Some echinoids were collected from the coast of Gangwon-do during the period from November 2008 to July 2011 and were identified on the basis of morphological characteristics and molecular analysis of cytochrome oxidase subunit I mitochondrial DNA. Among them,
(Sars, 1871) belonging to the family Strongylocentrotidae of the order Camarodonta is reported for the first time in Korea and is redescribed. The genetic differences ranged from 0.038 to 0.139 between
and four other species of genus
, but ranged from 0.002 to 0.005 between Korean specimens and GenBank data of
. This species is widely distributed in cold sea water along the western part of the North Pacific and the Northwest Atlantic.
of the family Strongylocentro-tidae consists of nine species globally (Smith, 2005; Kroh and Mooi, 2011):
S. djakonovi, S. droebachiensis, S. franci-scanus, S. intermedius, S. nudus, S. pallidus, S. polyacan-thus, S. pulchellus and S. purpuratus.
were reported in Korea (Shin and Rho,1996; Shin, 1998, 2011). The former inhabits only the coast of East Sea but the latter inhabits all coastlines of South Korea except Jeju-do Island (Shin, 2011). Some echinoids were collected from Daejin to Imwon harbors in Gangwon-do and were identified. Among them,
is newly re-ported in Korea. Mortensen (1943) reported that morphologi-cal characteristics of
were not distinctly distin-guished from the adjacent species such as
Recently, these two species were recorded as synonymous with
by morphological and mole-cular evidences (Jensen, 1974; Tatarenko and Poltarous, 1992; Bazhin, 1998). Therefore, we examined thoroughly morphological characteristics of Korean
species and analyzed the molecular differences between Ko-rean species and Genbank data of
and then other adjacent species using cytochrome oxidase subunit I (COI) mitochondrial DNA (mtDNA).
MATERIALS AND METHODS
- Sample collection and identification
The specimens of
were collected using fishing nets at depths of 50-190 m from nine coastal areas in Gangwon-do from November 2008 to July 2011 (
). Specimens were preserved in 95% methyl alcohol, and their important morphological characters were photographed by light- and stereo-microscopes (Nikon Eclipse 80
, Nikon SMZ1000; Nikon Co., Tokyo, Japan). Identification of spe-cimens referred to Mortensen (1943), Southward and Camp-bell (2006) and Shin (2011).
- DNA amplification and sequencing
Genomic DNA was extracted from the gonad tissues of echinoids using by DNeasy blood and tissues kit (Qiagen, Hilden, Germany) and the COI gene was amplified using primers of Knott and Wray (2000): ECO1a (5′-ACCATGC AACTAAGACGATGA-3′) and ECO1b (5′-GGTAGTCTGAGTATCGTCGWG-3′). PCR amplification chemistry con-taining 1.5 μL of genomic DNA, 2.5 μL of 10× PCR buffer (contained MgCl2), 1.0 μL of 2.0 mM dNTPs and each pri-mer, 0.3 μL of nTaq DNA polymerase (Enzynomics, Seoul, Korea) and add up to 25.0 μL with distilled water, and the following conditions: initial denaturation of 2 min at 95℃, 30 cycles of 95℃ 30 sec; 52℃ 1 min; and 72℃ 1 min and a final elongation of 7 min at 72℃. DNA fragments were sequ-enced on an ABI 3730XL sequencer (Applied Biosystems Inc., Forster City, CA, USA) using the ABI Prism Bigdye Terminator v3.1 (Applied Biosystems Inc.).
- Molecular data analysis
The mitochondrial COI gene was mostly sequenced for this study, but the sequences data of four species which are not distributed in Korea obtained from GenBank (
Examined materials of Korean echinoidsAll collection sites locate in Gangwon-do except Jeju Island ofH. cras-sispina. S., Strongylocentrotus; H., Heliocidaris.
Examined materials of Korean echinoids All collection sites locate in Gangwon-do except Jeju Island of H. cras-sispina. S., Strongylocentrotus; H., Heliocidaris.
List of taxa included in this study higher taxonomic placement and GenBank accession numbers for their gene sequencesStrongylocentrotus pallidus(A)-(E) were indicated inTable .3COI, cytochrome oxidase subunit I.aObtained from GenBank.
List of taxa included in this study higher taxonomic placement and GenBank accession numbers for their gene sequences Strongylocentrotus pallidus (A)-(E) were indicated in Table .3 COI, cytochrome oxidase subunit I. aObtained from GenBank.
sequences were checked and aligned using by BioEdit v.7.0 (Hall, 1999) and genetic distances were calculated accord-ing to the Kimura 2-parameter model (Kimura, 1980) using by MEGA5 (Tamura et al., 2011). The phylogenetic rela-tionship of the samples was drawn by using the neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian infer-ence (BI). The NJ tree (Saitou and Nei, 1987) was inferred from Kimura 2-parameter genetic distance with bootstrapp-ed 1,000 times using by MEGA5, and the ML analysis with the GTR+G model, determined with jModeltest 0.1.1 (Guin-don and Gascuel, 2003; Posada, 2008), with 1,000 bootstrap replications using by PhyML v3.0 (Guindon and Gascuel, 2003) also BI analysis with same model and analyzed by MrBayes 3.1 with 1×10
generation repeats with the nu-cleotide model 4by4, Nst=6, rates=gamma, Ngammacat=6, and Burnin=2.5×10
(Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003; Ronquist et al., 2005).
Korean name: 1*연약둥근성게 (신칭)
- Systematic notes
Strongylocentrotus pallidus (Sars, 1871)
Toxopneustes pallidus Sars
, 1871: 25.
Bidenkap, 1899: 112; Jensen, 1974: 119; Vader et al., 1986: 10; Southward and Camp-bell, 2006: 144; Kroh and Mooi, 2011: 124324.
Class Echinoidea Leske, 1778
Subclass Euechinoidea Bronn, 1860
Order Camarodonta Jackson, 1912
Infraorder Echinidea Kroh and Smith, 2010
Family Strongylocentrotidae Gregory, 1900
Genus Strongylocentrotus Brandt, 1835
Strongylocentrotus pallidus. A Dorsal side; B Ventral side; C Lateral side; D Dorsal side of denuded test; E Ventral sideof denuded test; F-I Lateral side of denuded test; J Apical system; K-M Amburacral plates; N Interamburacral plates; O Globiferous pedicellaria; P Q Large and small tridentate pedicellaria; R Ophiocephalous pedicellaria; S Triphyllous pedicellaria; T Spicules of tube-feet. Scale Bars: A-I=2.5 cm J-N=0.5 cm O P=300 nm Q R=200 nm S T=100 nm.
Molphological characteristics of species of Strongylocentrotus in Korea(A)-(E) ofS. pallidusused for the molecular analysis. Morphological data ofS. intermediusandS. nudusare referred from Shin (2011). D, diameter; H, height; P, peristome.
Molphological characteristics of species of Strongylocentrotus in Korea (A)-(E) of S. pallidus used for the molecular analysis. Morphological data of S. intermedius and S. nudus are referred from Shin (2011). D, diameter; H, height; P, peristome.
Döderl-ein, 1906: 517.
A Agassiz and HL Clark, 1907: 122; HL Clark, 1912: 360; Mortensen, 1943: 219; Downey, 1968: 82.
HL Clark, 1912: 353; Mor-tensen, 1943: 215; Kroh and Mooi, 2011: 513826.
Strongylocentrotus drøbachiensis sachalinica
D’yakonov, 1938: 470, 496.
Test form vertically very variable (
); flattened (
G), low-hemispherical (
F, H) and hemi-spherical forms (
I). Outline of test roundly pentagonal forms (
D). Margin of oral side slightly sunken towards peristome. Test of largest specimen 59 mm in diameter. Six ambulacral pore-pairs presented in an erected arc (
K-M). Madreporite has convexed form, larger than genital plates (
J). Ocular plates composed five plates, three plates has pentagonal form, stuck between genital plates, like wedges, the other two plates have hexagonal form, situ-ated between genital plates, bordering surnal parts. Surnal parts consist of numerous small plates, with anus situated beside center, surrounded with small blunt spines (
J). Globiferous pedicellaria has single slender apical tooth, without lateral tooth (
O). Tridentate pedicellaria has two different sizes, large and small form; large ones about five times larger than small one (
P, Q). Ophiocephal-ous pedicellaria with fountain pore-pattern valves (
R). Triphyllous pedicellaria apple-like shaped, with upraised radial pore-pattern (
S). Spicule of tube-foot usually elongated arch form, rarely twisted form, which has trifur-cated end, of which inside node slender, tapered to tip (
Korea (Gangwon-do), Japan (Siaukhu Bay), Bering Sea, Okhotsk Sea, North Pacific (Kuril island, Sakah-lin), Northwest Atlantic (Norway, U.K.).
Key to the species of genusStrongylocentrotusin Korea
1. Ambulacral pore-pairs five in number ∙∙∙∙∙∙S. intermediusAmbulacral pore-pairs six in number ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 2
2. Primary spines long and stout ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙S. nudusPrimary spines short and not stout ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙S. pallidus
- Molecular analysis
DNA sequence features.
A total of 1,214 base pairs (bp) of the COI mtDNA were obtained from five specimens of
and the other Korean echinoids such as
S. intermedius, S. nudus
). But, GenBank sequences were shorter than our sequences and so 818 bp COI mtDNA were analyzed in this study. In a group of
, only 4 bp of the 818 bp was different from each.
. The phylogenetic relationships of the COI mtDNA from
species were analyz-ed by BI, ML and NJ method. We appointed
as the outgroup, and analyzed with
S. droe-bachiensis, S. pallidus, S. polyacanthus
obtained from GenBank. In the phylogenetic trees, BI, ML and NJ analyses represented the non-discrimenatory phylogenetic branches (
). Korean specimens of
of GenBank, and our phylogenetic reconstruction suggests that the genus
is paraphyletic, but
is closely related to
and its taxonomical position still obscure. Therefore the genus
Kimura 2-parameter genetic distance between
Maximum likelihood (ML) and neighbor joining (NJ) combined tree generated from the COI mtDNA dataset. Node valuesfollowing ML/NJ. S. Strongylocentrotus; A. Allocentrotus; H. Heliocidaris; COI cytochrome oxidase subunit I; mtDNA mito-chondrial DNA.
Bayesian inference (BI) tree generated from the COI mtDNA dataset. S. Strongylocentrotus; A. Allocentrotus; H. Helio-cidaris; COI cytochrome oxidase subunit I; mtDNA mitochondrial DNA.
(GenBank) ranged from 0.002 to 0.005 (
) distance average of the
group is 0.062 and excepted
group is 0.041. Average of between
is 0.045, which was lower than average of the
group, and between
Interspecific pairwise (p) distance values among six species of genus Strongylocentrotus with an outgroup (Heliocidariscrassispina) based on partial sequences of mtDNA COI gene which determined by the Kimura 2-parameter modelS., Strongylocentrotus; A., Allocentrotus;COI, cytochrome oxidase subunit I; mtDNA, mitochondrial DNA.aObtained from GenBank.
Interspecific pairwise (p) distance values among six species of genus Strongylocentrotus with an outgroup (Heliocidariscrassispina) based on partial sequences of mtDNA COI gene which determined by the Kimura 2-parameter model S., Strongylocentrotus; A., Allocentrotus; COI, cytochrome oxidase subunit I; mtDNA, mitochondrial DNA. aObtained from GenBank.
tance (=0.039) was lower than the
Two species of
) were previously reported (Shin, 2011). In this study,
is newly reported, which has distinct characteri-stics compared with two species: external figure, pedicella-riae and number of pore-pairs in an arc.
has various range of test height; the range of hei-ght/diameter of Korean specimens is 38.2-50.0% and range of peristome/diameter is 31.6-35.7% (
). However, Mortensen (1943) described the range of peristome/diame-ter as 31.6-45.0%. Thus, we divided the three groups based on the test height: Group-1 (flattened form)=
(A), Group-2 (low-hemispherical form)=
(B)- (D) and Group-3 (hemispherical form)=
(E). Korean speci-mens of
revealed as identical with
of GenBank. According to the results of phylogenetic analysis and genetic distance (
). In the phylogene-tic tree,
group divided by two clades (
(A), (E) and
(B)-(D). But, that is not a significant cladogram, because the intraspecific
-distance average value is only 0.003 and is much lower than other interspecific
is very similar with
and can only be discriminated by rather impal-pable differences (Vasseur, 1951; Swan, 1962; Jensen, 1974;Vader et al., 1986). For that reason, it has been included in
(Mortensen, 1943). In these phylogenetic results,
showed rather close relationships (
-distance value is only 0.039, which is lower than average of
(0.062) and in phylogenetic tree,
were branched off close to
. Thus, echinoid specimens are unrecord-ed species examined in Korea by determining on the basis of the morphological and molecular evidences. Recently, they were divided from each other on the basis of morphological characters within populations and between geographical districts, genetic differences and capacity for hybridization (Vasseur, 1951; Jensen, 1974; Vader et al., 1986; Biermann et al., 2003).
Mortensen (1943) reported that
is very unlike any of the true species of
. However, previous studies (Strathmann, 1979; Biermann et al., 2003) and our phylogenetic results (
) show rather close relationships between
and the species of
. The taxonomical position of
needs to be consider in a further study.
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