J. Eukaryot. Microbiol., 55(4), 2008 pp. 331–342
r 2008 The Author(s)
Journal compilation r 2008 by the International Society of Protistologists
DOI: 10.1111/j.1550-7408.2008.00332.x
Identification of Three Highly Confused Marine Loxophyllum (Ciliophora:
Pleurostomatida) with a Key to Seven Congeners from the China Sea
XIAOFENG LIN,a KHALED A. S. AL-RASHEID,b SALEH A. AL-QURAISHY,b SALEH A. AL-FARRAJb and WEIBO SONGa,c
a
Laboratory of Protozoology, College of Life Science, South China Normal University, Guangzhou 510631, China, and
b
Zoology Department, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia, and
c
Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, China
ABSTRACT. Three highly confused Loxophyllum helus-like morphotypes (i.e. Loxophyllum rostratum Cohn, 1866, Loxophyllum
sinicum n. sp., and Loxophyllum simplex Kahl, 1933) found in mariculture waters near the coast of Qingdao, China, were investigated with
emphasis on their live morphology and infraciliature. Comparative descriptions of these three organisms are presented and synonyms are
critically discussed. The validity of L. sinicum n. sp. is confirmed mainly by the combination of the distribution of extrusomes, features of
general living morphology, morphometric data, and the characters of the somatic ciliature. Two previously reported organisms under the
name of L. helus by Dragesco (1966, variety a) and by Dragesco and Dragesco-Kernéis (1986) are discussed and are believed to be
synonyms of L. sinicum. Furthermore, two isolates described by Dragesco (1960) and Ozaki and Yagiu (1943) under the name of L. helus
are very likely, in our opinion, misidentifications, and might be two unknown forms. In the light of the current study, a key is presented to
the seven clearly defined marine Loxophyllum species found in the coastal areas of north China Sea.
Key Words. Loxophyllum choii, Loxophyllum jini, Loxophyllum qiuianum, Loxophyllum shini, new species, synonymy.
T
he ciliate species of the pleurostomatid genus Loxophyllum
are common inhabitants of marine and freshwater habitats,
and are found worldwide (Dragesco 1954, 1960, 1965; Foissner et
al. 1995; Kahl 1931, 1933, 1935; Lin et al. 2005; Lin, Song, and Li
2007; Song 1993; Song and Wilbert 1989). In terms of the
separation and identification of species, however, there has always
been a great confusion despite the fact that these organisms are
relatively simple in structure. There are several reasons for this: (i)
Loxophyllum species, like most of the genera of Pleurostomatida,
are still poorly studied compared with other well-known groups;
(ii) most early studies were based almost exclusively on live
observations where useful information necessary for species
identification and taxa separation were frequently insufficiently
described or unreported; and (iii) many species possess rather
similar morphologies, especially in terms of body shape and size,
extrusomes, nuclear apparatus, and contractile vacuole, which
have led to many misidentifications (Foissner et al. 1995; Lin
et al. 2005, 2007; Petz, Song and Wilbert 1995; Song 1993; Song
and Wilbert 1989).
In many early studies, the marine morphotypes similar to the
freshwater congener Loxophyllum helus were usually considered
to be marine varieties of the latter, which led to greater confusions
in the identification of these forms (Dragesco 1960, 1965, 1966;
Kahl 1931, 1935). Foissner (1978) and Song (1993) clarified the
differences between Loxophyllum helus and Loxophyllum rostratum. Three marine isolates of L. helus were regarded, by Song
(1993), as synonyms of L. rostratum. However, at least four other
populations of L. helus are still in confusion (Dragesco 1960,
1966; Dragesco and Dragesco-Kernéis 1986; Ozaki and Yagiu
1943). Based on detailed investigations of four morphologically
similar isolates, the species circumscription for these similar
organisms are presented in this study. A key to seven clearly
defined marine Loxophyllum species found in north China is also
presented.
Two populations of Loxophyllum sinicum n. sp. were sampled
on the Yellow Sea coast near Qingdao. Population 1 was collected
from a fish-culturing pond (November 25, 2001) with water
temperature ca 16 1C and salinity ca 30%; Population 2 was
collected from an abalone-culturing pond (30 September 2002)
with water temperature ca 18 1C and salinity ca 31%.
Loxophyllum simplex was collected from an abalone-culturing
pond (18 October 2004) on the coast of the Yellow Sea near
Qingdao with water temperature ca 16 1C, salinity 30%, and pH
ca 7.9.
After isolation, living cells were observed using bright field and
differential interference contrast microscopy (Nikon 80i, Tokyo,
Japan) at 100–1,000X magnification. The infraciliature was revealed by the protargol impregnation method according to Wilbert
(1975). Counts and measurements of stained specimens were
carried out with an ocular micrometer. Drawings of stained
specimens were performed at 1,250X with the aid of a camera
lucida. Terminology mainly follows Corliss (1979) and Lin et al.
(2005).
Deposition of slides. One voucher slide (No. Lin-02-10-4) of
protargol-impregnated specimens of L. rostratum is deposited in
the Laboratory of Protozoology, OUC, China.
One holotype slide of protargol-impregnated specimens of L.
sinicum n. sp. is deposited in the Natural History Museum,
London, U.K., with registration number 2006:6:6:4. Two paratype
slides (Nos. Lin-01-11-25 and Lin-02-9-30) are deposited in the
Laboratory of Protozoology, OUC, China.
One neotype slide of protargol-impregnated specimens of L.
simplex is deposited in the Natural History Museum, London,
U.K., with registration number 2006:6:6:6. One paraneotype slide
(No. Lin-04-10-18) is deposited in the Laboratory of Protozoology, OUC, China.
RESULTS
Redescription of Loxophyllum rostratum Cohn, 1866
(Table 1 and 2 and Fig. 1–30)
MATERIALS AND METHODS
Sample collection, observation, and identification. Loxophyllum rostratum was collected from a shrimp-culturing pond
(October 4, 2002) on the Yellow Sea coast near Qingdao with
water temperature ca 20 1C, salinity ca 29%, and pH 7.8.
Corresponding Author: W. Song, Laboratory of Protozoology, Ocean
University of China, Qingdao 266003, China—e-mail: wsong@ouc.
edu.cn
A population was briefly redescribed by Song (1993). Considering the confusion in identification and absence of data based
on modern methods, a reinvestigation is, in our opinion, still
necessary.
Morphology and infraciliature. The body is elongated with
cells size 100–300 40–70 mm in vivo, mostly 200–250 mm long
(Table 1). The body is slightly contractile, with the posterior end
331
332
J. EUKARYOT. MICROBIOL., 55, NO. 4, JULY–AUGUST 2008
Table 1. Morphometrical data of Loxophyllum rostratum (first line),
Loxophyllum sinicum (Pop. 1, second line; Pop. 2, third line) and Loxophyllum simplex (fourth line).
Characters
Body length
Min Max Mean
124
188
188
160
Body width
40
60
38
32
16
No. of right somatic kinetiesa
18
16
14
6
No. of left somatic kinetiesb
10
7
7
No. of macronuclear nodules (Ma)
2
2
2
2
Length of Ma
20
36
30
25
Width of Ma
13
30
13
16
No. of micronuclei
1
–
1
1
Length of micronuclei
4
–
4
4
Length of extrusomes
5
8
5
6
No. of warts on dorsal margin
6
5
6
0
Length of nemato-desmata
56
–
–
80
252
428
408
340
72
132
76
56
19
27
23
19
9
12
10
10
2
2
2
2
40
66
52
42
32
56
33
26
1
–
1
1
12
–
10
7
8
10
8
9
13
15
15
0
136
–
–
144
199.1
304.9
266.4
268.5
54.3
80.6
49.0
44.9
17.3
22.4
19.4
16.7
7.2
10.7
8.5
7.9
2
2
2
2
29.8
49.8
41.4
31.2
20.0
37.2
21.2
21.3
1
–
1
1
6.6
–
5.6
4.9
5.9
8.4
6.2
7.7
9.2
10.7
11.4
0
97.5
–
–
117.0
M
220
300
260
280
56
80
48
44
17
22
19
17
7
10
8
7
2
2
2
2
32
50
40
28
21
38
22
20
1
–
1
1
7
–
5
5
6
8
6
8
9
10
12
0
120
–
–
112
SD
n
37.3
25
66.6
25
51.2
25
41.1
25
7.9
25
16.5
25
8.9
5
6.1
25
1.4
5
2.2
25
1.7
25
1.1
25
0.8
25
0.8
6
0.7
24
1.1
14
0
25
0
25
0
25
0
25
5.5
25
8.2
25
6.1
25
4.6
25
4.5
23
6.0
25
3.9
25
2.9
25
0
3
–
–
0
20
0
25
1.6
23
–
–
1.4
20
0.9
25
0.9
25
0.7
25
0.6
25
0.8
25
2.0
25
2.5
25
2.1
25
0
425
21.1
19
–
–
–
–
19.9
25
Data based on protargol-impregnated specimens. All measurements in
micrometers.
a
Perioral kinety 2, 3 included;
b
Perioral kinety 1 and dorsal brush kinety included.
M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n,
sample size; SD, standard deviation.
bluntly pointed and the neck region about 20%–40% of body
length (Fig. 1, 3, 8). It is laterally compressed about 3–4:1; the
right side is flat, and the left is slightly vaulted in the main body,
with three or four longitudinal broad ridges, which are very
conspicuous at low magnification (Fig. 1, 8). The ciliary rows
on the left side are marked by the presence of longitudinal,
shallow grooves that appear as white lines on the cell surface at
high magnifications using differential interference contrast microscopy (Fig. 9, 10, arrows). The right side is densely ciliated with
cilia 7–10 mm in length; the cilia on the left side are difficult to
detect in life.
The pellicle is thin, with densely packed tiny cortical granules,
rice-shaped, and dark gray colored, ca 1 mm in length (Fig. 2, 10,
arrowheads). The cytoplasm is grayish, often with many greasily
shining globules (2–4 mm across), which render the main part of
body more or less opaque (Fig. 8–10). The body has often a
well-defined hyaline fringe. Extrusomes are bar-shaped, slender,
straight to slightly curved, 5–8 mm long, and evenly distributed along entire ventral margin. They are clustered together to
form 6–12 conspicuous warts (Wa) along the dorsal margin,
although they are absent from the anteriormost region, while
some scattered in cytoplasm (Fig. 4–6, 15). One contractile
vacuole (CV), about 6–10 mm in diam., is subterminally positioned (Fig. 1, 8).
Two macronuclear nodules are ovoid to ellipsoid, about 20–
40 10–30 mm in size, and located in the equatorial region. They
usually appear as two large transparent areas in life. The single
micronucleus is ovoid to ellipsoid, 4–12 mm in length, and situated
between two macronuclear nodules (Fig. 1, 3, 7, 8, 12, 13, 15).
Movement is by gliding on the substrate, usually with the body
stretching and contracting and the anterior neck region seldom
swaying from side to side; or by swimming with slow rotation
around the longitudinal axis.
Infraciliature is illustrated in Fig. 5–7, 11–16. The perioral
kinety (PK1), left of the oral slit, consists of densely arranged
basal body pairs in the anterior half and monokinetids in the
posterior half (Fig. 5, 6, 12, arrowheads). Two perioral kineties
(PK2,3) right of cytostome are densely ciliated: PK2 with dikinetid
nearly reaches the posterior end of cell, but PK3 is entirely of
monokinetids (Fig. 6, 7). There are 16–19 kineties (mean 17) on
the right side including PK2,3; the right somatic kineties are
densely arranged and terminate anteriorly along PK3 (Fig. 7).
The left side is sparsely ciliated with 6–9 kineties (mean 7),
including PK1 and the dorsal brush kinety; the left somatic
kineties terminate anteriorly along PK1 (Fig. 5, 6, 14). The dorsal
brush kinety (DB) is composed of basal body pairs extending to
near the middle body and it continues to the posterior end of cell
as monokinetids (Fig. 5, 6, 11). Nematodesmata are well developed, extend along the cytopharynx into the cytoplasm, and are
about 56–136 mm long; the cytostome is marked by nematodesmata about one-third of body length (Fig. 6, 16).
Loxophyllum sinicum n. sp.
(Tables 1 and 2 and Fig. 31–58)
Morphology and infraciliature. Slender leaf-shaped cells are
about 200–450 40–70 mm in vivo, and mostly 200–400 mm long
(morphometric data see Table 1). The body is slightly contractile,
with beak-like anterior end and bluntly pointed posterior end (Fig.
31, 36, 40, 46). The body is laterally compressed about 3–4:1. The
right side is flat; the left side is slightly vaulted in the main body
and has no longitudinal ridge (Fig. 35). The body often has welldefined thin and hyaline fringe (Fig. 31, 36, 40). The right side is
densely ciliated, cilia are ca 4–5 mm long; the left somatic cilia are
difficult to detect in vivo.
The pellicle is thin, and has densely spaced colorless dot-like
cortical granules (o0.5 mm across) among ciliary rows of both
sides (Fig. 33, 53). The cytoplasm is grayish, often has numerous
tiny (1–5 mm across) greasily shining globules, which render the
main part of the body opaque (Fig. 46–49). The extrusomes are
thin bar-shaped, about 7–10 mm long, distributed along the hyaline
edge of body. They are evenly spaced along entire ventral margin
and the posterior 1/2–1/3 of dorsal margin, and clustered to form
ca 10 warts (Wa) at the anterior 1/2–2/3 of dorsal margin, and
usually only several anterior ones are conspicuous and densely
spaced (Fig. 31, 32, 34, 41, 50, 51). The single contractile vacuole
333
LIN ET AL.—IDENTIFICATION OF THREE LOXOPHYLLUM SPECIES
Table 2. Comparison of some closely related congeners with Loxophyllum sinicum n. sp.
Species
Body length
in vivo (mm)
Position of
contractile
vacuole
No. of left and
right kineties
Distribution
of data source
Ridges on
left side
Biotope
Data source
L.
L.
L.
L.
L.
L.
L.
L.
100–300
150–250
150–250
130–250
ca. 150 ?
200–450
120–300
75–300
Subterminal
Subterminal
Subterminal
Subterminal
Subterminal
Subterminal
Subterminal
Subterminal
6–9, 16–19
6–7, 16–18
8–10, 13–23
–, –
–, 11–12
10–12, 18–27
–, 15–25
–, 15–25
Entire margin
Entire margin
Entire margin
Entire margin
Part of margin
Part of margin
Part of margin
Part of margin
Present
Present
Present
Present
Present
Absent
Absent
Absent
Marine
Marine
Marine
Marine
Marine
Marine
Marine
Marine
L. helus
150–220
–, 15–25
Entire margin
Absent
L.
L.
L.
L.
400
260
ca. 75
130–150
Subterminal,
near dorsal margin
Near dorsal margin
Along dorsal margin
Subterminal
Subterminal
–, ca. 20
–, 11–12
–, 15–16
18, 36
Part of margin
Part of margin
Part of margin
Entire margin
Present
Absent
Present
Present
Fresh
water
Marine
Marine
Marine
Marine
Present work
Song (1993)
Petz et al. (1995)
Kahl (1931)
Dragesco (1966) (var. b)
Present work
Dragesco (1966) (var. a)
Dragesco and
Dragesco-Kernéis (1986)
Foissner (1978)
rostratum
rostratum
rostratum
helusa
helusa
sinicum
helusb
helusb
perihoplophorum
acutum
helusc
helusc
Buddenbrock (1920)
Dragesco (1965)
Dragesco (1960)
Ozaki and Yagiu (1943)
a
Misidentified, synonyms of L. rostratum;
Misidentified, populations of L. sinicum;
Misidentified, species identity uncertain.
?, based on the illustration; –, data not available.
b
c
is subterminally located, about 15–30 mm in diam. (Fig. 31, 34–
36, 40, 47, 48).
Two macronuclear nodules are ovoid to ellipsoidal, about 30–
60 15–30 mm in size, and located in the equatorial region. They
are usually detectable in vivo and appear as two large transparent
areas (Fig. 31, 40). The single micronucleus is ca 4–6 mm in
length, and situated between two macronuclear nodules (Fig. 34).
Movement is by gliding moderately fast on the substrate with
the body continually stretching and contracting (Fig. 36), or by
swimming with slow rotation around the longitudinal axis.
Infraciliature is illustrated in Fig. 37–39, 42–45, 55–58. There
are three perioral kineties (PK1–3) around the oral slit: PK1, left of
cytostome, is consisted of kinetosome pairs and extend to the
posterior end of cell (Fig. 37, 38, 57, arrows); PK2 and PK3 locate
at the right of cytostome; PK2 consists of dikinetids, with closely
spaced basal body pairs extending to the posterior end of cell; PK3
is entirely of monokinetids (Fig. 39, 57). There are 18–27 (mean
22) right kineties (including PK2,3); the somatic kineties densely
ciliated and terminate anteriorly along PK3 (Fig. 39). The left side
is also densely ciliated, and has 10–12 (mean 11) kineties (including PK1 and the dorsal brush kinety). The left somatic kineties
terminate anteriorly along PK1 (Fig. 37, 38, 58). The dorsal brush
kinety (DB) extends to nearly half body-length as densely spaced
dikinetids and continuing posteriorly as a row of closely spaced
monokinetids which become more widely spaced posteriorly (Fig.
37, 38, 56). No nematodesmata is detected.
Redescription of Loxophyllum simplex Kahl, 1933
(Table 1 and 3 and Fig. 59–77)
Since the original report of Kahl (1933), no studies using
modern methods have been carried out and hitherto the infraciliature of this organism was unknown. Thus, a detailed redescription based on current study is presented here.
Morphology and infraciliature. The body is elongated and
slender leaf-shaped. Cells size are 150–450 40–70 mm in vivo,
mostly about 200–350 mm in length (for morphometrics see Table
1). The body is slightly contractile. The posterior end of body is
bluntly rounded, and the neck region is about 20%–30% of body
length (Fig. 59, 67–69). The body is laterally compressed about 3–
4:1. The right side is flat, and the left side is slightly vaulted in the
main body, with three to four longitudinal broad ridges, which are
very conspicuous even at low magnification, and the ciliary rows
are located between them (Fig. 59, 70, 71, arrows). The right side
is densely ciliated, the cilia are 8–10 mm in length; cilia on the left
side are difficult to detect in life.
The pellicle is thin; no cortical granule was detected. The
cytoplasm is colorless, and the body is nearly transparent under
low magnification, usually has some tiny shining globules, which
render the main part of the body more or less opaque (Fig. 67–71).
The body has well-defined hyaline fringe (Fig. 67–70). The
extrusomes are slender bar shaped, straight to slightly curved,
6–9 mm long, densely and uniformly arranged along both ventral
and dorsal margins, and some scattered in cytoplasm (Fig. 63–65,
74–76). One to several contractile vacuoles (CV) are subterminally positioned, and the largest one is about 12–15 mm in diam.,
other smaller ones are located anterior to it (Fig. 59, 63, 64, 67, 68,
72, 73).
Two macronuclear nodules are ovoid-shaped, about 25–
40 15–25 mm in size, locating in the middle region. They
usually appear as two large transparent areas in life under
differential interference contrast microscopy and detectable at
low magnifications; one ellipsoidal micronucleus, ca 4–7 mm in
length, is situated between two macronuclear nodules (Fig. 59–64,
67–73, 77).
Movement is by gliding moderately fast on the substrate,
usually with the body stretching and contracting frequently, or
by swimming with rotation around its longitudinal axis. They
often prey on other smaller ciliates, such as the scuticociliates.
Infraciliature is shown in Fig. 60–65, 72–77. There are three
perioral kineties (PK1–3): PK1, left of oral slit, is consisted of
densely arranged basal body pairs in anterior half and monokinetids in posterior half (Fig. 60, 65, 75); PK2,3 is at the right of
cytostome; the anterior two-fifth of PK2 consists of more densely
spaced dikinetids and monokinetids in posterior part; PK3 is
also with densely arranged dikinetids in anterior one-fourth
and continues to the posterior end of cell as monokinetids
(Fig. 60, 62).
The right side has 14–19 (mean 17, including PK2,3) densely
arranged kineties, which terminate anteriorly along PK3 (Fig. 62,
74, arrows). The left side is sparsely ciliated, and consist of 7–10
kineties (mean 8, including PK1 and dorsal brush kinety); the left
somatic kineties terminate anteriorly along PK1 (Fig. 60, 65). The
334
J. EUKARYOT. MICROBIOL., 55, NO. 4, JULY–AUGUST 2008
Fig. 1–7. Morphology and infraciliature of Loxophyllum rostratum from life (1, 2) and after protargol impregnation (3, 7). 1. Left view of a typical
individual. Note the longitudinal ridges. 2. Cortical granules. 3. Shape variants. 4. Lateral view. 5. Infraciliature of the anterior part of left side. 6.
Infraciliature of left side. 7. Infraciliature of right side. CV, contractile vacuole; DB, dorsal brush; Ex, extrusome; Ma, macronucleus; Mi, micronucleus;
PK1–3, perioral kinety 1, 2 and 3; Wa, wart. Scale bars 5 50 mm.
dorsal brush kinety (DB) is composed of densely arranged basal
body pairs in anterior two-fifth and continues to the posterior end
of cell as more loosely spaced monokinetids (Fig. 60, 65, 75,
arrows). The well-developed nematodesmata extend along the
cytopharynx into the cytoplasm, about 110 mm long; the cytostome marked by nematodesmata is about two-fifth of body length
(Fig. 64, 76).
DISCUSSION
The identification of many L. helus-like species or populations
is difficult and confusing (Fig. 17–30) (Buddenbrock 1920; Cohn
1866; Dragesco 1960, 1966; Dragesco and Dragesco-Kernéis
1986; Foissner et al. 1995; Kahl 1931; Ozaki and Yagiu 1943;
Petz et al. 1995; Song 1993). Most of them are discussed in the
LIN ET AL.—IDENTIFICATION OF THREE LOXOPHYLLUM SPECIES
335
Fig. 8–16. Photomicrographs showing the infraciliature of Loxophyllum rostratum from life (8–10) and after protargol impregnation (11–16). 8. Left
view of a typical individual at low magnification. Note the longitudinal ridges. 9, 10. Left views, arrows mark the ciliary rows that appear as white lines on
the cell surface at high magnifications under differential interference contrast microscopy, arrowheads show the cortical granules. 11. Infraciliature of
anterior part of left side, arrowheads mark the perioral kinety, arrows show the dorsal brush. 12, 13. Right views, arrows mark the micronucleus. 14.
Infraciliature of left side, arrows mark the somatic kineties. 15. Macronucleus, arrowheads indicate the extrusomes. 16. Arrowheads show the developed
nematodesmata. Scale bars 5 50 mm.
following comments. Some of them are considered to be synonymies, and some of them are misidentifications.
Comments on Loxophyllum rostratum Cohn, 1866.
Loxophyllum rostratum was initially found in the marine
environment by Cohn (1866) and was defined as having warts
along the dorsal margin but no extrusomes within them (Fig. 21).
Because of this special character, many similar marine morphotypes with extrusomes in warts were reported as marine varieties
or populations of the freshwater congener, L. helus Stokes, 1884
(Dragesco 1960, 1966; Dragesco and Dragesco-Kernéis 1986;
Kahl 1931; Ozaki and Yagiu 1943; Penard 1922). When the
infraciliatures of both L. helus and L. rostratum were revealed
(Foissner 1978; Petz et al. 1995; Song 1993), the confusion of
these congeners was clarified and indicated: (i) the warts of L.
rostratum were supported as well by extrusomes, which were,
nevertheless, overlooked previously (Song 1993); (ii) L. helus and
L. rostratum can be clearly separated by the structure of the right
somatic kineties (anteriorly shortened along two longitudinal
somatic kineties near the dorsal margin, arrows in Fig. 30 vs.
anteriorly shortened along the perioral kinety, Fig. 7, 26) and the
habitat (freshwater vs. marine) (Foissner 1978; Petz et al. 1995;
Song 1993); (iii) the marine forms of L. helus reported by Penard
(1922), Kahl (1931), and Dragesco (1966, variety b) (Fig. 20)
should be synonymized with L. rostratum (Song 1993).
Our isolate corresponds perfectly to the populations of Cohn
(1866) and Song (1993) in all the characters considering both the
living morphology and infraciliature (Fig. 21, 27) (Table 2).
The population that Dragesco (1960) reported as a small variety
of L. helus (misidentification, Fig. 17) has conspicuous longitudinal ridges on the left side and is similar to L. rostratum. Its
identity awaits further studies because no data about its infraciliature are available. However, it can be clearly separated from
L. rostratum as live cells are smaller in size (ca 75 vs. 100–
300 mm) and show a different distribution of extrusomes (i.e.
clustered and forming warts in the anterior portion while evenly
spaced in the posterior part along dorsal margin vs. forming warts
along the entire dorsal margin in L. rostratum). Hence, they
should not be conspecific.
The Japan isolate reported by Ozaki and Yagiu (1943) (Fig. 22,
23) under the name of L. helus, is also similar to L. rostratum
regarding its general morphological characters (i.e. body size,
distribution of extrusomes and warts, number and position of
336
J. EUKARYOT. MICROBIOL., 55, NO. 4, JULY–AUGUST 2008
Fig. 17–30. Loxophyllum species related to Loxophyllum rostratum. 17. Loxophyllum helus sensu Dragesco, 1960. 18. Loxophyllum helus sensu
Dragesco & Dragesco-Kernéis, 1986. 19, 20. Loxophyllum helus sensu Dragesco, 1966, variety a (19) and variety b (20). 21. Loxophyllum rostratum
Cohn, 1866. 22, 23. Loxophyllum helus sensu Ozaki and Yagiu, 1943. 24. Loxophyllum helus sensu Kahl, 1931. 25, 26. Loxophyllum rostratum, from Petz
et al., 1995. 27. Loxophyllum rostratum, from Song, 1993. 28. Loxophyllum perihoplophorum Buddenbrock, 1920. 29, 30. Loxophyllum helus, from
Foissner et al., 1995.
contractile vacuole, and the feature of nuclear apparatus) (Table
2). However, the number of somatic kineties on both sides is much
larger than our population (left/right: 18/36 vs. 6–9/16–19) and
other populations reported (Table 2). Thus, it should be clearly
separated from L. rostratum. Very likely, the Japanese morphotype is a new member of the genus, which needs, nevertheless,
further investigations to reveal both infraciliature and other details
of its morphology.
Based on previous reports (Cohn 1866; Kahl 1931; Song 1993)
and the present investigations, an improved diagnosis of L.
rostratum is suggested here.
Order: Pleurostomatida Schewiakoff, 1896
Family: Litonotidae Kent, 1882
Loxophyllum rostratum Cohn, 1866
Diagnosis. Marine Loxophyllum with wide hyaline margins,
100–300 mm long in vivo; three or four conspicuous longitudinal ridges on left side; two macronuclear nodules and
one micronucleus; one contractile vacuole subterminally
located; extrusomes bar shaped, evenly distributed along whole
ventral margin, about 6–13 conspicuous warts with clustered extrusomes along dorsal margin; 6–9 left and 16–19 right
kineties.
337
LIN ET AL.—IDENTIFICATION OF THREE LOXOPHYLLUM SPECIES
Table 3. Comparison of Loxophyllum simplex Kahl, 1933 with some related species whose dorsal extrusomes are evenly spaced.
Species
Body length
in vivo (mm)
No. of
macronuclei
No. and position of
contractile vacuoles
No. of left and
right kineties
Ridges on
left side
Biotope
Data source
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
150–450
150–200
80–100
–
60–160
300–600
280
ca. 450
140–160
ca. 270
100–260
2
2
2
2
2
8–17
6–7
Many
Many
Many
5–7
1–3, subterminal
1–3a, subterminal
1–4a, subterminal
1, subterminal
1, subterminal
3–7, along dorsal margin
4–5, along both margins
5a, along dorsal margins
3, near ventral margin
2, near dorsal margin
1, near dorsal margin
7–10, 14–19
–, –
–, 8
–, –
5–8, 9–11
9–14, 13–20
ca. 10, 14–16
–, –
–, 10
–, –
–, –
Present
Present
Absent
Absent
Present
Absent
Absent
Absent
Present
Present
Absent
Marine
Marine
Marine
Marine
Marine
Marine
Marine
Marine
Salt water
Marine
Marine
Present work
Kahl (1933)
Dragesco (1965)
Kahl (1931)
Lin, Song and Li (2007)
Lin et al. (2005)
Dragesco (1965)
Dragesco (1965)
Kahl (1931)
Dragesco (1960)
Dragesco (1954)
simplex
simplex
vitraeum
pyriforme
shini
jini
compressum
psammophyllus
multinucleatum
laevigatum
variabilis
a
Data based on the illustration.
–, data not available.
Synonymy. Loxophyllum helus sensu Penard, 1922, Loxophyllum helus sensu Kahl, 1931, and Loxophyllum helus sensu Dragesco, 1966 (variety b) (Song 1993).
Comments on Loxophyllum sinicum n. sp.
The arrangement of extrusomes along the dorsal margin is an
important character to distinguish species in the genus Loxophyllum (Foissner et al. 1995; Lin et al. 2005, 2007; Song and Wilbert
1989). As a new species, L. sinicum is distinguished from its
closely related congeners mainly by the distribution of extrusomes
along dorsal margin: clustered to form warts in the anterior part
while evenly spaced in the posterior part. Among the known
species, to our knowledge, only the marine forms Loxophyllum
perihoplophorum Buddenbrock, 1920 (Fig. 28) and Loxophyllum
acutum Dragesco, 1965 have a similar arrangement of extrusomes
(Buddenbrock 1920; Dragesco 1965). However, L. sinicum can be
clearly differentiated from L. perihoplophorum by (i) the number
and position of contractile vacuoles (single, subterminal vs.
several, along dorsal margin in L. perihoplophorum) and (ii) the
absence of longitudinal ridges on the left side of body (vs. present
in L. perihoplophorum) (Table 2).
Loxophyllum sinicum differs from L. acutum mainly in (i) the
number of macronuclear nodules (invariably 2 vs. 10–14) and (ii)
the number and position of contractile vacuoles (single, subterminal vs. several, along dorsal margin) (Table 2).
Considering the general morphology (i.e. body size, macronucleus, contractile vacuoles, somatic kineties, and habitat), L.
sinicum is also similar to the well-defined L. rostratum (see
above). Our new species, however, can be clearly distinguished
from the latter by the absence of the longitudinal ridge on left side
(vs. with conspicuous ridges in L. rostratum), the shape and size of
cortical granules (i.e. dot-like and o0.5 mm across vs. rice-shaped
and ca 1 mm in length), and the distribution of extrusomes along
dorsal margin (i.e. clustered to form warts in anterior part while
evenly spaced in the posterior part vs. forming conspicuous warts
along entire dorsal margin in L. rostratum) (Table 2).
Loxophyllum sinicum resembles the freshwater form, L. helus
Stoke, 1884 (Fig. 29, 30) in general appearance (Foissner 1978).
However, the former can be separated from the latter by (i) the
distribution of extrusomes along the dorsal margin (i.e. clustered
to form warts in anterior part while evenly spaced in the posterior
part vs. forming warts along entire dorsal margin in L. helus), (ii)
the pattern of right somatic kineties (i.e. terminating anteriorly
along the perioral kinety vs. terminating anteriorly along two
somatic kineties near dorsal margin, arrows in Fig. 30), and (iii)
the habitat (i.e. marine vs. freshwater).
As mentioned above, several L. helus-like marine morphotypes
were misidentified as L. helus (Dragesco 1960, 1966; Dragesco
and Dragesco-Kernéis 1986; Kahl 1931; Ozaki and Yagiu 1943;
Penard 1922). Among them, two organisms reported by Dragesco
(1966, variety a) and Dragesco and Dragesco-Kernéis (1986) (Fig.
18, 19) match our new species rather well in all the morphological
characters (i.e. body size, macronucleus, position of contractile
vacuole, the absence of ridges on left side of body, the distribution
of extrusomes, and the number of right somatic kineties) (Table
2). Thus, both forms are synonymized with L. sinicum.
The isolate that Dragesco (1960) reported under the name L.
helus is very likely a misidentification, and is morphologically
similar to L. sinicum in the distribution of extrusomes (Fig. 17).
However, it can be clearly separated from the latter by much
smaller size (i.e. ca 75 vs. 200–450 mm in L. sinicum), the
presence of conspicuous longitudinal ridges on left side of body
(vs. absent in L. sinicum), and fewer right somatic kineties (i.e.
15–16 vs. 18–27 in L. sinicum) (Table 2). So, it is not likely
conspecific with L. sinicum.
We present therefore the diagnosis of the new species L.
sinicum below.
Order: Pleurostomatida Schewiakoff, 1896
Family: Litonotidae Kent, 1882
Loxophyllum sinicum n. sp.
Diagnosis. Marine Loxophyllum about 200–450 mm long in
vivo, with wide hyaline margins; no ridges on left body side; two
macronuclear nodules and one micronucleus; one subterminally
located contractile vacuole; extrusomes bar-shaped, evenly
spaced along whole ventral and posterior part of dorsal margins,
while clustered to forming conspicuous dorsal warts in anterior
portion; about 10 left and 16–27 right kineties.
Synonymy. Loxophyllum helus sensu Dragesco, 1966 (variety
a), and Loxophyllum helus sensu Dragesco and Dragesco-Kernéis,
1986.
Comments on Loxophyllum simplex Kahl, 1933.
Loxophyllum simplex was briefly reported by Kahl (1933) (Fig.
66) and was subsequently mentioned in his monographs (Kahl
1935). The infraciliature has been hitherto unknown. Fortunately,
the original description and illustration presented the most important features for the recognition of this species: (i) leaf-shaped
body, about 150–200 mm in length, (ii) with wide hyaline fringe
containing evenly spaced extrusomes along both margins, (iii) two
macronuclear nodules and one to three contractile vacuoles subterminally located, (iv) conspicuous longitudinal ridges on the left
side of body, and (v) marine habitat (Table 3).
The Qingdao population corresponds very well with original
report. Some individuals of our isolate were somewhat larger (up
to 450 vs. 150–200 mm), but we believe this to be within the range
338
J. EUKARYOT. MICROBIOL., 55, NO. 4, JULY–AUGUST 2008
Fig. 31–39. Morphology and infraciliature of Loxophyllum sinicum n. sp. from life (31–33, 35, 36) and after protargol impregnation (34, 37–39). 31.
Left view of a typical individual. 32. Extrusomes. 33. Cortical granules. 34. Left view, to show the nuclear apparatus, contractile vacuole, and the
distribution of extrusomes. 35. Lateral view. 36. Shape variations of the same individual. 37. Infraciliature of the anterior part of left side. 38.
Infraciliature of left side. 39. Infraciliature of right side. CV, contractile vacuole; DB, dorsal brush; Ex, extrusome; Ma, macronucleus; PK1 3, perioral
kinety 1, 2 and 3; Wa, wart. Scale bars 5 100 mm in 31, 34, 35, 36, 39; 4 mm in 32, 33; 50 mm in 37.
LIN ET AL.—IDENTIFICATION OF THREE LOXOPHYLLUM SPECIES
339
Fig. 40–58. Photomicrographs showing the infraciliature of Loxophyllum sinicum n. sp. (Pop. 1, 40–45; Pop. 2, 46–58) from life (40, 41, 46–54) and
after protargol impregnation (42–45, 55–58). 40. Left view of a typical individual, arrow marks the contractile vacuole. 41. Anterior view of left side,
arrows mark the warts. 42. Anterior part of left side, arrows mark the extrusomes forming the warts. 43. To show the distribution of extrusomes, the
contractile vacuole (arrow), and the macronucleus. 44. The infraciliature of anterior part of right side. 45. Right side, extrusomes forming warts. 46–48.
Left views of typical individuals, arrows show the contractile vacuole, arrowheads mark the warts. 49. Anterior part lateralized, to show the right–left
compressed body. 50, 51. Anterior part of left side, arrowheads mark the warts. 52. Left side, to show the longitudinal ‘‘white line’’ like shallow furrows
(arrowheads), arrow marks the warts. 53. Right side, to show the distribution of cortical granules. 54. To show the extrusomes (arrows) distributed along
the ventral margin. 55. Infraciliature of left side, arrows mark the extrusomes, arrowheads show the left perioral kinety. 56. Anterior part of left side,
arrows mark the dorsal brush kinety. 57. Ventral view, arrows mark the left perioral kinety, arrowheads mark the right perioral kinety. 58. Left somatic
kineties. Scale bars 5 100 mm.
340
J. EUKARYOT. MICROBIOL., 55, NO. 4, JULY–AUGUST 2008
Fig. 59–66. Morphology and infraciliature of Loxophyllum simplex from life (59, 66) and after protargol impregnation (60–65). 59. Left view of a
typical individual. Note the longitudinal ridges. 60. Infraciliature of left side. Note the structure of oral apparatus. 61. Extrusomes. 62. Infraciliature of
right side, arrows mark the anterior end of somatic kineties. 63. The extrusomes and the developed nematodesmata. 64. To show the nuclear apparatus,
contractile vacuoles, and the distribution of extrusomes, note the anterior area of dorsal margin that extrusomes absent (arrows). 65. Infraciliature of the
anterior part of left side. 66. Loxophyllum simplex from Kahl, 1933. CV, contractile vacuole; DB, dorsal brush; Ex, extrusome; Ma, macronucleus; Mi,
micronucleus; PK1 3, perioral kinety 1, 2 and 3; Wa, wart. Scale bars 5 100 mm.
of variability for species of Loxophyllum. Therefore, the identification of the Qingdao population is rather certain.
To our knowledge, among the known nominal Loxophyllum
spp., at least nine congeners were reported with extrusomes that
are evenly distributed along entire dorsal margin: Loxophyllum
vitraeum Dragesco, 1965, Loxophyllum pyriforme Gourret and
Roeser, 1886, Loxophyllum shini Lin et al., 2007, Loxophyllum
jini Lin et al., 2005, Loxophyllum compressum Dragesco, 1965,
Loxophyllum psammophyllus Dragesco, 1954, Loxophyllum multinucleatum Kahl, 1928, Loxophyllum laevigatum Sauerbrey,
1928, and Loxophyllum variabilis Dragesco, 1954 (Dragesco
1954, 1960, 1965; Kahl 1931; Lin et al. 2005, 2007).
Like L. simplex, the former three have two macronuclear
nodules and one to several contractile vacuoles subterminally
located. However, L. vitraeum differs from L. simplex by its
smaller size (i.e. 80–100 vs. 150–450 mm in length), fewer right
somatic kineties (i.e. 8 vs. 14–19), and the absence of longitudinal
ridges on left side of body (vs. present in L. simplex).
As a rare species that has been forgotten for a long time, L.
pyriforme can be separated from L. simplex by its typical pyriform
body shape (vs. elongated leaf shape) and the absence of longitudinal ridges on the left side of body (vs. present in L. simplex).
By contrast, L. shini has also longitudinal ridges on the left side
like those in L. simplex, but it can be identified by its smaller size
(i.e. 60–160 vs. 150–450 mm in length), fewer somatic kineties
(i.e. left/right: 5–8/9–11 vs. 7–10/14–19), and/or the absence of
ridges on the left side of body (Lin et al. 2007).
The remaining six congeners have more than two macronuclear
nodules. In addition, they can be clearly distinguished from L.
simplex by a combination of body size and the number/position of
the contractile vacuoles (Table 3).
Based on the Qingdao population of L. simplex, we supply here
an improved diagnosis, which matches the original description by
Kahl (1933) rather well.
Order: Pleurostomatida Schewiakoff, 1896
Family: Litonotidae Kent, 1882
Loxophyllum simplex Kahl, 1933
Diagnosis. Marine Loxophyllum about 150–450 mm long in
vivo; left side with three or four conspicuous longitudinal ridges;
two macronuclear nodules and one micronucleus; one to three
contractile vacuoles subterminally located; extrusomes bar
shaped, evenly distributed along both ventral and dorsal margins;
7–10 left and 14–19 right kineties.
LIN ET AL.—IDENTIFICATION OF THREE LOXOPHYLLUM SPECIES
341
Fig. 67–77. Photomicrographs showing the infraciliature of Loxophyllum simplex from life (67–71) and after protargol impregnation (72–77). 67, 68.
Left views of typical individuals, arrows mark the contractile vacuoles. 69. Left view, arrows mark the macronuclear nodules. 70, 71. Left views, arrows
mark the longitudinal ridges. 72, 73. To show the nuclear apparatus and contractile vacuoles (arrows). 74. Infraciliature of the anterior part of right side,
arrows mark the anterior end of somatic kineties. 75. Infraciliature of the anterior part of left side, arrows indicate dorsal brush kinety, and arrowheads
point to the left perioral kinety. 76. To show extrusomes (arrowheads) and nematodesmata (arrow). 77. Nuclear apparatus, arrow marks the micronucleus.
Scale bars 5 100 mm.
Key to seven Loxophyllum species isolated from the north
China seas
To date, seven marine Loxophyllum species, which were isolated from the Bohai and Yellow Sea in China, have been
identified and described based on silver impregnation methods
(Lin et al. 2005, 2007; Song 1993). A key to them is supplied
here.
ACKNOWLEDGMENTS
This work was supported by ‘‘The Natural Science Foundation
of China’’ (project numbers 40506033, 40676076, and 30430090)
and Rural Affairs and a grant from the Center of Excellence in
Biodiversity, King Saud University.
LITERATURE CITED
1 Extrusomes along dorsal margin, clustered to form warts . . . . . . . . .
1 0 Extrusomes along dorsal margin, evenly distributed, not
forming warts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 All extrusomes on dorsal margin forming warts . . . . . . . . . . . . . .
2 0 Only extrusomes in anterior part of dorsal margin forming warts . .
3 Warts conspicuous, clearly detectable in vivo . . . . . . . . . . . . . . . .
3 0 Warts inconspicuous, not forming swelled humps . . . . . . . . . . . .
4 Spines along dorsal margin, no longitudinal ridge on left side . . .
4 0 No spine along dorsal margin, longitudinal ridges present on
left side . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Invariably two macronuclear nodules . . . . . . . . . . . . . . . . . . . . . . .
5 0 Numerous macronuclear nodules . . . . . . . . . . . . . . . . . . . . . . . . .
6 Tips of extrusomes protruding from body surface . . . . . . . . . . . . .
6 0 Tips of extrusomes not protruding from body surface . . . . . . . . .
2
5
3
L. sinicum
4
L. qiuianum
L. choii
L.
6
L.
L.
L.
rostratum
jini
shini
simplex
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Received: 11/27/07, 02/29/08; accepted: 03/05/08