restricted to South America and the western Atlantic. Roseolithon indicum has the broadest range, spanning Africa, Asia, the Indian Ocean islands, Southeast Asia, Australia, and the Americas. Roseolithon littorale and R. sabulosum are confined to Asia, whereas R. louisianense, R. occidentaleatlanticum, and R. rhodolapidosum are restricted to the western Atlantic. Roseolithon superpositum is known from Africa, South America, Australia and the western Atlantic while R. tupii is limited to South America. In contrast, R. mirabile remains poorly documented, with limited or unspecified geographic records. Among these, R. indicum has been reported from Korea, originally identified as Lithothamnion indicum Foslie (Lee 2008) based on morphological evidence without supporting molecular data.

During this study, we collected four unidentified crustose samples resembling Roseolithon from intertidal to 10 m subtidal zones around Jeju Island, Korea. In this study, we identified all specimens as R. sabulosum through morpho-anatomical analysis, along with rbcL and psbA sequence data, marking the first record of this species in Korea.

2. MATERIALS AND METHODS

2.1. Molecular analyses

DNA was extracted from 4 silica gel-dried specimens using the NucleoSpin Plant II Kit (Macherey- Nagel, Düren, Germany). The rbcL region was amplified via polymerase chain reaction (PCR) using Helix Amp Ready-2x-Go premix from NanoHelix Co., Ltd. (Daejeon, Korea). The primer sets used for rbcL were F57-R753, F577-R1150, and F993-RrbcSstart (Freshwater and Rueness 1994;Kim et al. 2023), and those for psbA following Jeong et al. (2021). PCR was carried out on a Veriti 96-well Thermal Cycler (Applied Biosystems, Waltham, MA, USA). Three rbcL and four psbA sequences from the coralline algae obtained in this study have been submitted to GenBank. All sequences were aligned using Geneious Prime^® (v.2023.0.1; Biomatters Ltd., New York, NY, USA). Genetic distances were calculated using the p-distance method in Mega 11 (Tamura et al. 2021). To determine the best partition scheme and model evolution for the rbcL and psbA, we used PartitionFinder 2 (Guindon et al. 2010;Lanfear et al. 2012, 2017). Phylogenetic analyses were performed in RAxMLGUI v2.0 (Edler et al. 2021) using the maximum likelihood method with the General Time-Reversible model of nucleotide substitution with invariant sites and gamma-distributed rates for the variable sites (GTR+ G+I) model with 1,000 bootstrap replicates. Bayesian inference was carried out with MrBayes 3.2.6 (Ronquist et al. 2012) involving Markov chain Monte Carlo runs performed for 2,500,000 generations with one cold and three heated chains, utilizing the GTR+G+I evolutionary model. Trees were sampled every 1,000 generations, and summary trees were created with a burn-in value of 25%.

2.2. Morphological analyses

Samples were collected from intertidal and subtidal zones along the coast of Jeju Island, Korea. The specimens were photographed to capture their external morphological features using a waterproof digital camera (Nikon COOLPIX AW100; Nikon Corp., Tokyo, Japan). Silica gel-dried specimens were fractured with a razor blade or a chisel and a small hammer. The resulting fragments were mounted on aluminum stubs with doublesided conductive carbon tape (Nisshin EM Co., Ltd., Japan) and sputter-coated with gold for 5 minutes using a digital ion coater (SPT-20; COXEM Co., Ltd., Korea). Observations were carried out with a COXEM EM-30 PLUS+ scanning electron microscope (Mini SEM; COXEM Co., Ltd., Korea). Morphometric measurements of anatomical and reproductive structures were obtained using ImageJ software (Schneider et al. 2012). Representative voucher specimens from this study were deposited in the herbarium of Chosun University (CUK) and the Marine Biodiversity Institute of Korea (MABIK).

3. RESULTS

3.1. Phylogenetic analyses

The 1,387 base pair (bp) sequence of the rbcL gene and an 812 bp sequence of the psbA region were obtained from coralline algal specimens collected in Korea. Phylogenetic trees were constructed from rbcL (Fig. 1) and psbA (Fig. 2) by aligning the newly generated sequences with those obtained from GenBank, respectively. Sporolithon tenue Bahia, Amado-Filho, Maneveldt & W.H. Adey; and S. ptychoides Heydrich were selected as outgroups for both datasets. The rbcL phylogenetic analysis (Fig. 1) placed the Korean coralline non-geniculate specimens within the genus Roseolithon, forming a strongly supported clade (99 BS/0.99 PP) with R. sabulosum from Japan, including the holotype (LC822439). No sequence divergence (0%) was detected between the Korean and Japanese R. sabulosum specimens. In the psbA phylogenetic analysis (Fig. 2), the Korean specimens formed a fully supported clade (100 BS/1 PP) with the holotype of R. sabulosum from Japan (LC822418). The intraspecific genetic variation between the Korean specimens and the holotype of R. sabulosum was 0-0.2%.

3.2. Morphological observations

Class Florideophyceae Cronquist, 1960

Order Hapalidiales W.A.Nelson, J.E.Sutherland, T.J.Farr & H.S. Yoon, 2015

Family Hapalidiaceae J.E.Gray 1864

Genus RoseolithonL.M.Coutinho & Barros-Barreto, 2021 (국명신칭: 장미돌말속)

Roseolithon sabulosum Min-Khant-Kyaw, A.Kato & M.Baba 2024 (국명신칭: 모래장미돌말) (Fig. 3A-J)

Holotype. SAP 115691 (tetrasporophyte), 21 April 2023, coll. Min-Khant-Kyaw & A.Kato.

Type Locality. 32°37.46ʹN, 130°15.06ʹE; intertidal; Shirasu, Minami-Arima, Minami-Shimabara City, Nagasaki Prefecture, Japan.

Specimens examined. MABIK AL00103225 (=CUK 16975), CUK16984 (tetrasporophyte), and CUK16985 (Biyangdo-gil, Hallim-eup, Jeju-si, Jeju-do, South Korea, subtidal at 10 m, collected by T.O.Cho and S.Y.Jeong, Mar. 13, 2016); CUK17432 (Sanho Beach, Udo-myeon, Jeju City, Jeju Island, South Korea, intertidal, collected by T.O.Cho and S.Y.Jeong, Oct. 07, 2016).

Vegetative structure. Plants are purplish red to rosepink (Fig. 3A), non-geniculate, presenting from warty to fruticose forms, formed completely free-living rhodoliths with spheroidal to sub-spheroidal shapes, and up to 4.7 cm in width. In fruticose specimens, protuberances are cylindrical, either dichotomous branching or anastomosis, apically swollen (2.8-5.2 mm wide), and up to 7.1-7.7 mm in length. Thalli are monomerous with a multistratose, non-coaxial (plumose) hypothallus composed of filaments running parallel to the substrate. Cells of hypothallial filaments are elongate, 12.1- 13.9 μm in length, and 5.7-8.0 μm in diameter. Cells of perithallial filaments are square to elongate, 13.9-15.9 μm in length, and 6.2-7.7 μm in diameter (Fig. 3B). Secondary pit connections are absent, and cell fusions occur between adjacent cells. Subepithallial initials (intercalary meristematic cells) are elongate, ranging from 3.5- 11.4 μm in length and 6.1-10.5 μm in diameter, relative to their immediate inner derivatives (Fig. 3C). Epithallus is composed of a single layer of flattened cells with flared and trapezoidal outer walls, measuring 1.6-5.8 μm in length and 5.0-8.4 μm in diameter (Fig. 3C). Trichocytes are not observed.

Reproductive structures. Tetra/bisporangial conceptacles are multiporate and protruded above the thallus surface, lacking differentiation between the peripheral rim and pore plate (Fig. 3D, E). The chambers of tetra/ bisporangial conceptacles are 71-118.1 μm in height and 163-333 μm in diameter. Each pore opening is bordered by 4-7 rosette cells situated in depressions, giving the pore plate a pitted appearance in surface view (Fig. 3E-G). In longitudinal section, the conceptacle roofs are composed of three cell layers, including epithallial cells. The pore canals are lined with filaments of 2-3 cells, formed by rosette cells with disintegrated roofs, and underlain by elongate and/or wedge-shaped cells (Fig. 3H, I). Some conceptacles are buried and filled with vegetative cells (Fig. 3J).

Habitat. Found as free-living rhodoliths in the intertidal to subtidal zone.

Geographic distribution. Korea and Japan.

4. DISCUSSION

Our molecular data and morphological observations confirm the presence of Roseolithon sabulosum in Korea. Phylogenetic analyses of rbcL and psbA sequences show that the Korean specimens clustered into a highly supported clade (99 BS/0.99 PP) in rbcL and a completely supported clade (100 BS/1 PP) in psbA, along with Japanese R. sabulosum, including the holotype. Genetic differences between the two regions are minimal, with no variation in rbcL (1,387 bp) and only 0- 0.2% divergence in psbA (812 bp), further supporting their conspecific status. Morphologically, the Korean specimens have all the key characteristics of R. sabulosum, including a monomerous thallus with a plumose hypothallus, flared epithallial cells, variable subepithallial cells, cell fusions without secondary pit connections, and the absence of trichocytes. They also feature multiporate tetra/bisporangial conceptacles with pitted surfaces created by disintegrating rosette cell roofs. The conceptacles are raised above the thallus surface, surrounded by 4-7 rosette cells around each pore (as seen from the surface), while the pore canals are lined with 2-3-cell filaments (in longitudinal view) that have an elongated or wedge-shaped form, matching the descriptions of R. sabulosum from Japan.

The Korean specimens of Roseolithon are further validated by their distinct morphological traits, which clearly set this genus apart from both Boreolithothamnion and Lithothamnion. In Roseolithon, epithallial cells are consistently flared, whereas Lithothamnion shows considerable variability in epithallial cell shape (Gabrielson et al. 2023). Trichocytes, which are frequently present in Lithothamnion, have not been observed in Roseolithon and are generally absent or unreported in fieldcollected specimens of Boreolithothamnion (Gabrielson et al. 2023;Min-Khant-Kyaw et al. 2024). Subepithallial initials in Roseolithon also exhibit greater size variation whereas in Boreolithothamnion and Lithothamnion they are typically as long as or longer than their inward derivatives (Coutinho et al. 2021;Gabrielson et al. 2023). A key distinguishing feature of Roseolithon is its pore canals, which are surrounded by rosette cells in noticeable depressions, producing a distinctly pitted surface of the tetra/bisporangial conceptacle due to the rosette cell roofs being sunken or disintegrated. In contrast, in Boreolithothamnion, the rosette cells are typically flush with the surface (Coutinho et al. 2021;Gabrielson et al. 2023).

Roseolithon sabulosum was recently described from subtidal environments in Japan by Min-Khant-Kyaw et al. (2024). Although R. sabulosum shares some morphological traits with other species within Roseolithon, it is distinguishable by distinct molecular markers and subtle anatomical differences. In our rbcL-based phylogeny, R. sabulosum forms a closely related lineage to the lectotype specimen of R. indicum, which was originally described as a Lithothamnion species from Australia. Morphologically, Australian R. indicum resembles to R. sabulosum, but it differs in having larger epithallial cells, squarish to rectangular subepithallial initials, and much larger tetra/bisporangial conceptacle chambers (Keats et al. 2000;Min-Khant-Kyaw et al. 2024). Specifically, the tetra/bisporangial conceptacle chambers of R. indicum measure around 150-170 μm in height and 450-600 μm in diameter (Wilks and Woelkerling 1995), while those of R. sabulosum from Korea are notably smaller, ranging from 71-118 μm in height and 163- 333 μm in diameter. Therefore, R. indicum can be distinguished by its larger multiporate tetra/bisporangial conceptacle chambers (Min-Khant-Kyaw et al. 2024). Roseolithon indicum has been identified as Lithothamnion indicum Foslie in Korea based on morphology (Lee 2008). Although R. indicum was previously the only species of the genus reported in Korea, its identification now requires re-evalutation using molecular data. Our study expands the Korean algal flora with the addition of Roseolithon sabulosum, confirmed by both morphological and molecular data. The Korean specimens from Jeju Island clustered with high support with the Japanese R. sabulosum holotype in rbcL and psbA phylogenetic analyses, supporting conspecificity.

Roseolithon species occur in intertidal or subtidal rhodolith beds or grow epilithically on bedrock or shells in intertidal to shallow subtidal zones (Gabrielson et al. 2023, 2024;Min-Khant-Kyaw et al. 2024). Collectively, these records demonstrate that the genus spans a wide ecological spectrum, from intertidal crusts to deepwater rhodolith beds, and includes both cosmopolitan species and regionally restricted endemics. Molecular evidence has confirmed the presence of several Roseolithon species across the Atlantic, Indian, and Pacific oceans (Fig. 4). Roseolithon crispatum has been validated from the Atlantic coast of Brazil and the Adriatic Sea in Croatia (Coutinho et al. 2021). In the Western Atlantic, R. goytacae, R. potiguarae, R. purii, R. tamoioi, R. tremembei, R. tupii, and R. karaiborum have been molecularly recorded from Brazil (Coutinho et al. 2021), while R. purii, R. rhodolapidosum, R. louisianense, and R. occidentale atlanticum were confirmed from the northwestern Gulf of Mexico (Richards et al. 2022). In the Indo-Pacific, R. indicum has been sequenced from southeastern Australia (Gabrielson et al. 2023), and R. mirabile was recently reassigned based on DNA sequencing of the lectotype from Corner Inlet, Victoria (Gabrielson et al. 2024). In southern Africa, R. superpositum was confirmed from Port Alfred, South Africa (Gabrielson et al. 2023). In East Asia, R. littorale and R. sabulosum have been confirmed in Japan (Min-Khant- Kyaw et al. 2024), and this study also records R. sabulosum from Jeju Island, Korea. Collectively, these molecularly confirmed records demonstrate that the genus includes both cosmopolitan species (e.g., R. crispatum) and regionally restricted taxa (e.g., R. goytacae in Brazil, R. superpositum in South Africa) and highlight that some are known only from their type or a single DNAconfirmed collection (e.g., R. mirabile, R. superpositum) (Fig. 4). Beyond molecularly confirmed records, Roseolithon has been reported from a wide range of marine regions; however, its cryptic morphology and pronounced phenotypic plasticity mean that many floristic and taxonomic records still require molecular validation (Jesionek et al. 2016;Coutinho et al. 2021).