Fissipedicella orientalis gen. et sp. nov. (Ralfsiales, Phaeophyceae), a new crustose brown alga from Korea based on molecular and morphological analyses

DNA extraction and amplification

From 2018 to 2020, we collected samples from intertidal areas along the coastlines of Korea (Supplementary Table S1). Genomic DNA was extracted using the NucleoSpin Plant II Kit from Macherey-Nagel (Düren, Germany), following the manufacturer’s instructions. The rbcL and COI-5P were amplified using polymerase chain reaction with HelixAmp Ready-2x-Go premix from NanoHelix Co., Ltd. (Daejeon, Korea), following the manufacturer’s protocol. The primer sets used for the amplifying rbcL and COI-5P were as specified in a previous study by Oteng’o et al. (2021).

Sequence analyses

The sequence datasets were assembled by combining previously published sequences from GenBank with 12 newly generated rbcL and 11 COI-5P sequences from this study (Supplementary Table S1). We analyzed two datasets: one consisting of 57 rbcL sequences and the other with 57 concatenated sequences (rbcL + COI-5P). We considered sequences that were unavailable in GenBank as missing data. We included sequences from Cladostephus spongiosus (Hudson) C. Agardh and Microzonia phinneyi (E. C. Henry & D. G. Müller) Camacho & Fredericq for the outgroup. The sequences were aligned using ClustalW (Thompson et al. 1994) and manually edited using Geneious Prime (v.2023.0.1; Biomatters Ltd., Auckland, New Zeland). To determine the best partition scheme and model evolution, we used PartitionFinder 2 (Lanfear et al. 2016). For the maximum likelihood (ML) analysis, RAxMLGUI v1.5 (Silvestro and Michalak 2012) was employed with the General Time Reversible (GTR) + Γ + I model with 1,000 bootstrap replications. We conducted Bayesian inference 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 GTR + Γ + I evolutionary model. Trees were sampled every 1,000 generations. Summary trees were generated with a burn-in value of 25%. The convergence was assessed using Tracer v.1.7.2 (Rambaut et al. 2018). Phylogenetic trees were constructed using the ML method in RAxML and MrBayes and visualized FigTree v.1.4.0 (Rambaut 2012). Genetic distances were calculated using the p-distance method in Mega X (Kumar et al. 2018).

Morphological analyses

To examine external morphological characteristics, samples were photographed using a waterproof digital camera (Nikon COOLPIX AW100; Nikon Corp., Tokyo, Japan). Before morphological observation, the samples were carefully detached from the substrate using a single-edged blade. We either squashed or prepared microtome sections of portions of each sample mounted on microscopy slides to observe internal morphology. The collected samples were embedded in a matrix (OCT; CellPath Ltd., Newtown, Wales, UK) to prepare microtome-sectioned samples. These embedded samples were then sectioned to a thickness of 8–10 μm using a freezing microtome (Shandon Cryotome FSE; Thermo Shandon Ltd., Loughborough, UK). The sliced and squashed samples underwent staining using a 1 : 1 combination of aqueous aniline blue and acetic acid for better visualization and analysis. The staining process enhanced the visibility of certain structures and details. The stained sections were mounted in 50% corn syrup and observed under a microscope. Photographs of samples were taken using a DP-71 camera (Olympus, Tokyo, Japan) mounted on a BX-51TRF microscope (Olympus). To ensure clarity and improve visual presentation, the digitized images were edited 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 National Institute of Biological Resources (NIBR), Korea.

Phylogenetic analyses

A total of 12 rbcL sequences (984 bp) and 11 COI-5P (654 bp) sequences were generated from newly collected samples from Korea (Supplementary Table S1). The phylogenetic tree inferred from concatenated dataset (rbcL + COI-5P) indicated that the unidentified Ralfsia-like crustose algae from Korea form a distinct and previously unknown genus, Fissipedicella orientalis gen. et sp. nov., within the family Ralfsiaceae (Fig. 1). Phylogenetic analyses based on the rbcL sequences showed overall agreement with concatenated phylogeny (Supplementary Fig. S1). Through our multi-gene sequence analyses, significant genetic differences were observed in rbcL (7.4–8.9%) and COI-5P (17.5–19.3%) sequences between Fissipedicella and Ralfsia within Ralfsiaceae (Table 1).

Fig. 1

Phylogenetic tree of Ralfsiales inferred from the concatenated dataset (rbcL + 5′ region of cytochrome c oxidase subunit I [COI-5P]). The value above branches = maximum likelihood bootstrap (BS) values in % ≥50, Bayesian posterior probabilities (BPP) ≥0.75. Values of <BS 50 or BPP 0.75 are indicated by hyphens (−). Values of BS 100 or BPP 1.00 are indicated by asterisks (*). GenBank accession numbers of each sequence data set are shown in Supplementary Table S1. Species analyzed in this study are marked in bold font. Branch lengths are proportional to substitution rate.

Table 1

Gene sequence divergence value (%) of rbcL and COI-5P between genera of Ralfsiaceae using pairwise distance

Morphological observations

Fissipedicella A. O. Oteng’o, B. Y. Won & T. O. Cho gen. nov

Description

Thalli are crustose with circular to irregular outlines, firmly attached to the substratum, and lacking rhizoids. They comprise two main parts: the hypothallial basal layer and the perithallial erect filament. The hypothallial basal layer is formed by one to several prostrate filaments, which are made up of elongated cells that give rise to perithallial erect filaments. The perithallial erect filaments exhibit branching, straight, or upward curvature, gradually tapering towards the surface, and are firmly adjoined. The chloroplast is parietal and one per cell. Tufts of hair in pits frequently arise from the lower to middle portions of the perithallial erect filaments. The plurangial reproductive structure is intercalary, composed of 1–2 reproductive filaments, and terminated by 1–3 sterile cells. Unangia originate from perithallial erect cells that branch to produce the paraphysis and stalk. The stalk serves as a supportive structure for the terminal unangium. The stalks are composed of vertically or obliquely cleaved cells.

Type species

Fissipedicella orientalis.

Etymology

The name “Fissipedicella” refers to unangial pedicels (stalks) formed by vertical cell division.

Korean name

갯바위딱지속.

Fissipedicella orientalis A. O. Oteng’o, B. Y. Won & T. O. Cho sp. nov. (Fig. 2)

Fig. 2

Morphology of Fissipedicella orientalis sp. nov. (A) Thalli on the rock forming epilithic greenish-brown crusts. (B) Thalli with circular confluent crusts. (C & D) Longitudinal section of vegetative young thallus (C) and mature thallus (D) composed of erect perithallial filaments (EF) and hypothallial basal layer (BL). (E) Erect perithallial filaments showing a single chloroplast (arrows) per cell chloroplasts. (F) Tufts with hairs (Ha) in pits developed from perithallial erect filaments. (G) Longitudinal section view of reproductive thallus showing protrusion of plurangial sorus. (H) Longitudinal section showing reproductive thallus with plurangia (Pl), vegetative filaments (VF), and hypothallial basal layer (BL). (I) Plurangial reproductive structure composed of plurangial filaments (PF) and terminal sterile cells (SC). (J) Longitudinal section of reproductive thallus showing protrusion of unangial sorus. (K) Longitudinal section of reproductive thallus showing unangia (arrow) among paraphyses (Pa). (L) Unangia (Un) on stalks (St) with vertical divisions (arrows) and paraphyses (Pa). Scale bars represent: A, 0.5 cm; B, 0.2 cm; C, D & K, 50 μm; E, 10 μm; F, H, I & L, 25 μm; G & J, 100 μm.

Description

Thalli are epilithic crusts, exhibiting a range of colors from greenish or reddish-brown to dark brown, which become very dark brown when dry. They have a circular to irregular outline and are often mere together, with no distinct lighter margins (Fig. 2A & B). Thalli are 0.2–4.5 (−7.5) cm across, 85–421 μm thick, firmly attached to the substratum, with inconspicuous growth lines and mostly smooth surface, with rust-red undersurface, and lacking rhizoids (Fig. 2A & B).

The hypothallial basal layer comprises one to several cell layers in which cells are 4–12 μm wide and have a width-to-length ratio of 1 : 1.8–3.8, giving rise to erect perithallial filaments (Fig. 2C & D). The erect perithallial filaments are branched, straight, or upwardly curved, tapering towards the surface, and firmly adjoined with one another to form pseudoparenchymatous tissue (Fig. 2D). Cells of the erect perithallial filaments are 4–25 μm long and 4–16 μm wide. The chloroplast is parietal and one per cell (Fig. 2E). Tufts of hair in pits arise from the basal to mid parts of erect perithallial filaments (Fig. 2F). Reproductive portions of the plurangial and unangial sori form slightly elevated areas on the same or different thalli (Fig. 2G & J). Plurangial reproductive structures are intercalary, 43–202 μm long, composed of 1–2 reproductive filaments, and terminated by 1–3 sterile cells (Fig. 2H & I). Unangial reproductive thalli give rise to produce two types of filaments, both originating from erect perithallial cells. These filaments branch out to form paraphyses, and there are also shorter biseriate stalk cells that support terminal unangia (Fig. 2K & L). Unangia are obovoid to ellipsoid to oblong, 47–116 μm long, and 7–40 μm wide, and terminally produced on 1–6 celled stalks that are vertically or obliquely cleaved (Fig. 2L). The paraphyses are clavate, 92–148 μm long, 2–12 μm wide, and composed of 5–10 cells (Fig. 2L).

Holotype

CUK18754, Mar 3, 2018, coll. by B. Y. Won and T. O. Cho deposited in the CUK herbarium, Korea.

Type locality

Pado-ri, Sowon-myeon, Taean-gun, Chungcheongnam-do, Korea (36°43′05.65″ N, 126°07′ 33.79″ E).

Isotypes

NIBROR0000004864 (CUK18765) at the National Institute of Biological Resources (NIBR), Korea.

Etymology

The specific epithet, orientalis, refers to the Orient (Far East Asia) region where the alga grows naturally.

Korean name

갯바위딱지.

DNA sequences of type specimens

For the holotype, OK031043 (rbcL) and OK031055 (COI-5P).

Paratypes

CUK18821, CUK18822, CUK18825 (Apr 1, 2018, Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea); CUK19178A (Oct 8, 2018, Gijang, Ilgwang-myeon, Gijang-gun, Busan, Korea); CUK19219B (Nov 3, 2018, Pado-ri, Sowon-myeon, Taean-gun, Chungcheongnam-do, Korea); CUK19509C, CUK19510C, CUK19512B, CUK19519 (Apr 19, 2019, Seongsan Ilchul Peak, Seongsan-eup, Seogwipo-si, Jeju Island, Korea); CUK19606C (May 1, 2019, Seongsan Ilchul Peak, Seongsan-eup, Seogwipo-si, Jeju Island, Korea); CUK19696, CUK19699, CUK19703, CUK19706, CUK19707B, CUK19709, CUK19710 (Jun 6, 2019, Yeraehaean-ro, Seogwipo-si, Jeju Island, Korea); CUK20040D, CUK20041D (Oct 26, 2019, Bomok Port, Bomok-dong, Seogwipo-si, Jeju Island, Korea); CUK20066A (Oct 28, 2019, Hwasam-ri, Yongnam-myeon, Tongyeong-si, Gyeongsangnam-do, Korea); CUK20331, CUK20332 (May 8, 2020, Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea); CUK20753A, CUK20754, CUK20755, CUK20759E, CUK20760A (Aug 17, 2020, Seongsan Ilchul Peak, Seongsan-eup, Seogwipo-si, Jeju Island, Korea); CUK20771, CUK20772 (Oct 17, 2020, Dolsan Port, Dolsan-eup, Yeosu-si, Jeollanam-do, Korea).

Reproductive phenology

Plants with both plurangial and unangial reproductive structures have been collected during March to June and also in November.

Distribution and habitat

This species has been observed in various localities along the western coast (Taean), southern coast (Busan, Tongyeong, and Wando), and Jeju Island, Korea. It is found on hard substrates, including pebbles and rocks in the intertidal zone.

Key to genera within Ralfsiaceae

In the classification of crustose brown algae, the detailed morphology of unangia has been used as a character of various taxonomic ranks within the Ralfsiales (Tanaka and Chihara 1981b, León-Alvarez and González-González 2003, Lim et al. 2007, León-Alvarez et al. 2017, Parente et al. 2021). Due to the variation in detailed morphologies of unangia, such as stalk-like filaments, paraphyses, and position of unangia, these reproductive structures may hold taxonomic significance within different groups at various levels within the Ralfsiales. There are two main types of unangia: those with paraphyses and those without. Unangia with paraphyses has been observed in the Neoralfsiaceae Lim & Kawai in Lim, Sakaguchi, Hanyuda, Kogame, Phang & Kawai, Pseudoralfsiaceae Patente, Fletcher & G. W. Saunders, Sungminiaceae, and Ralfsiaceae (Hollenberg 1969, Lim et al. 2007, Parente et al. 2021, Oteng’o et al. 2022). Fissipedicella orientalis produces unangia from stalks with paraphyses, similar to some species of Ralfsia (Ralfsia confusa Hollenberg, R. hesperia Setchell & N. L. Gardner, R. huanghaiensis Li & Li, R. integra Hollenberg, and R. pedicellata J. Tanaka & Chihara) within Ralfsiaceae (Hollenberg 1969, Tanaka and Chihara 1981a, Xiyi and Junfeng 1993). However, F. orientalis differs in that it has biseriate stalks formed through vertical or oblique divisions, whereas Ralfsia species have uniseriate stalks formed only through transverse divisions. The development of stalk by the vertical division has not been reported in any other genera within the Ralfsiaceae.

Applying molecular analyses to crustose brown algae has significantly broadened our comprehension of the phylogenetic relationships among related families within Ralfsiales, leading to valuable insights into their taxonomic positions. In our molecular analyses, Fissipedicella orientalis is more closely related to incertae sedis (“Ralfsia longicellularis” and Endopluralean sp. 1GWS) than other genera within Ralfsiaceae. “Ralfsia longicellularis” was originally described from Peter the Great Bay, Far East Russia by Perestenko (1980) and was subsequently reported to Korea based on molecular and morphological evidence by Oteng’o and Won (2020). “Ralfsia longicellularis” is clearly distinguished as a different species from other species of Ralfsia in our phylogenetic trees. Endopluraean sp. 1GWS has been reported as a closely related taxon with Endoplura based on molecular data by Parente and Saunders (2019). The sequence divergences for rbcL and COI-5P between Fissipedicella orientalis and the incertae sedis (“Ralfsia longicellularis” and Endopluralean sp. 1GWS) were 6.7–6.8 and 17.8%, respectively (Table 1). The sequence divergence values between genera within Ralfsiaceae have been shown to be ≥5.5 and ≥17.5% for rbcL and COI-5P, respectively (Table 1). In the future, if more information becomes available about the clade of incertae sedis, it may be recognized as a distinct taxonomic group within Ralfsiaceae. Additionally, none of unpublished and non-identified sequences in GenBank included in these analyses have been related to our new genus, Fissipedicella orientalis gen. et sp. nov.