INTRODUCTION
Farlow (1881) was the first to describe the Ralfsiaceae, which has been recognized as the largest family within the order Ralfsiales. Ralfsiaceae has been characterized by rounded to irregularly spreading crust, basal layer composed of radiating appressed filaments, and tightly appressed erect vegetative filaments, single or few chloroplasts per cell, plurangial reproductive structures topped with sterile cell(s), unangia accompanied by paraphysis on the terminal part of erect filaments, diplohaplontic and isomorphic or heteromorphic life history (Farlow 1881, Setchell and Gardner 1924, Abbott and Hollenberg 1976, Womersley 1987, Parente and Saunders 2019, Oteng’o et al. 2023b). Currently, the family Ralfsiaceae comprises five recognized genera: Analipus Kjellman, Endoplura Hollenberg, Fissipedicella A. O. Oteng’o, B. Y. Won & T. O. Cho, Heteroralfsia Kawai, and Ralfsia Berkeley (Lim et al. 2007, Silberfeld et al. 2014, Parente and Saunders 2019, Guiry and Guiry 2023, Oteng’o et al. 2023b). Traditionally, the recognition of genera within Ralfsiaceae has been based on morphological characters such as thallus type, number of sterile cells on plurangia, number of chloroplasts per cell, and the development of unangia (Tanaka and Chihara 1981b, Lim et al. 2007, León-Álvarez et al. 2017, Oteng’o et al. 2021, Parente et al. 2021). However, due to the morphological simplicity and external similarities among them, the classification of these genera has been challenging. Recent molecular analyses are increasing the taxonomic resolution of these genera within Ralfsiaceae, providing a clearer depiction of their phylogenetic relationships (Lim et al. 2007, Silberfeld et al. 2014, Parente and Saunders 2019, Oteng’o and Won 2020, Oteng’o et al. 2021, 2023a, 2023b).
In recent studies conducted by Oteng’o et al. (2021, 2022, 2023a, 20233b), using molecular analyses, new findings about crustose brown algae in Korea have been revealed: Fissipedicella (F. orientalis A. O. Oteng’o, B. Y. Won & T. O. Cho), Sungminia Oteng’o, Won & T. O. Cho (Sungminia gladiata Oteng’o, Won & T. O. Cho, S. pyriformis Oteng’o, Won & T. O. Cho and S. asiatica Oteng’o, Won & T. O. Cho), and four new species within Endoplura (E. jejuensis A. O. Oteng’o, T. O. Cho & B. Y. Won, E. koreana A. O. Oteng’o, T. O. Cho & B. Y. Won, E. gyeokpoensis Oteng’o, B. Y. Won & T. O. Cho, and E. limpeticola Oteng’o, B. Y. Won & T. O. Cho). Also, Ralfsia longicellularis Perestenko has been added as a new record in Korea based on morphological and molecular analyses (Oteng’o and Won 2020). These findings showed the possibility that Korea may be one of the hotspots for crustose brown algal biodiversity (Oteng’o et al. 2023b).
We gathered several unidentified samples of crustose brown algae resembling Ralfsia from the coastal shores of Korea and samples labeled as Ralfsia longicellularis collected from both Korean and Far East Russian coastal shores. This study employed a dual approach involving morphological characterization and the molecular analyses of rbcL and the 5’ region of the cytochrome c oxidase subunit I (COI-5P) gene sequences. As a result, we propose Ramipedicella gen. nov. including Ramipedicella miniloba sp. nov. and Ramipedicella longicellularis comb. nov.
MATERIALS AND METHODS
DNA extraction and amplification
We collected samples from intertidal areas of Korea and Far East Russia from 2013 to 2020 (Supplementary Table S1). Genomic DNA extraction was performed using the NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. Extracted DNA was stored at −20°C and used to amplify the rbcL and COI-5P genes. Polymerase chain reactions were conducted following the protocol for the HelixAmpTM Ready-2x-Go premix (NanoHelix Co., Ltd., Daejeon, Korea). The primers used to amplify rbcL and COI-5P were the same as those used in previous study by Oteng’o et al. (2021).
Sequence analyses
The nucleotide sequence used in this study was compiled from existing published sequences in GenBank along with newly generated sequences, consisting of 26 rbcL and 7 COI-5P sequences (Supplementary Table S1). The sets of 78 concatenated sequences (rbcL + COI-5P) and 78 rbcL were aligned. The unavailable COI-5P sequences in GenBank were regarded as missing data in our analysis of the concatenated dataset. Cladostephus spongiosus (Hudson) C. Agardh and Microzonia phinneyi (E. C. Henry & D. G. Müller) Camacho & Fredericq were chosen as outgroup taxa. The alignment of sequences was conducted using ClustalW (Thompson et al. 1994), followed by manual editing using Geneious Prime (v.2023.0.1; Biomatters Ltd., Auckland, New Zealand). We used PartitionFinder 2 (Lanfear et al. 2017) to determine the best partition scheme and model selection for the concatenated sequences. Maximum-likelihood (ML) analysis was estimated by the GTR + Γ + I model with 1,000 bootstrap replications using RAxMLGUI v1.5 (Silvestro and Michalak 2012). Bayesian inference (BI) was conducted with MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003). Markov chain Monte Carlo runs were executed for 2,000,000 generations with one cold chain and three heated chains, using the GTR + Γ + I evolutionary model. Trees were sampled every 1,000 generations. Summary trees were generated using a 25% burn-in value. Convergence was evaluated with Tracer v.1.7.2 (Rambaut et al. 2018). For the analysis of rbcL sequences, we used the same outgroup and Mega X (Kumar et al. 2018) to determine the optimal model. ML analysis was estimated by the Tamura 3-parameter + Γ + I model with 1,000 bootstrap replications using Mega X (Kumar et al. 2018). BI was performed using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003). Markov chain Monte Carlo runs were carried out for 2,000,000 generations, with one cold chain and three heated chains, using the T92 + Γ + I evolutionary model. Trees were sampled every 1,000 generations. Summary trees were generated using a burn-in value of 25%. Phylogenetic trees were visualized using FigTree v.1.4.0 (Rambaut 2012). Genetic distances were determined using the p-distance method in Mega X as described by Kumar et al. (2018).
Morphological analyses
A waterproof digital camera (Nikon COOLPIX AW100; Nikon Corp., Tokyo, Japan) was used to photograph the external morphological characteristics of the samples. Using a single-edged blade, the samples were carefully detached from the substrate. Microtome sections were then prepared from selected portions from each sample and mounted on microscopy slides to facilitate observation of their internal morphology. In the microtome-sectioned preparations, samples were embedded in a matrix (OCT; CellPath, Ltd., Newtown, Wales, UK) and then sectioned with a freezing microtome (Shandon Cryotome FSE; Thermo Shandon, Ltd., Loughborough, UK) at a thickness of 8–10 μm.
The sliced and squashed samples were stained with a mixture of aqueous aniline blue and acetic acid in a 1 : 1 ratio to enhance visualization and analysis. Following staining, the sections were mounted in 50% corn syrup and examined under a microscope. Photographs of the samples were captured using a DP-71 camera (Olympus, Tokyo, Japan) mounted on a BX-51TRF microscope (Olympus). Adobe Photoshop software v.6.1 (Adobe Systems Inc., San Jose, CA, USA) was used for editing the digitized images. Voucher specimens were stored in the herbarium collections of Chosun University (CUK) and the Marine Biodiversity Institute of Korea (MABIK), Korea.
RESULTS
Phylogenetic analyses
A sum of 26 rbcL sequences, with a length of 984 base pairs (bp), and 6 COI-5P sequences, with a length of 654 bp, were obtained from recently collected samples originating from Korea and the Far East of Russia (Supplementary Table S1). The phylogenetic tree inferred from concatenated dataset (rbcL + COI-5P) indicated that both the unidentified Ralfsia-like crustose algae from Korea and Ralfsia longicellularis from Korea and Far East Russia formed a distinct clade, Ramipedicella gen. nov., within the family Ralfsiaceae (Fig. 1). Phylogenetic analyses based on the rbcL sequences showed overall congruency with concatenated phylogeny (Supplementary Fig. S1). The two lineages within Ramipedicella are recognized as distinct species, Ramipedicella miniloba sp. nov. and Ramipedicella longicellularis comb. nov. The significant genetic differences were observed in rbcL (6.7–9.3%) and COI-5P (15.5–20.8%) sequences between Ramipedicella and other genera within Ralfsiaceae (Table 1). The genetic divergence between R. miniloba and R. longicellularis was 1.6–1.7% for rbcL and 7.7–8.7% for COI-5P.
Morphological observations
Ramipedicella A. O. Oteng’o, B. Y. Won & T. O. Cho gen. nov
Description
Thalli are epilithic or epizoic, crustose with circular to irregular outline, firmly attached to the substratum with or without rhizoids. They are multistratose owing to superimposed thalli. They comprise two main parts: the hypothallial basal layer and the perithallial erect filament. Hypothallial layer is formed by several prostrate cells, giving rise to firmly adjoined, assurgent, and usually branched perithallial erect filaments. Chloroplasts are one per cell. Phaeophycean hairs are present or absent. Sori develop in elevated patches. Plurangial reproductive structures are uniseriate, occasionally biseriate, subterminal on erect filaments, and terminated by one to several sterile cells. One to several unangia arise on branched stalks with paraphyses or on lateral-basal region to the paraphyses. Paraphyses arise laterally on stalks.
Ramipedicella miniloba A. O. Oteng’o, B. Y. Won & T. O. Cho sp. nov. (Fig. 2)
Description
Thalli are thick, epilithic or epizoic crusts, light to dark brown (almost black in dry state) with lighter margins, circular to irregular in outline, with external concentric zones and radial lines, with small lobes mostly towards the center making the surface uneven, up to 15 cm across, and 300–1,650 μm thick (Fig. 2A & B). Thalli are multistratose owing to superimposed ones composed of 1–4 plant layers (Fig. 2C). The hypothallial basal layer consists of multiple cell layers, where the cells are 7–15 μm wide and have a width-to-length ratio of 1 : 1–12. These cells give rise to erect perithallial filaments. The erect perithallial filaments are branched, straight, or upwardly curved, tapering towards the surface. They are tightly adjoined to each other, forming pseudoparenchymatous tissue (Fig. 2D). Cells of erect perithallial filaments are 3–12 μm wide and with a width-to-length ratio of 1 : 1–16. The chloroplast is parietal and one per cell, mostly on the apical part of the cell. Tufts of hairs in sunken curved cryptostome-like pits are numerous and arise from the mid to basal parts of the erect perithallial filaments (Fig. 2E). Rhizoids are unevenly scattered, consisting of multiple cells with 6–15 μm in width (Fig. 2F). The reproductive portions, including plurangial and unangial sori, form elevated areas on different thalli (Fig. 2G, H & J). Plurangial reproductive structures are intercalary, measuring 65–153 μm in length, composed of 1–2 reproductive filaments, and terminated by 1–4 sterile cells (Fig. 2G & I). Sterile cells are elongated rod-shaped, 4–12 μm wide, and 1.5–4 times as long as the width (Fig. 2I). The unangial reproductive sori have two types of filaments, paraphyses and irregularly branched filaments with unangia arising sequentially from mainly unicellular stalks (Fig. 2J & K). The paraphyses arise from the same filaments that produce the unangia. They are clavate, composed of 14–20 cells, 114–363 μm long, and 3–16 μm wide. Mature unangium has a single-celled stalk supported by a cell that branches and also produces another unicellular stalk with a terminal unangium primordium (Fig. 2K). Unangia are oblong, 90–180 μm long, 20–46 μm wide, and positioned centrally among surrounding filaments (Fig. 2J & K).
Type locality
Hwasam-ri183-7, Yongnam-myeon, Tongyeong-si, Gyeongsangnam-do, Korea (34°51′08.07″ N, 128°27′03.36″ E).
Paratypes
CUK13559 (Jan 22, 2015; Bangeojin, Bangeo-dong, Dong-gu, Ulsan, Korea); CUK18162A, CUK18164A (Jul 8, 2017; Cheongsapo, Jung-dong, Haeundae-gu, Busan, Korea); CUK18395, CUK18396, CUK18397, CUK18398, CUK18399 (Nov 18, 2017; Daecheon Beach, Sinheuk-dong, Boryeong-si, Chungcheongnam-do, Korea); CUK18400, CUK18401, CUK18402, CUK18404, CUK18405 (Nov 17, 2017; Chaeseokgang, Buan-gun, Jeollabuk-do, Korea); CUK18428A, CUK18432, CUK18440A, CUK18445A, CUK18449, CUK18452 (Nov 3, 2017; Dala park, Sanyang-eup, Tongyeong-si, Gyeongsangnam-do, Korea); CUK18457, CUK18458, CUK18459, CUK18460 (Nov 4, 2017; Yeonhwa-ri, Gijang-eup, Gijang-gun, Busan, Korea); CUK18729B, CUK18730, CUK18731 (Mar 1, 2018; Cheongsapo, Jung-dong, Haeundae-gu, Busan, Korea); CUK18737A, CUK18738B (Mar 1, 2018; Yeonhwa-ri, Gijang-eup, Gijang-gun, Busan, Korea); CUK18751, CUK18753, CUK18755A, CUK18768 (Mar 3, 2018; Pado-ri, Sowuon-myeon, Taean-gun, Chungcheongnam-do, Korea); CUK18823 (Apr 1, 2018; Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea); CUK18883 (Apr 29, 2018; Bangjukpo, Dolsan-eup, Yeosu-si, Jeollanam-do, Korea); CUK18972 (May 18, 2018; Seosang, Seo-myeon, Namhae-gun, Gyeongsangnam-do, Korea); CUK19150 (Oct 3, 2018; Chaeseokgang, Byeonsan-myeon, Buan-gun, Jeollabuk-do, Korea); CUK19215A, CUK19217A, CUK19218A, CUK19219A, CUK19222A, CUK19225A (Nov 3, 2018; Pado-ri, Sowuon-myeon, Taean-gun, Chungcheongnam-do, Korea); CUK19794 (Jul 26, 2019; Gyeokpo Port, Byeonsan-myeon, Buan-gun, Jeollabuk-do, Korea); CUK19813A, CUK19823 (Aug 1, 2019; Oeyeondo, Ocheon-myeon, Boryeong-si, Chungcheongnam-do, Korea); CUK20066D (Oct 28, 2019; Hwasam-ri, Yongnam-myeon, Tongyeong-si, Gyeongsangnam-do, Korea); CUK20165, CUK20166 (Feb 8, 2020; Hwasam-ri183-7, Yongnam-myeon, Tongyeong-si, Gyeongsangnam-do, Korea); CUK20578, CUK20585, CUK20589 (Aug 5, 2020; Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea); CUK20613A (Aug 12, 2020; Chunghwa-dong Port, Baekryeongdo, Incheon, Korea); CUK20649 (Aug 12, 2020; Dumunjin Port, Baekryeongdo, Incheon, Korea); CUK20666C, CUK20673A, CUK20676A, CUK20677 (Aug 12, 2020; Gobong Port, Baekryeongdo, Incheon, Korea).
Reproductive phenology
Plants with plurangial and unangial reproductive structures were collected during January and February.
Distribution and habitat
This species has been observed in various localities along the eastern coast (Busan and Ulsan), western coast (Baekryeongdo, Boryeong, Buan, and Taean) and southern coast (Namhae, Tongyeong, Wando, and Yeosu), Korea. Plants are found on hard substrates, including pebbles and rocks and seldom on shells of molluscs in the intertidal zone.
DISCUSSION
Oteng’o et al. (2023b) recognized a unique and unidentified clade within Ralfsiaceae based on specimens collected in Korea and Far East Russia. They classified this clade as incertae sedis. In this study, we have focused on researching a greater number of samples belonging to the incertae sedis clade. We suggest the establishment of Ramipedicella gen. nov. based on morphological and molecular examinations to incorporate this unidentified group. The genetic divergences between Ramipedicella and other genera in the Ralfsiaceae have been determined to be ≥6.7–9.3% for rbcL and ≥15.5–20.8% for COI-5P, surpassing the corresponding values observed among the already acknowledged genera (Oteng’o et al. 2023b). Our phylogenetic analyses based on both the concatenated (rbcL + COI-5P) and rbcL datasets indicate that Ramipedicella is closely related to Endoplura and Fissipedicella, forming a sister relationship (Fig. 1, Supplementary Fig. S1). Although Endoplura shows a sister relationship with our Ramipedicella, it is distinguished from Ramipedicella by having several chloroplasts per cell, sessile unangia and 2–5 plurangial reproductive filaments (Hollenberg 1969, Tanaka and Chihara 1981b, Lim et al. 2007, Oteng’o et al. 2021, 2023a). Also, Fissipedicella is distinguished from Ramipedicella by having unistratose thalli and unangia on stalks with vertically or obliquely cleaved cells (Oteng’o et al. 2023b). Our new genus, Ramipedicella, is distinguished from the other genera within the Ralfsiaceae by having multistratose thalli and one to several unangia arising from unicellular stalks in specialized branched filaments or on lateral-basal region to the paraphyses (Table 2). A combination of characters is often used in the identification of different taxa within the Ralfsiales.
Key to genera within Ralfsiaceae
1. Plants with heteromorphic thalli, crustose portion bearing plurangia, and erect sporophytic thalli bearing unangia ........................................................... Heteroralfsia
1. Plants with isomorphic thalli, either crustose or erect thalli bearing both plurangia and unangia ...................... 2
3. Chloroplasts per cell several, plurangial reproductive filaments 2–5 ...................................................... Endoplura
3. Chloroplast per cell single, plurangial reproductive filaments 1–2 ............................................................................ 4
4. Thalli multistratose, one to several unangia on specialized branched filaments or lateral-basal region to the paraphyses ………………………......…. Ramipedicella
4. Thalli unistratose or multistratose, one unangia on unbranched filament or lateral-basal region to the paraphyses .......................................................................... 5
5. Tufts of hair in pits present, stalk composed of 1–6 vertically or obliquely cleaved cells .................. Fissipedicella
5. Tufts of hairs in pits absent, stalk composed of 1–2 horizontally cleaved cells ............................................... Ralfsia
The detailed morphology of unangia has been recognized as a crucial characteristic in the classification of crustose brown algae, particularly within the Ralfsiales. It has been applied in delineating various taxonomic ranks within this group (Tanaka and Chihara 1981b, León-Alvarez and González-González 2003, Lim et al. 2007, León-Alvarez et al. 2017, Parente et al. 2021, Oteng’o et al. 2023b). The diverse morphological features observed in unangia, including stalk-like filaments, paraphyses, and the positioning of unangia, suggest that these reproductive structures could carry taxonomic importance within distinct groups at different levels within the Ralfsiales (Oteng’o et al. 2023b). According to Oteng’o et al. (2023b), there are two main types of unangia: those with paraphyses and those without. A paraphysis is considered a cell or filament arising from a vegetative or reproductive filament adjacent to the reproductive structures and its probable function is providing protection (León-Álvarez and Norris 2005). Apart from the paraphyses, the presence or absence, as well as the morphology of stalks, represents another important aspect. Ramipedicella produces unangia with paraphyses, similar to species of Endoplura, Fissipedicella and Ralfsia within Ralfsiaceae (Hollenberg 1969, Tanaka and Chihara 1981a, Xiyi and Junfeng 1993, Oteng’o et al. 2021, 2023a, 2023b). However, Ramipedicella differs in that it has one to several unangia on specialized branched filaments, whereas the latter three genera have sessile unangia or unangia on uniseriate stalks. With the exception of Ramipedicella, there are no reports of branched stalks in any other genera within the Ralfsiaceae.
Our phylogenetic analyses reveal that the new genus, Ramipedicella, is composed of two distinct species: R. miniloba sp. nov. and “Ralfsia longicellularis”. Although the two species share similarities, R. miniloba is distinguished from Ralfsia longicellularis by crusts with small lobes, numerous hair tufts, plurangia terminating with 1–4 sterile cells, and large oblong unangia. Ralfsia longicellularis was originally described from Russia by Perestenko (1980) and has only been described and reported from Russia (Kozhenkova 2009). Recently, this species was added as new recorded species in list of Korean macroalgal flora by Oteng’o et al. (2020) based on comparison of rbcL genetic analyses and morphology between Korean and Vladivostok samples. However, phylogenetically, “Ralfsia longicellularis” was clearly distinguished as a different species from other species of Ralfsia (Oteng’o et al. 2023b) and reported as Endopluraean sp. 1GWS, positioning it within a closely related taxon with Endoplura by Parente and Saunders (2019). Oteng’o et al. (2023b) clarified that it may belong to a novel genus, representing a distinct unidentified taxon (incertae sedis) comprised of “Ralfsia longicellularis” and Endopluralean sp. 1GWS within the Ralfsiaceae. On the basis of both the concatenated (rbcL + COI-5P) and rbcL gene phylogeny and morphological features, we hereby transfer Ralfsia longicellularis to Ramipedicella.
Ramipedicella longicellularis (Perestenko) A. O.Oteng’o, B. Y. Won & T. O. Cho comb. nov. (Fig. 3)
Basionym
Ralfsia longicellularis Perestenko 1980: 193, figs 308, 309 [The Seaweeds of Peter the Great Bay. Akademia NAUK SSSR, Leningrad, Russia, 231 pp.].
Specimens examined
CUK10442 (Oct 24, 2013; Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea); CUK17342 (May 20, 2016; Sa-ri, Heuksan-myeon, Sinan-gun, Jeollanam-do, Korea); CUK18766 (Mar 3, 2018; Pado-ri, Sowuon-myeon, Taean-gun, Chungcheongnam-do, Korea); CUK18819, CUK18824 (Apr 1, 2018; Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea); CUK19221, CUK19224 (Nov 3, 2018; Pado-ri, Sowuon-myeon, Taean-gun, Chungcheongnam-do, Korea); CUK19270 (Jan 26, 2019; Chaeseokang, Byeonsan-myeon, Buan-gun, Jeollabuk-do, Korea); CUK20022, CUK20023, CUK20025, CUK20026, CUK20027, CUK20028, CUK20029, CUK20030, CUK20031, CUK20032 (Oct 10, 2019; Ulitsa Leytenanta Shmidta, Vladivostok, Russia); CUK20611A, CUK20616A, CUK20617 (Aug 12, 2020; Chunghwa-dong Port, Baekryeongdo, Incheon, Korea); CUK20650B, CUK20653B, MABIK AL00100601(= CUK20659) (Aug 12, 2020; Dumunjin Port, Baekryeongdo, Incheon, Korea); CUK20664A, CUK20664C (Aug 12, 2020; Gobong Port, Baekryeongdo, Incheon, Korea).
Reproductive phenology
Plants with plurangial reproductive structures were collected from the Korean coast in March. Unangia bearing plants were collected from Vladivostok, Russia in October.
Distribution and habitat
This species grows in various localities along the western coast (Baekryeongdo, Buan, Sinan, and Taean) and southern coast (Wando), Korea. Outside Korea, the species is found in the Canadian Pacific (as Endopluralean sp._1GWS) and Far East Russia (Fig. 4). Plants grow on hard substrates, including pebbles, small boulders and rocks in the intertidal zone.
The genetic distances between Ramipedicella miniloba and R. longicellularis were shown to be 1.6–1.7 and 7.7–8.7% for rbcL and COI-5P, respectively, values that are comparable to genetic divergences between other species in the Ralfsiales (Parente and Saunders 2019, Parente et al. 2021, Oteng’o et al. 2023a). Although the two species show resemblance to one another, Ramipedicella longicellularis differs from R. miniloba by having generally smooth crusts, absence of phaeophycean hairs, plurangia terminated by 1–2 apical sterile cells, and smaller mostly oblanceolate unangia.
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