The type species of
After the first record of "
To clarify the entry of
According to previous characters of "
DNA extraction, amplification, and sequencing
Genomic DNA was extracted from silica-gel samples using the G-spinTM Iip genomic DNA extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea). The
Alignment and phylogenetic analyses
All generated sequence data of
Thalli grow on rocky areas in the intertidal zone. Thalli grow up to 7 cm high, form dense tufts (Fig. 1A), and consist of erect and prostrate axes. Branching is pseudodichotomous (Fig. 1B) to trichotomous and, rarely, tetrachotomous. Branches are formed at intervals of 10-13 axial cells in the main and lateral axes. Adventitious branches develop from periaxial cells. Erect axes have forcipulate apices (Fig. 1B). The mature cortex is complete. Twelve periaxial cells are cut off obliquely from each axial cell and remain at the nodes (Fig. 1E & F). All periaxial cells (except the abaxial one) produce 3 corticating initials (Fig. 1G & I). Each corticating initial divides into cortical filaments contributing to the cortex. The first cortical initial acropetally cuts off a spine and a cortical cell (Fig. 1G), a gland cell and a cortical cell (Fig. 1H), or two cortical cells. The second cortical initial cuts off one cell acropetally and a filament basipetally by transverse division (Fig. 1G-I). The third cortical initial cuts off a filament basipetally (Fig. 1G-I). Acropetal cortical filaments are two cells long, including the cortical cell initial. Whorled spines occur at each node (Fig. 1B & C). Each spine is straight and three cells long (Fig. 1G) including the cortical initial. A gland cell is obliquely cut off from the first cortical initial and is ovoid in shape (Fig. 1H & I). Rhizoids are numerous, uniseriate, and multicellular and are produced from periaxial cells on the prostrate (Fig. 1D).
[Fig. 1.] Specimen (TC6721) of Centroceras gasparrinii from Baekdongri, Imhoemyeon, Jindo, Jeollanamdo, Korea. (A) Thallus. (B) Apical region. (C) Whorled arrangement in middle part of thallus. (D) Lower part of thallus showing rhizoid produced from periaxial cells. (E-F) Cross section views through cortical nodes of upper (E) and middle (F) parts of thallus. (G) Cortical unit showing the first cortical initial bearing a spine and a cortical cell. (H) Cortical unit showing the first cortical initial with both an ovoid gland cell (arrow) and a cortical cell. (I) Mature cortication showing cortical filaments with gland cell (arrows) developed from periaxial cells. (J) Tetrasporic thallus. (K-L) Cortical nodes of upper (K) and middle (L) parts showing tetrasporangia with involucral branchlets (arrows) in the abaxial side of axis. (M) Cortical nodes of lower part showing tetrasporangia with involucral branchlets (arrow) in whorl around the axis. Scale bars represent: A, 1 cm; B, C, K, 100 μm; D, 250 μm; E, F, L, M, 50 μm; G, H, I, 25 μm; J, 1 mm. Ax, Axial cell; C1?3, Sequence of cortical initials; P, Periaxial cell; T, Tetrasporangium; R, Rhizoid.
[Fig. 2.] Phylogeny of the Centroceras based on the rbcL sequences inferred from Maximum Likelihood analyses using the General Time Reversible model (GTR) (Rodriguez et al. 1990) + G (Gamma distribution). The parameters were as follows: assumed nucleotide frequencies A = 0.3235, C = 0.1361, G = 0.2043, T = 0.3361; substitution rate matrix with A-C substitutions = 1.3647, A-G = 3.9246, A-T = 2.3629, C-G = 0.9594, C-T = 18.2459, G-T = 1.0000; proportion of sites assumed to be invariable = 0 and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.1547. Bootstrap proportion values (> 50%) for maximum likelihood (500 replicates).
Tetrasporangia are distributed along upper cortical nodes of the erect axes (Fig. 1J). They are produced initially on the abaxial side (Fig. 1K & L), then on the adaxial side resulting in a whorl around the axis (Fig. 1M). A tetrasporangium is tetrahedral, spherical to ellipsoidal, and 42-55 μm in size. It is surrounded by involucral branchlets that are produced from the first cortical initials or are transformed from spines (Fig. 1K-M). Male and female thalli were not found in our collections.
A 1412-bp portion of the 1467-bp
Specimens from Korea correspond in their detailed morphological characters to the description of
To identify each species in Centroceras, Won et al. (2009) indicated that the vegetative features of Centroceras such as the shape of the acropetal cortical cells, the number of acropetal cortical cells and the shape of gland cells and spines are more important than previously used features. The importance of gland cell features has been viewed as one of the diagnostic characters in Centroceras, and its potential taxonomic merit was reported by Gallagher and Humm (1983) and Won et al. (2009). Two different shapes of gland cells were recognized: ovoid in C. gasparrinii, C. hyalacanthum, C. micracanthum versus flattened in C. clavulatum, C. rodmanii, C. tetrachotomum (Won et al. 2009). Won et al. (2009) also described two different types of reproductive structure: tetrasporangia with involucral branchlets in C. gasparrinii and without involucral branchlets in C. clavulatum, C. hyalacanthum, C. rodmanii and C. tetrachotomum. Involucral branchlets rarely occur in tetrasporic plants, but are commonly exhibited in female plants in Ceramiaceae. Although tetrasporangial involucral branchlets have not been emphasized as diagnostic characters for Centroceras species, they may be key characters for C. gasparrinii. Although C. gasparrinii is similar to C. clavulatum in that they both have the dichotomous branching pattern, the arrangement of cortical filaments, the straight spines and the ovoid cortical cell, the former is distinguished from the latter by having ovoid gland cells and tetrasporangia with involucral branchlets. Centroceras species with dichotomous habit from Korea has been recognized as C. clavulatum (e.g., Okamura 1936, Kang 1966, Boo and Lee 1985, Lee 2008). However, the results in this study show that all Centroceras collections with dichotomous branching patterns from Guryongpo, Gampo, Jindo, and Jeju, Korea are not C. clavulatum, but C. gasparrinii having ovoid gland cells and tetrasporangia with involucral branchlets.
In addition, newly generated sequences of rbcL gene reveal that all taxa of Centroceras collected from Korea are C. gasparrinii. Five sequences of Centroceras species from Korea are clustered in a group and form a monophyletic group with sequences of C. gasparrinii downloaded from GenBank. There is 0-0.07% sequence divergence among specimens of C. gasparrinii in Korea.
Centroceras gasparrinii is widely distributed throughout the Pacific Ocean (including Korea), Atlantic Ocean, Indian Ocean, the Gulf of Mexico, and Caribbean Sea, while C. clavulatum is restricted to northern Chile, Peru, Southern California, S. Australia, and New Zealand (Won et al. 2009). In Korea, Centroceras gasparrinii was reported from the east coast of Korea (Won et al. 2009) and its distribution range extends to the south coast and Jeju Island in this study.