Two urostylid ciliates,
Anteholosticha pulchra
(Kahl, 1932) Berger, 2003 and
Metaurostylopsis struederkyp-keae
Shao et al., 2008, new to Korea, were collected from the Yellow Sea and the East Sea, Korea, respec-tively. They were identified based on live observation and protargol impregnation. Taxonomical characters of
A. pulchra
are as follows: 190-300×30-55 μm size
in vivo
; contractile vacuole located on the left side of the posterior 1/4 of the cell; spherical-reddish granules at cirral bases and around dorsal bristles, somewhat sparsely distributed throughout the cell surface; four frontal and two frontoterminal cirri; four dorsal kineties;caudal cirri absent.
Metaurostylopsis struederkypkeae
is characterized as follows: 80-110×40-50 μm size
in vivo
; caudal cirri absent; two types of cortical granules: type 1, yellow-green arranged along the ventral cirral rows and dorsal kineties; type 2, small and reddish, with an irregular arrangement; four frontal, four to eight frontoterminal, and two to six transverse cirri; five to seven left and three to five right marginal rows. Sequences of small subunit ribosomal DNA were determined from both species, and pairwise distances with their relatives were analyzed.
INTRODUCTION
Genera
Anteholosticha
Berger, 2003 and
Metaurostylopsis
Song et al., 2001 belong to the family Urostylidae Bütschli, 1889. This family is characterized by having one or more marginal rows of left and right marginal cirri and the midventral cirri which arranged as a “zig-zag” file (Borror, 1979; Borror and Wicklow, 1983; Lynn, 2008).
Diverse and polyphyletic species in the
Holosticha
com-plex (Gao et al., 2010) were split up into four genera,
Holosti-cha
,
Anteholosticha
,
Caudiholosticha
and
Biholosticha
by Berger (2003). Recently Li et al. (2009) added another genus
Nothoholosticha
to this complex, and therefore there are five genera in the
Holosticha
complex at present. The genus
Ante-holosticha
can be distinguished from other genera in
Holo-sticha
complex by its lack of caudal cirri and several other apomorphic characters, e.g., anterior end of left marginal row curved rightwards, proximalmost membranelles widened (Berger, 2003). Recently a Chinese population of
A. pulchra
was reported by Li et al. (2007).
The genus
Metaurostylopsis
was established by Song et al. (2001). The characteristics of this genus are as follows: clear-ly differentiated frontal, buccal, transverse cirri and frontoter-minal cirral row present; caudal cirri absent; more than one left and right marginal row (Song et al., 2001).
Metaurosty-lopsis struederkypkeae
was newly established by Shao et al. (2008).
Here, we redescribe two urostylid ciliates,
A. pulchra
and
M. struederkypkeae
, collected from Korea. For precise iden-tification and description, we conducted live observation, protargol impregnation and small subunit ribosomal DNA(SSU rDNA) sequence analysis.
MATERIALS AND METHODS
Anteholosticha pulchra
was isolated from Incheon Harbor (salinity, 30.6‰; temperature, -0.6 ̊C; 37 ̊ 6′N, 126 ̊ 35′E), Incheon, Korea at January of 2011.
Metaurostylopsis strue-derkypkeae
was collected from the lagoon of Youngrang (salinity, 14.8‰; temperature, 18.3 ̊C; 38 ̊13′N, 128 ̊ 35′E), Sokcho, Korea at June of 2011. These two populations were maintained in petri dishes with rice grains to enrich bacteria growth as a food resource. The specimens were observed
in vivo
under a light microscope (Leica DM2500, Wetzlar, Ger-many) at a magnification of 50× to 1,000×. Protargol im-pregnation was performed to reveal the infraciliatures (Foiss-ner, 1991). Drawing was performed under a light microscope at a magnification of 1,000×. Terminology and classification were mainly followed according to Lynn (2008).
DNA extraction, amplification and sequencing of nearly complete SSU rDNA were performed according to Jung et al. (2011). Our sequences and retrieved sequences from Gen-Bank were aligned using BioEdit (Hall, 1999), and pairwise genetic distance based on Kimura two-parameter inference was obtained using MEGA 4.0 (Kumar et al., 2004).
Comparison of morphometric data from Anteholosticha pulchra with Li et al. (2007)All data compared protargol-impregnated specimens of Korean population (Ko) and Chinese population (Ch).Frontal cirri including parabuccal cirrus (III/2).P, population; Min, minimum; Max, maximum; Mean, arithmetic mean; CV, coefficient of variation in %; n, number of individuals examined.
Comparison of morphometric data from Anteholosticha pulchra with Li et al. (2007) All data compared protargol-impregnated specimens of Korean population (Ko) and Chinese population (Ch). Frontal cirri including parabuccal cirrus (III/2). P, population; Min, minimum; Max, maximum; Mean, arithmetic mean; CV, coefficient of variation in %; n, number of individuals examined.
Korean name: 1*홍색옛전열하모충 (신칭)
RESULTS
-
Phylum Ciliophora Doflein, 1901
-
Class Spirotrichea Bütschli, 1889
-
Order Urostylida Jankowski, 1979
-
Family Urostylidae Bütschli, 1889
-
GenusAnteholostichaBerger, 2003
-
1*Anteholosticha pulchra(Kahl, 1932) Berger, 2003 (Table 1, Figs.1 A-E, 2)
Keronopsis pulchra
Kahl, 1932: 573, fig.104 ; Kahl, 1933:109, fig. 16.
Holosticha (Keronopsis) pulchra
: Berger, 2001: 36.
Anteholosticha pulchra:
Berger, 2003: 377; Berger, 2006: 435, fig.90 ; Li et al., 2007: 113, figs. 4-7
Description.
Live cell size usually 190-300×30-55 μm, slen-der in shape (
Figs.1
A,
2
A, B), flexible but not contractile, an-terior and posterior ends rounded, contractile vacuole approxi-
Morphology of Anteholosticha pulchra and Metaurostylopsis struederkypkeae from live (A-C F-J) and protargol impregnated (D E K L) specimens. A-E Anteholosticha pulchra: A Ventral view of live contractile vacuole (arrow); B C Pattern of cortical granules in dorsal and ventral views reddish cortical granules (arrow); D E Cirral base in ventral and dorsal views from protargol impregnation; F-L Metaurostylopsis struederkypkeae: F Ventral view of live contractile vacuole (arrow); G H Ventral and dorsal views yellow-green cortical granules (arrow) small reddish cortical granules (arrowhead); I J Ventral and dorsal views which show the arrangement of yellow-green cortical granules; K L Cirral base on ventral and dorsal views with protargol impregnation. AZM adoral zone of membranelles; BC buccal cirrus; DK dorsal kineties; EM endoral membrane; FC frontal cirri; FTC frontoterminal cirri; LMR left marginal row; Ma macronuclear nodules; MP midventral pairs; MVR midventral row; PM paroral membrane; RMRright marginal row; TC transverse cirri. Scale bars: A-E=100 μm F-L=50 μm.
Micrographs of Anteholosticha pulchra from live (A-E) and protargol impregnated (F-L) specimens. A B Live ventral (A) anddorsal (B) view arrangement of cortical granules (arrowhead); C Ventral view contractile vacuole (arrow); D Dorsal view reddishcortical granules (arrow); E Live ventral view of transverse cirri; F G Dorsal and ventral views of protargol-impregnated specimen; H Ventral view the frontal (arrows) and frontoterminal (arrowhead) cirri; I Buccal cirri (arrow); J Ventral view the transversecirri; K Dorsal view the dorsal kineties typically four; L Macronuclear nodules (arrow). Scale bars: A B F G=100 μm.
mately 10 μm in diameter, positioned on left side of posterior 1/4 of cell (
Figs.1
A,
2
B, C, arrow). Spherical-reddish corti-cal granules at cirral bases and around dorsal bristles,
ca.
1μm across, some sparsely distributed throughout cell surface (
Figs.1
B, C,
2
D, arrow). Four frontal cirri each located in anterior ventral area,
ca.
18 μm (
Fig.2
H, arrows), and two frontoterminal located near anterior end of right marginal row (
Fig.2
H, arrowhead) and seven to nine transverse cirri (
Fig.2
E, J), respectively
ca.
10 and 20 μm in length, with one left and right marginal row located on left and right side (left marginal cirri
ca.
56, right marginal cirri
ca.
61). Buccal cirrus positioned near paroral membrane (
Fig.2
I, arrow), cau-dal cirri absent. Four dorsal kineties extended from anterior to posterior area (
Fig.2
G, K, arrows) and approximately 49 macronuclear nodules scattered in body (
Figs.1
E,
2
L).
46-60 membranelles in adoral zone,
ca.
25-35% of body length, and thick buccal lip. Long midventral pairs extending to transverse cirri, in a “zig-zag” pattern, 27-34 pairs of cirri. Food vacuoles colorless and sparsely located on cell.
Distribution.
Germany (Kiel, the Baltic Sea), China (Laiz-hou, the Yellow Sea), and Korea (this study).
Remarks.
Polyphyly of genus
Anteholosticha
was shown by molecular phylogenetic analysis based on the SSU rDNA. Therefore this genus should be divided into subgroups acc-
Comparison of morphometric data from Metaurostylopsis struederkypkeae with Shao et al. (2008)All data compared protargol-impregnated specimens of Korean population (Ko) and Chinese population (Ch).P, population; Min, minimum; Max, maximum; Mean, arithmetic mean; CV, coefficient of variation in %; n, number of individuals examined.
Comparison of morphometric data from Metaurostylopsis struederkypkeae with Shao et al. (2008) All data compared protargol-impregnated specimens of Korean population (Ko) and Chinese population (Ch).P, population; Min, minimum; Max, maximum; Mean, arithmetic mean; CV, coefficient of variation in %; n, number of individuals examined.
ording to the relativeness of both morphology and phylogeny (Berger, 2006; Gao et al., 2010; Yi and Song, 2011). Among
ca.
40 species known from this genus, following five species showed closest relationship with
Anteholosticha pulchra
in morphology:
A. gracilis
,
A. xanthichroma
,
A. australis
,
A.sigmoidea
and
A. monilata.
Anteholosticha gracilis
is most similar to
A. pulchra
be-cause both species have four frontal and two frontoterminal cirri as marine ciliates. However
Anteholosticha pulchra
dif-fers from
A. gracilis
mainly in size (190-300×30-55 μm vs.100-150×30-40 μm
in vivo
), position of contractile vacuole (posterior 1/4 vs. anterior 1/3), color of cortical granules (re-ddish vs. yellow-greenish), number of dorsal kineties (four vs. three), adoral membranelles (46-60 vs. 24-30) and pair of midventral cirri (27-34 vs. 17-24) (Hu and Suzuki, 2004).
An-teholosticha pulchra
can be distinguished from other species by the position of contractile vacuoles: it locates around 1/4 region from the posterior margin in
A. pulchra
; around 1/3 from the anterior margin in
A. gracilis
; ahead of mid-body in
A. xanthichroma
,
A. australis
,
A. sigmoidea
and
A. monilata
(Foissner and Didier, 1981; Foissner, 1982; Wirnsberger and Foissner, 1987; Blatterer and Foissner, 1988; Hu and Suzuki, 2004).
Nothoholosticha fasciola
transferred from the genus
Ante-holosticha
by Li et al. (2009) because this species has no frontoterminal cirri. However two species are similar in fol-lowing characteristics, e.g., one buccal cirrus and long belt-like body shape. However,
A. pulchra
differs from
N. fasci-ola
in having frontoterminal cirri (vs. absent), four frontal cirri (vs. six), four dorsal kineties (vs. three), reddish cortical granules (vs. bright orange) and the contractile vacuole posi-tion (located around 1/5 region from the posterior margin in
N. fasciola
) (Li et al., 2009).
The Korean population corresponds well with Chinese population reported by Li et al. (2007), and showed high sim-ilarity each other in morphometric characteristics (
Table 1
).
The SSU rDNA sequence of Korean
A. pulchra
is 1,650 bp in length (GenBank accession no: JN880476). Inter-specific genetic variation among five species (
A. pulchra
,
A. gracilis
,
A. monilata
,
A. pseudomonilata
, and
N. fasciola
) are ranged from 3.3% to 6.6% (
Table 3
).
-
1*GenusMetaurostylopsisSong, Petz and Warren, 2001
-
2*Metaurostylopsis struederkypkeaeShao et al., 2008 (Table 2, Figs.1F-L, 3)
Metaurostylopsis struederkypkeae
Shao et al., 2008: 289, figs. 1-20
Description.
Live cell size usually 80-110× 40-50 ㎛ (
Figs.1
F, 3A-C), slender in shape and slightly contractile, with an-terior and posterior ends rounded, and contractile vacuole approximately 10㎛ in diameter located on left side of equa-torial level of cell (
Fig.3
C, arrow). Two types of cortical granules: one small and reddish, approximately 0.5㎛ across, spread irregularly on surface (
Figs.1
G, H, 3D, arrowhead), and other one yellow-green, approximately 1.5 ㎛ across, situated at base of cirri, with some cortical granules clustered along cirral rows and dorsal kineties (
Figs.1
G, H,
3
D, arrow). Color of cells appeared reddish under low magnification due to numerous small cortical granules. Lengths of transverse and frontal cirri 15 ㎛ and 13 ㎛, respectively. Other soma-tic cirri, midventral, left and right marginal cirri,
ca
. 10 ㎛ in length. Four frontal cirri and four to eight frontoterminal cirri located near distal portion of adoral membranelles (
Fig.3
H, arrow). Two to six transverse cirri, and one buccal cirrus located in middle of next paroral membrane (
Fig.3
H, arrow-head). Left and right marginal rows composed of five to seven and three to five, respectively (
Fig.3
F). 12-20 midventral pairs, and six to nine unpaired midventral cirri between mid-ventral pairs and transverse cirri, three dorsal kineties (
Fig.3
G, arrows), and separately two dikinetids (
Fig.3
G, arrow-heads) on anterior dorsal side. Macronuclear nodules, ovoid and ellipsoid shape, c
a.
82. Membranelles in adoral zone con-sisted of 25-36 membranelles, and portion of adoral zone is
ca.
35-45% in length. Paroral and endoral membrane are equal level in length.
Distribution.
China (Qingdao, the Yellow Sea) and Korea (this study).
Remarks.
There are six species in this genus:
Metaurostylop-sis marina
Song et al., 2001,
M. rubra
Song and Wilbert, 2002,
M. songi
Lei et al., 2005,
M. salina
Lei et al., 2005,
M. struederkypkeae
Shao et al., 2008, and
M. cheni
Chen et al., 2011.
Metaurostylopsis struederkypkeae
and
M. rubra
have red-dish cell color. However
M. struederkypkeae
is distinguished from
M. rubra
by the number of right marginal rows (3-5 vs. 6-7) and the type of cortical granules (two vs. one) and size (80-110 × 40-50 ㎛ vs. 150-300× 50-90 ㎛
in vivo
) (Song and Wilbert, 2002).
Metaurostylopsis struederkypkeae
is distinguished from
M. marina
by the body shape (slender vs. oval), the cell color (reddish vs. colorless) and the type of cortical granules (two vs. one) (Song et al., 2001).
The Korean population of
M. struederkypkeae
corresponds with the original description by Shao et al. (2008) and mor-phometric comparisons highly overlap (
Table 2
); however, there are a few differences. The Korean population has more
Micrographs of Metaurostylopsis struederkypkeae from live (A-D) and protargol impregnated (E-H) specimen. A B Live ven-traland dorsal views; C Contractile vacuole (arrow); D Yellow-green cortical granules (arrow) small reddish cortical granules (arrow-head); E Ventral view of protargol-impregnated specimen; F Ventral view left and right marginal rows; G Dorsal view typicallythree dorsal kineties (arrows) and two dikinetids (arrowheads); H Frontoterminal (arrow) and buccal (arrowhead) cirri. Scale bars: A-C E=50 ㎛.
Kimura two-parameter pairwise distances (%) among the five species in Anteholosticha-Nothoholosticha assemblage basedon small subunit ribosomal DNA sequences
Kimura two-parameter pairwise distances (%) among the five species in Anteholosticha-Nothoholosticha assemblage basedon small subunit ribosomal DNA sequences
number of left and right marginal rows, midventral pairs, and macronuclear nodules than those of the Chinese population (Shao et al., 2008).
The SSU rDNA sequence of
M. struederkypkeae
is 1,652 bp in length (GenBank accession no: JN880477). The inter-specific pairwise distances between
M. struederkypkeae
and its relatives (
M. cheni
and
M. salina
) are more than 2%, while intra-specific variation are less than 0.3% among two Korean and one Chinese
M. struederkypkeae
populations (
Table 4
).
Kimura two-parameter pairwise distance (%) based on small subunit ribosomal DNA sequencesaKorean population from Youngrang lagoon (this study),bKorean population from Taehwagang river (Li et al., 2011),cChinese population (Shao et al., 2008).
Kimura two-parameter pairwise distance (%) based on small subunit ribosomal DNA sequences aKorean population from Youngrang lagoon (this study), bKorean population from Taehwagang river (Li et al., 2011), cChinese population (Shao et al., 2008).
Korean name: 1*사미주하모충 (신칭), 2* 작은홍색사미주하모충 (신칭)
Acknowledgements
This study was supported by the Invasive Species Mana-gement Program in Marine Ecosystem, Korean Ministry of Land, Transport & Maritime Affairs of Korean Government, and also funded by the National Fisheries Research & Devel-opment Institute (NFRDI) of Korea and Polar Academic Pro-gram (PAP), KOPRI.
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