Journal 
1. INTRODUCTION
The crustose brown algal genus, Pseudolithoderma Svedelius is encompassed in the family Lithodermataceae Hauck and order Sphacelariales Migula (Guiry and Guiry 2024). Pseudolithoderma was described by Svedelius (Kjellman and Svedelius 1911) based on Lithoderma fatiscens sensu Kuckuck. This genus is characterized by following characters: 1) crustose thalli, relatively thin, horizontally expanded, attached firmly to the substratum, without rhizoids; 2) hypothallial basal layer of 1-3 cells and perithallial erect filaments simple or sparsely branched, strictly erect, loosely or firmly adjoined; 3) cells with several to many discoid chloroplasts without pyrenoids; 4) tufts of hairs occasionally arising from the basal layer; 5) plurangial reproductive structures arising terminally on erect filaments, without sterile cells; 6) unangia arise terminally on erect filament, without paraphyses (Kjellman and Svedelius 1911;Tanaka and Chihara 1981;Kaehler 1998;Poong 2014).
Currently, ten Pseudolithoderma species are recognized worldwide (Guiry and Guiry 2024), but none has been reported in Korea. Pseudolithoderma subextensum (Waern) S. Lund was first described as Lithoderma subextensum from Öregrund, Sweden (Waern 1949). It is mainly characterized by a thin, dark chestnut brown crust; hypothallial basal layer giving rise to strictly erect perithallial filaments; tufts of hairs occasionally arising from the basal layer; several discoid or irregularly shaped chloroplasts per cell; plurangia arising terminally on erect filaments, uniseriate and branched, without sterile cells; unangia arising terminally on erect filaments, elongated and cylindrical (Waern 1949;Tanaka and Chihara 1981;Kaehler 1998;Poong 2014). Lund (1959), transferred this species to the genus Pseudolithoderma based on their plurangial reproductive organs borne terminally on erect filaments, that differentiates them from the Lithoderma which have plurangia borne both laterally and terminally on erect filaments.
The global distribution of P. subextensum includes Europe, Western Atlantic, Middle East and Asia (Hong Kong and Japan) (Tanaka and Chihara 1981;Kaehler 1998;Guiry and Guiry 2024). Despite the proximity of Korea to both Japan and Hong Kong, P. subextensum has not yet been reported in Korea. Most of the encrusting brown algae in Korea have been identified as species of the genera Ralfsia or Neoralfsia based on their morphology (Lee and Kang 1986;Lee and Kang 2002;Lee 2008;Keum 2010). However, in their recent floristic surveys, Oteng’o et al. (2020a, 2021, 2022, 2023a, 2023b) reported Ralfsia longicellularis as a new record, described four new Endoplura species, and erected a new genus Sungminia within Sungminiaceae and genus Fissipedicella from Korea.
Two unidentified samples of Ralfsia-like crusts were collected from intertidal areas along southern coast of Korea. In this study, we examined their morpho-anatomy and analyzed COI-5P sequences, identifying them as Pseudolithoderma subextensum.
2. MATERIALS AND METHODS
2.1. Morphological analyses
Samples were collected from intertidal areas along the coastline of Korea. Specimens were photographed using a waterproof digital camera (Nikon COOLPIX AW100; Nikon Corp., Tokyo, Japan) for their external morphological characteristics related to characters, including color, outline, and surface morphology. Prior to the analysis of morphological observation, the specimens were detached from the substrate using a single- edged blade. Squashed and microtome-sectioned preparations were prepared for each sample. For the microtome-sectioned preparations, samples were embedded in a matrix (OCT; CellPath Ltd., Newtown, Wales, UK) and sectioned (8-10 μm thickness) using a freezing microtome (Shandon Cryotome FSE; Thermo Shandon Ltd., Loughborough, UK). The sectioned and squashed samples were stained with a 1 : 1 mixture of aqueous aniline blue and acetic acid. Sections were mounted in 50% corn syrup and photographed using a DP-71 camera (Olympus, Tokyo, Japan) mounted on a BX-51TRF microscope (Olympus). Digitized images were edited for clarity using Adobe Photoshop software v.6.1 (Adobe Systems Inc., San Jose, CA, USA). Representative voucher specimens examined in this study were deposited in the herbarium of Chosun University (CUK) and the Marine Biodiversity Institute of Korea (MABIK).
2.2. Molecular analyses
Genomic DNA was extracted using the NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The COI-5P region was amplified via polymerase chain reaction (PCR) with HelixAmp Ready-2x-Go premix from NanoHelix Co., Ltd. (Daejeon, Korea), adhering to the provided protocol. The primer sets for amplifying COI-5P were those specified in a previous study by Oteng’o et al. (2021). PCR was performed on a Veriti 96-well Thermal Cycler (Applied Biosystems, Waltham, MA, USA). The resulting PCR products were purified using the PCRquick-spinTM PCR Product Purification Kit (iNtRON Biotechnology, Inc., Seongnam, Korea). One COI-5P sequence from the brown crustose algae obtained in this study has been deposited in GenBank. The COI-5P sequence generated, along with those retrieved from GenBank, were aligned using Geneious Prime® (v.2023.0.1, Biomatters Ltd., Auckland, New Zealand). Phylogenetic analyses were conducted in Mega X (Kumar et al. 2018), employing the maximum likelihood method with the GTR+G+I model and 1,000 bootstrap replicates. Bayesian inference was executed using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003), with Markov chain Monte Carlo runs performed for 2,000,000 gen erations. This involved one cold chain and three heated chains, using the GTR+Γ+I evolutionary model. Trees were sampled every 1,000 generations, and summary trees were generated with a burn-in value of 25%. Genetic distances were calculated using the p-distance method in Mega X (Kumar et al. 2018).
3. RESULTS
3.1. Morphological observations
Class Phaeophyceae F.R. Kjellman, 1891 갈조강
Order Sphacelariales Migula, 1908 갯쇠털목
Family Lithodermataceae Hauck, 1883 바위부과 (신칭)
Genus Pseudolithoderma Svedelius, 1911 헛바위부속 (신칭)
Pseudolithoderma subextensum (Waern) S. Lund, 1959 헛바위부 (신칭) (Fig. 1A-F)
Basyonym.Lithoderma subextensum Waern
Holotype. UPS; Waern No. 1-2/49.
Type locality. Öregrund
Material examined. MABIK AL00100605 (=CUK 20782), Dolsan Port, Gunnae-ri, Dolsan-eup, Yeosu-si, Jeollanam-do, Korea (34°36ʹ52.38ʺN, 127°43ʹ09.55ʺE), October 19, 2020, T.O. Cho and B.Y. Won, at 1 m depth by hand; CUK20783, Dolsan Port, Gunnae-ri, Dolsaneup, Yeosu-si, Jeollanam-do, Korea (34°36ʹ52.38ʺN, 127°43ʹ09.55ʺE), October 19, 2020, T.O. Cho and B.Y. Won, at 1 m depth by hand.
Description. Thalli are epilithic crusts, dark chestnut brown to very dark brown, circular to irregular in outline and often confluent, without lighter margins, without growth lines and mostly smooth surface covered with thin gelatinous layer, 0.2-1.0 cm across, 65- 180 μm thick, firmly attached to the substratum (Fig. 1A), and lacking rhizoids (Fig. 1B). The hypothallial basal layer is composed of 1-2 cell layers in which cells are 10-20 μm wide and have a width-to-length ratio of 1 : 0.3-0.8 and give rise to erect perithallial filaments (Fig. 1B). Erect perithallial filaments are sparsely branched, straight, and firmly adjoined with one another to form pseudoparenchymatous tissue. Cells of the erect perithallial filaments are 8-12 μm long and 7-16 μm wide. Chloroplast are discoid and several per cell (Fig. 1C). Hairs in tufts arising from the basal layer were rarely observed. Reproductive portions of the unangial sori form slightly elevated areas on the thalli (Fig. 1D). Unangia are cylindrical to sub-globose, 27-50 μm long, and 10-15 μm wide, terminally arising on erect filaments (Fig. 1E and F).
Habitat. This species has been confirmed on the southern coast (Dolsan), Korea. It grows on hard substrates such as pebbles and rocks in the intertidal zone. World distribution. Europe, Western Atlantic, Middle East, Asia (Hong Kong and Japan) (Tanaka and Chihara 1981;Kaehler 1998;Guiry and Guiry 2024) and Korea (this study).
Identifier. Antony Otinga Oteng’o and Tae Oh Cho.
3.2. Phylogenetic analysis
The 545 base pair (bp) of the COI-5P region was sequenced from brown crustose samples collected in Korea. Phylogenetic tree was constructed by aligning the newly generated COI-5P sequences with those obtained from GenBank (Fig. 2). Cladostephus hirsutum (Linnaeus) Boudouresque & M. Perret-Boudouresque ex Heesch & al. and C. spongiosum (Hudson) C. Agardh were included as outgroup taxa. Phylogenetic analyses based on COI-5P sequences indicate that our Korean brown crustose algae are nested within the Pseudolithoderma clade of the Lithodermataceae (Fig. 2). There is a genetic divergence of 24.5% in the COI- 5P (545 bp) between Pseudolithoderma subextensum and P. roscoffense.
4. DISCUSSION
Our morphological and molecular analyses indicate that our two unidentified brown crustose samples are Pseudolithoderma subextensum (Sphacelariales, Phae- ophyceae). In Korea, there are six genera of crustose brown algae; Endoplura (Ralfsiaceae, Ralfsiales), Fissipedicella (Ralfsiaceae, Ralfsiales), Neoralfsia (Neoralfsiaceae, Ralfsiales), Ralfsia (Ralfsiaceae, Ralfsiales), Petroderma (Petrodermataceae, Ishigeales), and Sungminia (Sungminiaceae, Ralfsiales) (Oteng’o et al. 2020a, 2020b, 2021, 2022, 2023a, 2023b). Pseudolithoderma differs from Endoplura, Fissipedicella, Neoralfsia, Ralfsia, and Sungmnia by having thinner crustose thalli and cylindrical unangia. While Petroderma also has thin thalli, it is distinguished by cells with a single chloroplast and the presence of rhizoid, while Pseudolithoderma has cells with several chloroplasts and lacks rhizoids. These distinguishing characteristics are critical for accurate identification and classification within the diverse group of crustose brown algae found in Korea.
Pseudolithoderma subextensum (Waern) S. Lund was originally described by Waern (1949) from Sweden. It was characterized by simple erect threads, large chromatophores, thick gelatinous membrane, and elongated unilocular sporangia. Our Korean samples closely correspond to the majority of characteristics described in Waern’s protologue from Öregrund, Sweden (Waern 1949), showing only unangial reproductive structures on the thalli and hairs arising from the basal layer. In contrast, Waern’s protologue indicated that P. subextensum exhibited both unilocular and plurilocular reproductive structures on the same thalli, with hairs originating from the apices of erect filaments. However, Tanaka and Chihara (1981) pointed out that P. subextensum in Japan had only one type of reproductive organs born on per thallus and hairs arising from the basal layer. Similarly, Kaehler (1998) described P. subextensum as a new record in Hong Kong, based on the original description by Waern (1949) and the description from Japan by Tanaka and Chihara (1981), noting the presence of hair pits originating from the basal layer. These observations suggest variability in the reproductive structures and hair origin of P. subextensum across different geographical locations, highlighting the need for further taxonomic studies to understand its morphological diversity and distribution.
Our molecular analyses based on COI-5P indicate that Pseudolithoderma subextensum group together with P. roscoffense, although without robust support (Fig. 2). The genetic divergence between these two species is notably high at 24.5% for COI-5P sequences. In comparison, the divergence between our samples and other ‘Lithoderma sp.’ sequences from GenBank ranges from 17.1% to 21.0%, which is lower than the divergence observed with P. roscoffense. Despite higher sequence similarities between our samples and ‘Lithoderma sp.’, our morphological findings confirmed that our samples are actually Pseudolithoderma, not Lithoderma. Lund (1959) reestablished the genus Pseudolithoderma for species with terminal sporangia, a characteristic clearly observed in Pseudolithoderma subextensum (Fig. 1E and F).
In conclusion, our study makes an important contribution to the marine algal inventory of Korea by documenting the presence of the previously unreported crustose alga, Pseudolithoderma subextensum (Sphacelariales, Phaeophyceae). This discovery not only adds to the biodiversity records of the region but also underscores the importance of continued exploration and documentation of marine life. Consequently, we have provided new Korean names for the family, genus, and species to reflect this addition to the scientific and local community.
