phylogeny of three choreotrich genera (protozoa, ciliophora, spirotrichea), with morphological,...

Zoologica Scripta

Phylogeny of three choreotrich genera (Protozoa, Ciliophora,

Spirotrichea), with morphological, morphogenetic and

molecular investigations on three strobilidiid speciesWEIWEI LIU, ZHENZHEN YI, XIAOFENG LIN, ALAN WARREN & WEIBO SONG

Submitted: 20 November 2011Accepted: 19 February 2012doi:10.1111/j.1463-6409.2012.00542.x

ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian

Liu, W., Yi, Z., Lin, X., Warren, A. & Song, W. (2012). Phylogeny of three choreotrich

genera (Protozoa, Ciliophora, Spirotrichea), with morphological, morphogenetic and

molecular investigations on three strobilidiid species. —Zoologica Scripta, 41, 417–434.

The phylogenetic relationships among three strobilidiid genera, namely Strobilidium, Rimo-

strombidium and Pelagostrobilidium, are investigated using a combination of morphological,

morphogenetic and molecular data. The results indicate that all three genera belong to the

same lineage, in which Rimostrombidium evolved first and Strobilidium and Pelagostrobilidium

derived later. Improved genus diagnoses for Rimostrombidium and Pelagostrobilidium are sup-

plied. The curved kinety 2 and the caudal spiralling of some somatic kineties are confirmed

as generic characters for Pelagostrobilidium and Strobilidium, respectively. In addition, the

morphology and morphogenesis of three species, namely R. veniliae (Montagnes & Taylor,

1994) Petz et al., 1995, P. paraepacrum sp. n. and P. minutum sp. n., isolated from the

South China Sea are described. Pelagostrobilidium paraepacrum sp. n. is characterized by the

presence of six somatic kineties, 30–32 external and two internal membranelles. Pelagostro-

bilidium minutum sp. n. is characterized by its extremely small body size, four somatic kin-

eties, and in having one internal and 19–21 external membranelles. Rimostrombidium conicum

Kahl, 1932 is transferred to the genus Pelagostrobilidium as P. conicum (Kahl, 1932) comb. nov.

Corresponding author: Zhenzhen Yi, Laboratory of Protozoology, Key Laboratory of Ecology &

Environmental Science in Guangdong Higher Education, South China Normal University,

Guangzhou 510631, China. E-mail: [email protected]

Weiwei Liu and Weibo Song, Laboratory of Protozoology, Institute of Evolution & Marine Biodi-

versity, Ocean University of China, Qingdao 266003, China. E-mails: [email protected],

[email protected]

Alan Warren, Department of Zoology, Natural History Museum, Cromwell Road, London SW7

5BD, UK. E-mail: [email protected]

Xiaofeng Lin, Laboratory of Protozoology, Key Laboratory of Ecology & Environmental Science in

Guangdong Higher Education, South China Normal University, Guangzhou 510631, China.

E-mail: [email protected]

IntroductionMembers of the choreotrich family Strobilidiidae are

mostly small to medium-size, spheroid to conoid ciliates

that are characterized by the possession of somatic kineties

arranged in spiral or longitudinal rows and cortical flaps

covering the bases of the somatic cilia (Lynn & Montagnes

1988; Song et al. 1999; Agatha & Struder-Kypke 2007;

Lynn 2008; Xu et al. 2009). According to Lynn (2008),

there are three genera in the family Strobilidiidae, viz.

Strobilidium, Rimostrombidium and Pelagostrobilidium. Nev-

ertheless, their systematic positions are still ambiguous,

and in some studies, results based on molecular data are

Academy of Science and Letters,

not consistent with those based on morphology (Agatha &

Struder-Kypke 2007; Tsai et al. 2008; Kim et al. 2010).

The somatic ciliary pattern is considered an important

generic character for the family Strobilidiidae. Petz &

Foissner (1992), for example, supplied an improved diag-

nosis of Strobilidium based on the characteristic spiralling

of some ciliary rows in the caudal region of the cell.

Strobilidiids lacking such caudal spiralling of its ciliary

rows were transferred to the genus Rimostrombidium, the

original definition of which was simply ‘with ribbed cor-

tex’ (Jankowski 1978). Petz et al. (1995) established

the genus Pelagostrobilidium based on its possession of

41, 4, July 2012, pp 417–434 417

Phylogeny of three choreotrichous genera d Weiwei Liu et al.

transversely arched somatic kineties that do not form a

spiral at the posterior pole. Subsequently, Agatha et al.

(2005) and Kuppers et al. (2006) improved the diagnosis of

Pelagostrobilidium by emphasizing the location of the

arched kinety 2 relative to the other somatic kineties.

The different ciliary patterns in the family Strobilidiidae

were, however, considered as evolutionary convergences

and thus were rejected as a genus level character by Mon-

tagnes & Taylor (1994). This raises questions as to the

validity of Rimostrombidium and Pelagostrobilidium, both of

which were established based on their distinctive ciliary

patterns and the phylogenetic relationships among strobili-

diid species with different arrangements of somatic kin-

eties. Some studies have attempted to address these

questions, for example, the considerable genetic distance

between Pelagostrobilidium neptuni and Strobilidium cauda-

tum corroborated the morphological data and thus the

validity of the genus Pelagostrobilidium (Agatha et al. 2005).

Furthermore, three strobilidiid species each representing

one of these genera, that is, R. lacustris, P. neptuni and

S. caudatum form a monophyletic group in both morpho-

logical and gene trees (Agatha & Struder-Kypke 2007;

McManus & Katz 2009). However, because of the lack of

morphogenetic data and the low number of small subunit

ribosomal RNA (SSrRNA) gene sequences available for

strobilidiid species, taxonomic distinctions and phyloge-

netic relationships among these three genera remain

unclear (Deroux 1974; Petz & Foissner 1992; Agatha &

Riedel-Lorje 1998; Dale & Lynn 1998; Foissner et al.

1999; Agatha 2003; Agatha et al. 2005; Kuppers et al.

2006; Tsai et al. 2008; Kim et al. 2010; Liu et al. 2011).

In the present study, three strobilidiid species, that is,

Rimostrombidium veniliae (Montagnes & Taylor 1994) Petz

et al. (1995) and two previously unknown Pelagostrobilidium

species, were isolated from coastal waters of southern

China, providing an opportunity to investigate their mor-

phology, morphogenesis and SSrRNA gene sequences.

Based on these and previous data, the phylogenetic rela-

tionships among genera within the family Strobilidiidae

are analysed. In addition, improved diagnoses are supplied

for Rimostrombidium and Pelagostrobilidium.

Materials and methodsCollection, observation and identification

Rimostrombidium veniliae was collected from Daya Bay

(22�43¢N; 114�32¢E), Guangdong Province, China, on 29

April 2007. The water temperature was 24.5 �C, salinity

28.2 & and pH 8.4.

Pelagostrobilidium paraepacrum sp. n. was isolated from

Shekou Port (22�29¢N; 113�55¢E), Guangdong Province,

China, on 22 December 2008. The water temperature was

20.1 �C, salinity 27.8 & and pH 8.1.

418 ª 2012 The Authors d Zoologica S

Pelagostrobilidium minutum sp. n. was collected from

Daya Bay (22�43¢N; 114�32¢E), Guangdong Province,

China, on 8 November 2007. The water temperature was

23.0 �C, salinity 33.0 & and pH 8.0.

Plankton samples were collected using 20 lm mesh

plankton nets. The samples were then transferred to Petri

dishes and specimens were immediately isolated for further

study in the laboratory. No cultures were established. Live

cells were observed using bright-field and differential

interference contrast microscopy. The infraciliature was

revealed by protargol impregnation (Song & Wilbert

1995). Illustrations of live specimens were based on direct

observations and light micrographs, while those of protar-

gol-impregnated specimens were made with the help of a

camera lucida at 1000· magnification. Terminology and

systematics are according to Agatha (2004, 2011) and

Lynn (2008), respectively. The numbering of the somatic

kineties follows Deroux (1974) and Montagnes & Lynn

(1991). Briefly, the kineties are numbered in a clockwise

fashion when the cell is viewed from the posterior, the

kinety nearest the cytostomal region being kinety 1 (K1).

Extraction, amplification and sequencing of DNA

Four cells of each species were collected with a micropi-

pette and rinsed 3–5 times with autoclaved seawater to

remove other protists. They were then transferred to a

1.5-mL microfuge tube with the minimum possible vol-

ume of seawater (Huang et al. 2010). Extraction of geno-

mic DNA was performed according to Tsai et al. (2010)

and Yi & Song (2011).

The PCR reactions were carried out using the universal

eukaryotic primers EukA (5¢-AACCTGGTTGATCCTG

CCAGT-3¢) and EukB (5¢-TGATCCTTCTGCAGGTT-

CACCTAC-3¢) (Medlin et al. 1988) for amplification of the

small subunit rRNA gene. Cycling parameters were as fol-

lows: 5 min at 94 �C; 35 cycles of 1 min at 95 �C, 2 min at

56 �C and 2 min at 72 �C; and 15 min at 72 �C (Zhang et al.

2011). The PCR product was purified using the TIAN gel

Midi Purification Kit (Tiangen Bio. Co., Shanghai, China)

and inserted into a pUCm-T vector (Sangon Bio. Co.,

Shanghai, China). DNA from plasmids was harvested using a

QIAprep Spin Miniprep Kit (Tiangen Bio. Co.) and

sequenced (Invitrogen sequencing facility, Shanghai, China).

Phylogenetic analysis

The SSrRNA gene sequences of 75 ciliates were used for

the construction of phylogenetic tree. Coleps nolandi, Pror-

odon viridis and P. teres were used as the out-group taxa. In

addition to the new sequences of Rimostrombidium veniliae,

Pelagostrobilidium paraepacrum sp. n. and P. minutum sp. n.,

72 others were obtained from the GenBank database, the

accession numbers of which are listed in Fig. 1.

cripta ª 2012 The Norwegian Academy of Science and Letters, 41, 4, July 2012, pp 417–434

Fig. 1 Maximum Likelihood tree inferred from small subunit rRNA gene sequences indicating phylogenetic positions of Rimostrombidiumveniliae, Pelagostrobilidium paraepacrum sp. n. and P. minutum sp. n. (bold typeface). Numbers at the nodes represent support values in the

following order: Maximum Likelihood (ML) bootstrap values, Maximum Parsimony (MP) bootstrap values, and Bayesian inference (BI)

posterior probabilities. Nodes absent from one of the three phylogenies are indicated by a hyphen instead of a support value. The field in

green represents the family Strobilidiidae. The scale bar indicates the number of substitutions per 10 nucleotides.

Weiwei Liu et al. d Phylogeny of three choreotrichous genera

The SSrRNA gene sequences were aligned using Hmmer

v2.3.2 (Eddy 1998). The ends were trimmed and ambigu-

ously aligned sites were refined by eye using BioEdit (Hall

1999), yielding an alignment of 1570 characters for phylo-

genetic inferences. Bayesian inference (BI) was performed

with MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) using

the GTR+I (=0.3863) +G (=0.4432) evolutionary model

ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters,

indicated by MrModeltest v.2 (Nylander 2004). The pro-

gram was run for 2 500 000 generations with a sample

every 100th generation. A ‘burn-in’ of 5000 sampled trees

was discarded. The remaining trees were used to calculate

posterior probabilities using a majority rule consensus.

Maximum likelihood (ML) trees were constructed with the

PhyML v2.4.4 (Guindon & Gascuel 2003). The reliability

41, 4, July 2012, pp 417–434 419

Table 1 SSrRNA gene sequence similarities (lower triangle) and number of unmatched nucleotides (upper triangle) between sequenced

strobilidiid species

Rimostrombidium

veniliae R. lacustris

Strobilidium

caudatum

Pelagostrobilidium

minutum P. paraepacrum P. neptuni

R. veniliae 117 105 140 121 151

R. lacustris 93.36% 73 112 96 125

S. caudatum 94.07% 95.85% 105 90 114

P. minutum 91.73% 93.37% 93.78% 108 107

P. paraepacrum 93.16% 94.54% 94.91% 93.61% 116

P. neptuni 91.07% 92.60% 93.23% 93.65% 93.12%

Table 2 Results of AU and SH tests comparing trees that are

representative of alternative hypotheses about the phylogenetic

associations of groups of interest in this study

Hypothesis tested ln likelihood score AU SH

Best maximum likelihood tree (unconstrained) Best 1.000 1.000

Constraint: Lynnella clustered with oligotrichs 18684.22482 0.088 0.365

Constraint: monophyly of Choreotrichida 18712.96882 0.010 0.096

Constraint: monophyly of Strombidinopsidae 18680.67828 0.070 0.382

Constraint: monophyly of Rimobstrombidium 18698.73494 0.012 0.152


of internal branches was assessed using nonparametric

bootstrap method with 1000 replicates. Maximum parsi-

mony (MP) trees were constructed with PAUP* 4.0b 10

(Swofford 2002), including bootstrapping with 1,000 repli-

cates. TreeView v1.6.6 (Page 1996) and MEGA 4.0 (Tam-

ura et al. 2007) were used to visualize tree topology.

PAUP* 4.0b 10 was used to generate the constraint ML

trees under the GTR + I + G model to test the hypotheses

that: (i) Lynnella clusters with subclass Oligotrichia, (ii)

Choreotrichida is monophyletic, (iii) Strombidinopsidae is

monophyletic, and (iv) Rimostrombidium is monophyletic.

The best constrained trees, that is, those with the lowest-lnL

values, were compared with the unconstrained ML trees

using the approximately unbiased (AU) test and the Shimo-

daira-Hasegawa (SH) test (Shimodaira 2002) as implemented

in CONSEL package (Shimodaira & Hasegawa 2001).

ResultsSSrRNA gene sequence analyses

The SSrRNA gene sequences of Rimostrombidium veniliae,

Pelagostrobilidium paraepacrum sp. n. and P. minutum sp. n.

are 1768, 1767 and 1683 bp in length (including primers),

respectively. The sequences are deposited in GenBank

with accession numbers FJ876964, FJ876963 and

FJ876959, respectively. The pairwise sequence similarities

between P. minutum sp. n. and other sequenced strobilidi-

ids ranged from 91.73 to 93.78%, whereas those between

P. paraepacrum sp. n. and other sequenced strobilidiids

ranged from 93.12 to 94.91% (Table 1).

All three phylogenetic methods resulted in similar tree

topologies; thus, only the ML tree is shown with support

values for all three analyses listed at the nodes (Fig. 1).

The monophyly of the subclass Choreotrichia was con-

firmed with high support in ML (100%), MP (99%) and

BI (1.00) trees. Lynnella semiglobulosa branches separately

from the choreotrich clade with low support (58% ML,

69% MP, 0.61 BI). Together these form a sister group

with the oligotrichs. Within the subclass Choreotrichia,

the order Tintinnida forms a clade. The monophyly of the

order Choreotrichida was not recovered, because the fam-


ily Strobilidiidae groups with the tintinnids rather than

with the strombidinopsid genera Strombidinopsis and Para-

strombidinopsis (Fig. 1). Strombidinopsis branches basally

within the choreotrich clade, followed by Parastrombidinop-

sis. Thus, the monophyly of the family Strombidinopsidae

was not supported. Within the strobilidiid clade, P. minu-

tum sp. n. and P. neptuni cluster together with high sup-

port (98% ML, 99% MP, 1.00 BI) and form a moderately

well-supported clade with P. paraepacrum (65% ML, 79%

MP, 0.94 BI). In ML and MP trees, S. caudatum branches

basally to the Pelagostrobilidium group (33% ML, 42%

MP), followed by R. lacustris (Fig. 1). In all trees, R. veniliae

occupies the basal position within the Strobilidiidae clade

with moderate support (71% ML, 54% MP, 0.98 BI)

(Fig. 1). The monophyly of Choreotrichida and the possi-

bility that the two Rimostrombidium species cluster together

were both rejected by the AU tests (P = 0.010 and 0.012,

respectively) but were not rejected by the SH tests

(P = 0.096 and 0.152, respectively). No other hypothetical

phylogenetic associations were rejected (Table 2).

Description of Rimostrombidium veniliae

Genus Rimostrombidium Jankowski 1978

Rimostrombidium veniliae (Montagnes & Taylor 1994) Petz

et al. 1995 (Figs 2 and 3; Table 3).

Strobilidium veniliae Montagnes & Taylor 1994: 576–578,

Figs 4–6.

Rimostrombidium veniliae Petz et al. 1995: 144; Agatha &

Riedel-Lorje 1998: 16, Fig. 4.


A

B C D

E F G H

I J K L

Fig. 2 Rimostrombidium veniliae (Montagnes & Taylor 1994) Petz et al. 1995 from life (A) and after staining with protargol (B–L). ––A.

Lateral view of a typical specimen; arrows mark the kinetal lips. ––B, C. Right (B) and left (C) lateral views of the same specimen

showing the ciliary pattern and nuclear apparatus, note the different lengths of somatic kineties. ––D, E. Early dividers showing the

location of oral primordium (arrows) and the replication bands in the macronucleus (arrowheads). ––F, G. Apical (F) and aboral (G)

views showing the buccal apparatus, somatic kineties and macronucleus. ––H. Early middle divider, the polykinetids are differentiating

and the endoral membrane originates de novo (arrow). ––I–K. Cells in middle stage of division, the new polykinetids perform a distinct

spiral to form a funnel and then start to evaginate; arrowheads denote the replication bands in the macronucleus, arrow marks the

endoral membrane. ––L. Late divider, the new polykinetids splay out to form a closed circle; note the macronucleus condenses to an

ellipsoidal mass, and the somatic kineties split into two equal parts (arrowheads). E, endoral membrane; EM, external membranelles; IM,

internal membranelle; K1–9, somatic kineties 1–9; Ma, macronucleus; Mi, micronucleus. Scale bars–30 lm.


Remarks. This species was originally described as Stro-

bilidium veniliae by Montagnes & Taylor (1994) based on

scanning electron microscopy and observations of protar-

gol-impregnated specimens. Subsequently, Petz et al.

(1995) transferred it to the genus Rimostrombidium, and

Agatha & Riedel-Lorje (1998) reported a German popula-

tion based on protargol-impregnated specimens. However,

its live morphology remained unknown. An improved

diagnosis is here supplied based on previous and present

observations including data from live specimens from the

Chinese population.

Improved diagnosis. Size 35–55 · 35–50 lm in vivo; sub-

spherical with flattened anterior and rounded posterior

end. One micronucleus in dorsal indentation of C-shaped


macronucleus. About nine somatic kineties. Usually 20–23

external and one or two internal adoral membranelles.

Description of Chinese population

Cells mostly about 45 · 40 lm in vivo and 29–41 · 31–

54 lm in protargol preparations. Subspherical to cordiform

in shape, flattened anteriorly and rounded posteriorly

(Figs 2A and 3A–C). No frontal protrusion recognized, with

shallow ridges caused by kinetal lips (Fig. 2A, arrows).

Cytoplasm packed with colourless lipid droplets 2–4 lm

across, and food vacuoles 5–8 lm across, often containing

yellow algae (Figs 2A and 3B,C). Macronucleus C-shaped

with ventral gap, transversely oriented underneath external

membranelles, containing numerous globular nucleoli 1–3

lm across (Fig. 2B, C and G). One faintly impregnated micro-

41, 4, July 2012, pp 417–434 421

Table 3 Morphometric characterizations of Rimostrombidium

veniliae (Montagnes & Taylor 1994) Petz et al. 1995 (Chinese

population, first row), Pelagostrobilidium paraepacrum sp. n. (second

row) and P. minutum sp. n. (third row). All data based on

randomly selected protargol-impregnated specimens

Characters Min Max Mean SD CV n

Cell length in lm 29 41 34.8 3.3 9.8 17

61 81 70.6 5.7 8.2 18

16 27 20.0 3.2 16.0 17

Cell width in lm 31 54 43.8 7.2 16.2 17

52 71 60.2 4.7 7.7 18

12 19 15.4 2.6 16.8 17

External membranelles, number 20 21 20.1 0.3 1.7 17

30 32 30.8 0.7 2.3 21

19 21 20.4 0.7 3.4 17

Elongated external membranelles,

number

2 3 2.3 0.5 20.1 12

3 5 4.2 0.6 13.2 17

3 4 3.5 0.5 14.7 11

Internal membranelles, number 1 1 1.0 0 0 12

2 2 2.0 0 0 17

1 1 1.0 0 0 12

Somatic kineties, number 8 9 8.9 0.2 2.7 17

6 6 6.0 0 0 21

4 4 4.0 0 0 17

Macronucleus, number 1 1 1.0 0 0 17

1 1 1.0 0 0 21

1 1 1.0 0 0 17

Micronucleus, number 1 1 1.0 0 0 10

1 3 1.7 0.7 42.4 14

1 1 1.0 0 0 11

CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean; Min,

minimum; n, number of cells measured; SD, standard deviation.


nucleus in dorsal indentation of macronucleus (Fig. 2C). Nei-

ther contractile vacuole nor cytopyge observed.

Swimming behaviour typical of many strobilidiids with

adoral membranelles projecting radially. In motionless cells,

membranelles overlap like an iris diaphragm (Fig. 3D).

Somatic ciliature usually composed of nine longitudinal

kineties, most of which are conspicuously shortened at

both ends and evenly arranged around equatorial region of

cell (Figs 2B,C,G and 3F,G). Kineties (K) 3, 7 and 8 lon-

gest (about 22–28 lm); K1, 2 and 9 medium length (about

18–23 lm); K4–6 short, about 12–18 lm (Fig. 2B and C).

All kineties composed of closely spaced dikinetids, each

basal body bearing a cilium about 2 lm long (Figs 2B and

3I, arrows). Cilia with kinetal lips covering their proximal

portion (Fig. 2A).

About 20 external adoral membranelles, two or three of

which are elongated and extend into oral cavity; each

external membranelle composed of three basal body rows,

with cilia up to 30 lm long, bases of external membran-

elles 17–20 lm long (Figs 2F and 3E). Usually one very

small internal membranelle near cytostome (Figs 2F and

3D, arrow, E, J). Endoral membrane lying near right inner


wall of oral cavity (Fig. 2F). Near inner side of each exter-

nal membranelle are four fibres that are associated with

each other and directed inward (Figs 2F and 3H, arrow-

heads). One fibre underneath peristomial rim, parallel to

last elongated external membranelle and associated with

internal membranelle (Fig. 3J, arrowhead).

Morphogenesis

Several divisional stages were observed. Stomatogenesis

commences with the appearance of an anarchic field of

basal bodies on the left-dorsal side anterior to K4 and K5

(Figs 2D, E and 3K,L, arrows). A bulge develops at each

end of the macronucleus from which the replication bands

originate (Figs 2D and 3L, arrowheads). The oral primor-

dium enlarges and the individual polykinetids differentiate

gradually from the inner towards the outer portion

(Fig. 2H). The endoral originates de novo (Fig. 2H,

arrow). With the development of polykinetids, the distal

and proximal portions of the adoral zone both curve

towards each other forming a tube perpendicular to the

cell surface. The polykinetids then perform an anticlock-

wise twist when viewed facing the opisth’s cytostome

(Figs 2I and 3M). The replication bands of the macronu-

cleus gradually migrate from both ends towards the mid-

region (Figs 2I and 3M, arrowheads).

The polykinetids continue to rotate (Figs 2J and 3N)

and the new oral apparatus gradually evaginates as the

diameter of the membranellar zone increases (Figs 2K and

3O). The somatic kineties are lengthened by the intrakin-

etal proliferation of basal bodies. The macronucleus begins

to expand (Figs 2K and 3O). When the rotation of polyki-

netids has finished, the membranellar zone forms a closed

circle, and the short posteriormost polykinetid migrates to

the inner margin of the cytostome to become the internal

membranelle (Figs 2L and 3P). The macronucleus con-

denses to form a longitudinally oriented ellipsoidal mass

(Figs 2L, arrow and 3P). Each somatic kinety splits into

two parts, one each for the opisthe and proter, respectively

(Fig. 2L, arrowheads).

Comparison with other populations

Rimostrombidium veniliae was first described by Montagnes

& Taylor (1994) based on a population from British

Columbia, Canada (Fig. 7A,B). It was subsequently rede-

scribed by Agatha & Riedel-Lorje (1998) based on a

German population (Fig. 7C,D). The Chinese population

corresponds well with both previous descriptions with

respect to the basic infraciliature and the general morphol-

ogy. There are some differences, however, with the Chinese

population having: (i) slightly fewer external membranelles

(20–21 vs. 22–23) than that reported by Montagnes & Tay-

lor (1994); (ii) a somewhat smaller cell size in protargol


A B C D

E F G H

M N O P

I J K L

Fig. 3 Photomicrographs of Rimostrombidium veniliae (Montagnes & Taylor 1994) Petz et al. 1995 from life (A–D) and after staining with

protargol (E–P). ––A–C. Lateral views showing different body shapes. ––D. Apical view; the external membranelles arranged like an iris

diaphragm, note the internal membranelle (arrow). ––E. Oral apparatus. ––F, G. Lateral (F) and aboral (G) views showing the somatic

kineties. ––H. Detail of the oral field, arrowheads mark the fibre systems associated with the external membrane. ––I. Detail of the

somatic kinety showing the dikinetids (arrowheads). ––J. Detail of the oral apparatus showing the single internal membranelle and the

fibre stretching from the internal membranelles (arrowhead). ––K, L. Early dividers showing the oral primordium (arrows) and

the replication bands in the macronucleus (arrowhead). ––M–O. Middle dividers showing the spiralled new polykinetids, arrow marks the

endoral membrane of the opisthe arrowhead notes the replication bands in the macronucleus. ––P. Late divider with the new oral

membranelles splayed out onto the cell surface and the macronucleus condensing to an ellipsoidal mass. EM, external membranelles; IM,

internal membranelle; Ma, macronucleus; K1–4, somatic kineties 1–4. Scale bars–30 lm.


preparations (30–40 · 30–54 lm vs. 47–76 · 44–54 lm)

than that reported in the German population by Agatha &

Riedel-Lorje (1998) (Table 4A); (iii) all somatic kineties

extending to the posterior end of the cell, whereas in

the other populations, some somatic kineties are conspicu-

ously shortened (Montagnes & Taylor 1994; Agatha &

Riedel-Lorje 1998). Although we consider these to be

population-dependent variations, it is noteworthy that:


(i) the four ciliated kinetosomes on the inner side of each

external polykinetid reported in previous investigations

(Montagnes & Taylor 1994; Agatha & Riedel-Lorje 1998)

are here recognized as fibres associated with the external

polykinetid; (ii) the rod-like inclusion in the cell periphery

described by Agatha & Riedel-Lorje (1998) was found

neither in the Chinese population nor in the type specimens

even after careful re-examination. Furthermore, in their

41, 4, July 2012, pp 417–434 423

A B

C D

E

F

H

I J K L M

G

Fig. 4 Pelagostrobilidium paraepacrum sp. n. from life (A–D) and after staining with protargol (E–M). ––A. Lateral view of a representative

specimen, arrow marks the kinetal lip. ––B. The motionless cell with adoral membranelles forming a flame shape. ––C, D. Laterial views

showing different body shapes. ––E, F. Ventral (E) and dorsal (F) views of the same specimen showing the ciliary pattern and nuclear

apparatus. ––G. Oral apparatus. ––H. Nuclear apparatus. ––I, J. Early dividers showing the location of oral primordium (arrows). ––K, L.

Middle dividers with oral primordium rotated clockwise, arrowheads mark the endoral membrane originating de novo, arrow denotes the

replication bands in the macronucleus. ––M. Late divider, the new polykinetids form a closed circle and the macronucleus is condensed

(arrow). E, endoral membrane; EM, external membranelles; IM, internal membranelles; K1–6, somatic kineties 1–6; Ma, macronucleus;

Mi, micronuclei. Scale bars–40 lm.


species definition of R. veniliae, Montagnes & Taylor (1994)

reported a wide range of somatic kinety number, that is,

6–12, on average 10 (vs. usually 8, rarely 9, in the Chinese

population) (Table 4A). This degree of variation is beyond

what would normally be expected in a single population. It

is therefore possible that the original population contained

more than one morphologically similar species.

Comparison with related species

Considering its hemispherical body shape, Rimostrombidi-

um veniliae is similar to five congeners: R. orientale Song &

Bradbury 1998; R. undinum (Martin & Montagnes 1993)

Petz et al. 1995; R. sphaericum (Lynn & Montagnes 1988)

Petz & Foissner 1992; R. armeniensis (Zharikov 1987)


Foissner et al. 1999; and R. multinucleatum (Lynn & Mon-

tagnes 1988) Petz & Foissner 1992 (Fig. 7E–M;

Table 4A).

Rimostrombidium orientale (Fig. 7E,F; Table 4A) most

closely resembles R. veniliae. However, R. orientale can be

separated from latter by having: (i) a conspicuous frontal

protrusion (vs. absent); (ii) fewer (6 vs. 9 or 10 on average)

somatic kineties; and (iii) short somatic kineties that are

limited to the equatorial region (vs. longer somatic kin-

eties some of which extend nearly to the posterior pole)

(Song & Bradbury 1998).

Rimostrombidium veniliae is distinguished from R. undi-

num (Fig. 7G,H; Table 4A) by its larger body length after

protargol impregnation (30–76 lm vs. 16–29 lm), fewer


A B C ED

F G H I

J

P Q R

M N

O

S

K L

Fig. 5 Photomicrographs of Pelagostrobilidium paraepacrum sp. n. from life (A–G) and after staining with protargol (H–S). ––A–C. Lateral

views showing different body shapes. ––D. Lateral view of a motionless cell. ––E. Apical view showing the fully extended external

membranelles. ––F. Detail of the oral field showing the two internal membranelles (arrows). ––G. Cell surface showing the kinetal lip

(arrows). ––H. Detail of the oral apparatus showing the internal membranelle and the endoral membrane. ––I. Dorsal view showing the

micronuclei (arrows) lying within indentations of the macronucleus. ––J. Lateral view showing the somatic kineties. ––K, L. Ventral (K)

and dorsal (L) views of the same specimen showing the ciliary pattern. ––M, N. Early dividers showing the oral primordium (arrows).

––O–Q. Middle dividers with oral primordium rotated clockwise. Arrows note the replication bands in the macronucleus and arrowheads

mark the new endoral membrane. ––R–S. Later dividers showing the new oral membranelles and the macronucleus divided into two

parts. E, endoral membrane; IM, internal membranelles; K1–6, somatic kineties 1–6; Ma, macronucleus. Scale bars–40 lm.


internal membranelles (1–2 vs. 4–6), and the appearance of

the somatic ciliature (basal bodies extremely closely spaced

vs. the basal bodies widely separated) (Martin & Montagnes

1993).

Rimostrombidium sphaericum (Fig. 7I,J; Table 4A) differs

from R. veniliae in having more external membranelles


(24–30 vs. 20–23) and in the absence (vs. presence) of

internal membranelles (Lynn & Montagnes 1988).

Rimostrombidium armeniensis (Fig. 7K; Table 4A) can be

distinguished from R. veniliae by having more somatic kineties

(12 vs. 9), a globular (vs. C-shaped) macronucleus and the pres-

ence (vs. absence) of a contractile vacuole (Zharikov 1987).

41, 4, July 2012, pp 417–434 425

A

B CL O P QN

G I J KHM

D E F

Fig. 6 Pelagostrobilidium minutum sp. n. from life (A, B, G–K) and after staining with protargol (C–F, L–Q). ––A. Ventral view. ––B.

Swimming traces. ––C. Oral apparatus. ––D, E.Ventral (D) and dorsal (E) views of the same specimen showing the ciliary pattern and

nuclear apparatus. ––F. Middle divider with the new polykinetids forming a closed circle, note the new endoral membrane (arrow) and

internal membranelle (arrowhead). ––G–K. Lateral views showing different body shapes. ––L. Left lateral view showing the spiralled

somatic kinety 2. ––M. Oral apparatus, arrowhead marks the endoral membrane. ––N. Detail of the oral field, arrow notes the single

internal membranelle. ––O, P. Ventral (O) and dorsal (P) views showing the ciliary pattern. ––Q. Late divider with the new oral

membranelles and the condensed macronucleus. E, endoral membrane; EM, external membranelles; IM, internal membranelle; K1–4,

somatic kineties 1–4; Ma, macronucleus; Mi, micronucleus. Scale bars–10 lm (A, D–F, L), 20 lm (G–K).


Rimostrombidium veniliae can be easily separated from

R. multinucleatum (Fig. 7L,M; Table 4A) by having only

one macronucleus (vs. 11 spherical macronuclei), 9 (vs. 5)

somatic kineties, and 20–23 (vs. 18–20) external membran-

elles (Lynn & Montagnes 1988).

Description of Pelagostrobilidium paraepacrum sp. n.

Genus Pelagostrobilidium Petz et al. 1995

Pelagostrobilidium paraepacrum sp. n. (Figs 4 and 5;

Table 3).

Deposition of slides. A protargol slide with the holotype

specimen (marked with a blue circle) is deposited in the

Natural History Museum, London, with registration num-

ber NHMUK 2010.7.14.1. One paratype slide is deposited

in the Laboratory of Protozoology, SCNU, with registra-

tion number WW08122202.

Etymology. The specific epithet ‘paraepacrum’ refers to

the superficial similarity in body shape between this spe-

cies and Pelagostrobilidium epacrum.


Type location. Coastal waters of Shenzhen (22�29¢N;

113�55¢E), Guangdong Province, China.

Diagnosis. Obconical Pelagostobilidium, about 65 · 50 lm

in vivo; posterior end tapered to a point; one to three mi-

cronuclei lying within dorsal indentations of macronu-

cleus; constantly six somatic kineties: kineties 1, 3, 4, 5

and 6 oriented longitudinally, kinety 2 spiralled around

left posterior area of cell and terminating posteriorly

below kineties 3 and 4; 30–32 external and two internal

membranelles.

Description

Cell size 50–80 · 45–65 lm in vivo, 61–81 · 52–71 lm

after protargol impregnation. Body shape variable, obconi-

cal to cordiform (Figs 4C,D and 5A–C). Anterior end

truncated, posterior region narrowed and tapered to a

point (Figs 4A,B and 5A–C). Length:width ratio about

3:2–1:1. Cell surface often ridged because of kinetal lips

(Fig. 5G). Lip of kinety 2 causing an oblique flattening of


A B C D E F

G H I J K L M

N O P Q R S T

U V W X Y Z

Fig. 7 Morphologically similar species in Rimostrombidium (A–M) and Pelagostrobilidium (N–Z). ––A, B. R. veniliae (from Montagnes &

Taylor 1994); ––C, D. R. veniliae (from Agatha & Riedel-Lorje 1998); ––E, F. R. orientale (from Song & Bradbury 1998); ––G, H.

R. undinum (from Martin & Montagnes 1993); ––I, J. R. sphaericum (from Lynn & Montagnes 1988); ––K. R. armeniensis (from Zharikov

1987); ––L, M. R. multinucleatum (from Lynn & Montagnes 1988); ––N. P. epacrum (from Lynn & Montagnes 1988); ––O, P. P. wilberti

(from Kuppers et al. 2006); ––Q, R. P. neptuni (from Montagnes & Taylor 1994); ––S, T. P. neptuni (from Agatha et al. 2005); ––U, V.

P. simile (from Song & Bradbury 1998); ––W, X. P. spirale (from Lynn & Montagnes 1988); ––Y, Z. P. sp. (from Ota & Taniguchi 2003).

Scale bars–20 lm.


left posterior half, making cell asymmetrical (Figs 4A,

arrow and 5A). No frontal protrusion observed.

Cytoplasm colourless with numerous small globular

inclusions (2–4 lm across) that render cells almost dark

grey at low magnification (Fig. 4A,B). Food vacuoles

located in posterior portion of cell, 4–6 lm across, con-

taining some green algae (Figs 4A and 5A). Macronucleus

transversely oriented underneath external membranelles,

with numerous globular nucleoli 1–2 lm across (Fig. 4E,

F and H). Usually, two globular micronuclei, 1–2 lm in

diameter, each lying in a small, dorsally located indenta-

tion of macronucleus (Figs 4H and 5I, arrows). Neither

contractile vacuole nor cytopyge observed.

Swimming behaviour typical of strobilidiids, that is, cell

rotates while in stationary position with its anterior end

facing ahead interspersed by sudden ‘jumps’ of four to six

cell lengths. Jumps may be stimulated by physical agitation


or by changes in food concentration. In resting cells,

adoral membranelles are motionless and directed upwards

(Figs 4B and 5D).

Six somatic kineties each composed of closely spaced

monokinetids and cilia about 2–3 lm long (Figs 4E,F

and 5K,L). Apart from K2, somatic kineties commenced

about 5 lm below membranellar zone (Figs 4E,F and

5J–L). K1 and K6 longest (40–60 lm long), ventrally

positioned and extend longitudinally almost to posterior

pole (Figs 4E and 5K). K3 shortest (25–35 lm long),

extending longitudinally to mid-region of cell, terminat-

ing above posterior portion of K2 (Figs 4F and 5J,L).

K4 and K5 longer than K3 (35–45 lm long), extending

to posterior fifth of cell (Figs 4F and 5L). Length of

somatic kineties progressively increased clockwise from

K3 to K1 (when viewed aborally). K2 which starts in

anterior 2 ⁄ 5 of cell on left side, spirales around poster-

41, 4, July 2012, pp 417–434 427

Tab

le4

(A)

Mo

rph

olo

gica

lco

mp

aris

on

amo

ng

Rim

ostr

ombi

dium

spec

ies

wit

ha

hem

isp

her

ical

bo

dy

shap

e(B

)M

orp

ho

logi

cal

com

par

iso

nam

on

gP

elag

ostr

obil

idiu

msp

ecie

s

Spec

ies

Size

inl

m,

afte

r

prot

argo

lim

preg

natio

n

Exte

rnal

mem

bran

elle

s,N

o.

Inte

rnal

mem

bran

elle

s,N

o.

Som

atic

kine

ties,

No.

Mac

ronu

clei

,

No.

and

shap

eR

efer

ence

s

(A)

Rim

ostr

ombi

dium

veni

liae

29–4

1·

31–5

420

–21

18–

91,

C-s

hape

Pres

ent

stud

y

R.

veni

liae

14–4

0·

15–4

522

–23

1–2

ca.

101,

C-s

hape

Mon

tagn

es&

Tayl

or(1

994)

R.

veni

liae

47–7

6·

44–5

4ca

.20

1ca

.10

1,C

-sha

peA

gath

a&

Rie

del-L

orje

(199

8)

R.

orie

ntal

e21

–30

·21

–28

20–2

31

61,

C-s

hape

Song

&B

radb

ury

(199

8)

R.

undi

num

16–2

9·

15–2

321

–24

4–6

61,

C-s

hape

Mar

tin&

Mon

tagn

es(1

993)

R.

spha

eric

um40

–60

·40

–70

24–3

00

9–11

1,C

-sha

peLy

nn&

Mon

tagn

es(1

988)

R.

arm

enie

nsis

ca.

55–6

01ca

.31

–ca

.12

1,gl

obul

arZh

arik

ov(1

987)

R.

mul

tinuc

leat

um26

–32

·25

–35

18–2

00

511

,gl

obul

arLy

nn&

Mon

tagn

es(1

988)

(B)

Spec

ies

Size

2in

lm

Bod

ysh

ape

Exte

rnal

mem

bran

elle

s,N

o.

Inte

rnal

mem

bran

elle

s,N

o.

Som

atic

kine

ties,

No.

Arr

ange

men

tof

Kin

ety

2

Ref

eren

ce

Pela

gost

robi

lidiu

mep

acru

m60

–95

·35

–50

Con

ical

33–4

01

5Sp

iralle

dar

ound

body

and

post

erio

rpo

rtio

n

belo

wK

inet

ies

3an

d4

Lynn

&M

onta

gnes

(198

8)

P.ep

acru

m35

–55

·31

–47

Con

ical

30–3

12

5Sp

iralle

dar

ound

body

and

post

erio

rpo

rtio

n

belo

wK

inet

ies

3an

d4

Pett

igro

sso

(200

3)

P.m

inut

a16

–27

·12

–19

Con

ical

19–2

11

4Sp

iralle

dar

ound

body

and

post

erio

rlycl

ose

toK

inet

y3

Pres

ent

wor

k

P.ne

ptun

i40

–53

·40

–48

Subs

pher

ical

35–3

81–

25

Tran

sver

sely

arch

edan

dpo

ster

ior

port

ion

belo

wK

inet

ies

3an

d4

Aga

tha

etal

.(2

005)

P.pa

raep

acru

m61

–81

·52

–71

Con

ical

30–3

22

6Sp

iralle

dar

ound

body

and

post

erio

rpo

rtio

n

belo

wK

inet

y3

Pres

ent

wor

k

P.si

mile

36–5

6·

37–5

5Su

bsph

eric

al29

–31

15

Tran

sver

sely

arch

edan

dpo

ster

ior

port

ion

belo

wK

inet

ies

3,4

and

5

Song

&B

radb

ury

(199

8)

P.sp

irale

40–6

0·

40–5

2Su

bsph

eric

al33

–39

8–20

35

Tran

sver

sely

arch

edan

dpo

ster

ior

port

ion

belo

wK

inet

ies

3an

d4

Lynn

&M

onta

gnes

(198

8)

P.sp

.–

Subs

pher

ical

––

6Tr

ansv

erse

lyar

ched

and

post

erio

rpo

rtio

n

belo

wK

inet

ies

3,4

and

5

Ota

&Ta

nigu

chi

(200

3)

P.w

ilber

ti42

–77

·38

–53

Con

ical

25–3

22

6Sp

iralle

dan

dsh

ort,

post

erio

rlycl

ose

toK

3Ku

pper

set

al.

(200

6)

1B

ased

onin

vivo

spec

imen

s.2B

ased

onpr

otar

gol-i

mpr

egna

ted

spec

imen

s.3B

ased

ondi

ffer

ent

term

inol

ogy.

–,D

ata

unav

aila

ble.


428 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 41, 4, July 2012, pp 417–434

A B C

D E F

Fig. 8 Infraciliature of representative species in Strobilidiidae. ––

A, B. Rimostrombidium orientale (from Song & Bradbury 1998); ––

C, D. Strobilidium gyrans (from Deroux 1974), showing

numbering system for somatic kineties in choreotrichs; ––E, F.

Pelagostrobilidium neptuni (from Montagnes & Taylor 1994). Scale

bars–20 lm.


ior region of cell, and terminates near posterior pole

(Figs 4E, F and 5J–L).

About 30 external membranelles, the 3–5 most distal of

which become progressively elongate and extend into oral

cavity (Fig. 4G). Each membranelle composed of three

basal body rows, longest base about 15 lm. Cilia of

membranelles about 30 lm long in vivo, usually directed

upwards and extend slightly outward at top (Figs 4A and

5A,E). Two internal membranelles located within oral cav-

ity, right of elongated external membranelles (when viewed

dorsally), left internal membranelle with three-rowed kin-

eties, 5 lm long and extending to anterior rim of membra-

nellar zone; right internal membranelle with two-rowed

kineties and only 3 lm long (Figs 4G and 5F, arrows, H).

Endoral membrane single-rowed extending across right

side of peristomial field (Figs 4G and 5H). Oral cavity

funnel-shaped, acentric on anterior surface; some pharyn-

geal fibres extending posteriorly to halfway down cell

(Fig. 4E).

Morphogenesis

Several dividers were observed. Early dividers have a cune-

ate oral primordium on the left-dorsal side, above K2

(Figs 4I and 5M, arrows). With the development of basal

bodies, the polykinetids differentiate (Figs 4J and 5N,

arrows). Next, the oral primordium gradually rotates

clockwise to become inverted C-shaped while the inner

portions of the polykinetids begin to sink towards the cell

centre (Fig. 5O). Two replication bands form, one at each


end of the macronucleus (Fig. 5O, arrow). The endoral

originates de novo (Fig. 5O, arrowhead). With the rota-

tion of the oral primordium, the polykinetids perform an

anticlockwise spiral (Figs 4K and 5P). The distal end of

the oral primordium curves towards the proximal end to

form a closed circle and the membranellar zone forms a

funnel perpendicular to the cell surface (Figs 4L and 5Q).

Meanwhile, the new oral apparatus evaginates gradually

(Figs 4L and 5Q) and finally forms a closed circle

(Figs 4M and 5R,S). The replication bands migrate to the

mid-region of the macronucleus (Figs 4K and 5P, arrows)

and the endoral membrane lies transversely across the cen-

tre of the new oral zone (Figs 4K, L and 5P, arrowhead).

The somatic kineties lengthen by intrakinetal proliferation

of basal bodies. The macronucleus condenses into a trans-

versely oriented ellipsoidal mass (Figs 4M, arrow and 5R)

and then divides into two (Fig. 5S).


Agatha (1995) described a Rimostrombidium species from

the coast of northern Germany (Rimostrombidium sp.) that

corresponds closely with P. paraepacrum sp. n. with respect

to its infraciliature and general morphology. These are

therefore regarded as two populations of the same species.

To date, six species have been assigned to the genus Pel-

agostrobilidium: P. epacrum (Lynn & Montagnes 1988)

Agatha et al. 2005; P. neptuni (Montagnes & Taylor 1994)

Petz et al. 1995; P. simile Song & Bradbury 1998; P. spirale

(Lynn & Montagnes 1988) Petz et al. 1995; Pelagostrobilidium

sp. sensu Ota & Taniguchi (2003), and P. wilberti Kuppers

et al. 2006.

Pelagostrobilidium epacrum (Fig. 7N; Table 4B) most clo-

sely resembles P. paraepacrum sp. n. although it can be

separated from the latter by having: (i) a more slender cell

shape in protargol preparations (35–50 lm vs. 52–71 lm

in width); (ii) fewer somatic kineties (5 vs. 6); (iii) more

external membranelles (33–40 vs. 30–32); and (iv) fewer

internal membranelles (1 vs. 2). Although Pettigrosso

(2003) reported a population of P. epacrum with similar

numbers of external (30 or 31) and internal (two) mem-

branelles, it can easily be separated from P. paraepacrum

sp. n. by the possession of only 5 (vs. 6) somatic kineties

(Lynn & Montagnes 1988; Pettigrosso 2003).

With its conical body shape and six somatic kineties,

P. wilberti (Fig. 7O,P; Table 4B) also closely resembles

P. paraepacrum sp. n. However, it can be separated from

the latter by: (i) K2 conspicuously short and terminating

posteriorly close to K3 (vs. K2 longer than K3 and termi-

nating near posterior pole); (ii) contractile vacuole present

(vs. absent) (Kuppers et al. 2006).

Like P. paraepacrum sp. n., Pelagostrobilidium sp. sensu

Ota & Taniguchi (2003) has six somatic kineties

41, 4, July 2012, pp 417–434 429


(Fig. 7Y,Z; Table 4B); however, it can be separated from

the former by its transversely arched K2 which lies below

kineties 3–6 (vs. K2 spiralled around posterior body and

lying just below K3) and its subspherical (vs. conical) body

shape (Ota & Taniguchi 2003).

Pelagostrobilidium paraepacrum sp. n. can be easily sepa-

rated from P. neptuni, P. simile and P. spirale (Fig. 7Q–X;

Table 4B) by its conical (vs. subspherical) body shape, hav-

ing 6 (vs. 5) somatic kineties, and the position of K2

(arched transversely across the posterior region of the

body vs. spiralled around the posterior area of body, and

terminating near the posterior pole) (Lynn & Montagnes

1988; Montagnes & Taylor 1994; Petz et al. 1995; Song &

Bradbury 1998).

Description of Pelagostrobilidium minutum sp. n.

Genus Pelagostrobilidium Petz et al. 1995

Pelagostrobilidium minutum sp. n. (Fig. 6; Table 3).

Deposition of slides. A protargol slide with the holotype

specimen (marked with a blue circle) is deposited in the

Natural History Museum, London, with registration num-

ber NMHUK 2010.11.9.2. Three paratype slides are

deposited in the Laboratory of Protozoology, SCNU, with

registration numbers WW07110803-01, WW07110803-02

and WW07110803-03.

Etymology. The Latin word ‘minutum’ refers to the small

cell size of this species.

Type location. Coastal waters of Daya Bay (22�43¢N;

114�32¢E), Guangdong Province, China.

Diagnosis. Small Pelagostrobilidium about 25 · 20 lm in

vivo with tapered posterior end; one micronucleus located

in dorsal indentation of macronucleus; invariably four

somatic kineties: kinety 2 spiralled around left area of cell

and terminates near posterior pole; kinety 1 located below

kinety 2; kineties 3 and 4 slightly spiralled around

mid-body of right side; one internal and 19–21 external

membranelles.

Description

Cell size about 15–30 · 15–25 lm in vivo, 16–27 · 12–

19 lm after protargol impregnation. Cell shape variable,

global to cordiform, usually obconical (Fig. 6A and G–K).

Anterior end transversely truncated to slightly domed in

centre (Fig. 6A and G). Posterior region usually narrowed

with a tapered end, but sometimes rounded (Fig. 6G–K).

Widest slightly below adoral zone of membranelles. Kin-

etal lips conspicuous, spirally arranged (Fig. 6A and K).

Cells fragile and readily burst under cover glass. Cyto-

plasm colourless and opaque because of numerous lipid


droplets 1–2 lm across, and food vacuoles about 4 lm

across (Fig. 6A). Macronucleus transversely oriented under-

neath external membranelles, containing numerous globu-

lar nucleoli 1–2 lm across (Fig. 6D,E). One micronucleus

2 lm in diameter located in dorsal indentation of macro-

nucleus (Fig. 6E). Neither contractile vacuole nor cyto-

pyge observed.

Swimming behaviour typical of strobilidiids, that is, cell

rotates while in stationary position with its anterior end

facing ahead, interspersed by sudden ‘jumps’ of about five

cell lengths (Fig. 6B).

Four somatic kineties, each comprising a continuous

row of basal bodies, probably monokinetids, with cilia

about 2 lm long (Fig. 6D,E,O and P). Somatic kinety 2

(K2) longest, originating below oral cavity, spiralling anti-

clockwise (when viewed from oral aspect) around left pos-

terior region and terminating near posterior end of cell on

dorsal side (Fig. 6D,E and L); K1 shortest (about 6 lm

long), slightly curved, located in posterior half of

cell, below K2 (Fig. 6D and O); K3 and K4 10–12 lm

long, longitudinally oriented but slightly dextrally spi-

ralled, located in mid-region of cell (Fig. 6D,E,O and P).

About 20 external membranelles, four of which are

slightly elongated and extend into oral cavity (Fig. 6C and

N). Each membranelle probably 3-rowed with cilia about

10 lm long in vivo that are directed upwards with distal

ends bent outward slightly like a crown (Fig. 6A and G–I).

Bases of external membranes about 5 lm long. One inter-

nal membranelle lying on right of buccal cavity, close to

the longest external membranelle (Fig. 6C and N, arrow-

head). Endoral membrane extending across peristomial

field (Fig. 6C and M, arrowhead). Oral cavity rather shal-

low, located on anterior surface.

Morphogenesis

Stomatogenesis commences on the dorsal left side, above

K2 (Fig. 6F), and follows a similar pattern to that of

P. paraepacrum sp. n., that is, the oral primordium under-

goes a clockwise rotation forming a funnel and the distal

end of the oral primordium curves towards the proximal

end forming a closed circle (Fig. 6F). The endoral mem-

brane originates de novo and traverses the centre of the

new oral zone (Fig. 6F, arrow). The replication bands

migrate from both ends towards the mid-region of the

macronucleus, which gradually bulges to form a globular

mass (Fig. 6Q).


Pelagostrobilidium minutum sp. n. can clearly be separated

from its congeners by having: (i) a different arrangement of

somatic kineties 1, 3 and 4 with K3 and K4 slightly spiralled

(vs. longitudinally oriented) and K1 located below K2 (vs.



K1 to the right of K2); (ii) a smaller body size after protar-

gol impregnation (16–27 · 12–19 lm vs. larger than

30 · 30 lm); (iii) 4 (vs. >5) somatic kineties; and (iv) fewer

(19–21 vs. more than 29) external membranelles (Fig. 7N–

Z; Table 4B) (Lynn & Montagnes 1988; Montagnes &

Taylor 1994; Petz et al. 1995; Song & Bradbury 1998).

DiscussionEvolutionary relationships among strobilidiids

According to the evolution of ciliary patterns in choreo-

trichs proposed by Agatha & Struder-Kypke (2007), the

longitudinally oriented somatic kineties represent the ple-

siomorphic state. Of the three strobilidiid genera, only

Rimostrombidium has this feature (Fig. 8A,B); Strobilidium

and Pelagostrobilidium have spiralled or curved somatic kin-

eties which represent the apomorphic state (Fig. 8C–F).

However, previous phylogenetic analyses have consistently

shown that Rimostrombidium clusters with Strobilidium and

that Pelagostrobilidium branches basally, which appears to

disagree with the morphological analyses (Agatha &

Struder-Kypke 2007; Tsai et al. 2008; McManus & Katz

2009; Kim et al. 2010).

In our analyses, the SSrRNA gene sequence differ-

ences among the three newly sequenced species and

their congeners ranged from 5.09 to 8.93%. These find-

ings, along with the phylogenetic positions as revealed

in the SSrRNA gene trees, support the validity of these

three species. In addition, the two Rimostrombidium spe-

cies are located basally within the strobilidiid clade sug-

gesting that Rimostrombidium is representative of

ancestral forms in strobilidiids, which is consistent with

the morphological data. Moreover, the present study

reveals that R. veniliae has ciliated dikinetids, which is

recognized by Agatha & Struder-Kypke (2007) as more

ancestral than the ciliated monokinetids in other strobi-

lidiids. Although the two Rimostrombidium species do not

cluster together in our phylogenetic analyses, the possi-

bility that they cluster together was rejected by the AU

(P = 0.012) but supported by SH (P = 0.152) tests. The

monophyly of genus Rimostrombidium cannot be defini-

tively assessed at this point because too few species have

been sequenced.

Strobilidium is characterized by the spiralling of its cili-

ary rows in the posterior region of the cell (Fig. 8C,D). In

the SSrRNA gene trees, S. caudatum branches from the

strobilidiid clade after Rimostrombidium spp., whose ciliary

rows do not spiral posteriorly thus supporting the hypoth-

esis that the caudal spiral of somatic kineties represents

the apomorphic state (Agatha & Struder-Kypke 2007).

Although nodal support for the S. caudatum branch is low

in ML (33%) and MP (42%) trees and S. caudatum groups

with R. lacustris in the BI tree (data not shown) with low


support (0.66), the somatic kineties of R. lacustris are

slightly obliquely oriented (Foissner et al. 1988), which

indicates that it may represent an intermediate stage

between these two genera.

Unlike Strobilidium and Rimostrombidium, Pelagostrobilidi-

um has a curved K2 on the left side of the body

(Fig. 8E,F). The validity of the genus Pelagostrobilidium is

also supported by the SSrRNA gene analyses as the three

sequenced Pelagostrobilidium species form a clade in our

trees. Furthermore, the Pelagostrobilidium group branches

separately from the strobilidiid clade which is consistent

with the view that the curved K2 is a derived character, as

suggested by Agatha & Struder-Kypke (2007). In addition,

different curvatures of K2 seem to suggest different evolu-

tionary relationships as follows: (i) the transversely arched

K2 of P. neptuni, P. simile and P. spirale represents the

derived state in Pelagostrobilidium species; (ii) P. wilberti,

with its short and slightly curved K2, occupies an interme-

diate position between Rimostrombidium and Pelagostrobili-

dium, which was also suggested by Kuppers et al. (2006);

(iii) P. epacrum, P. paraepacrum sp. n. and P. minutum sp.

n. each has a spiralled K2 that extends to the posterior

end of the cell and thus probably evolved after P. wilberti

but before P. neptuni. However, this proposed evolutionary

sequence needs to be confirmed by further molecular phy-

logenetic analyses with gene sequence data for additional

Pelagostobilidium species.

Circumscription and definition of the genera

Rimostrombidium and Pelagostrobilidium

The patterns of the somatic ciliature in Pelagostrobilidium,

Rimostrombidium and Strobilidium can be used to separate

these genera from each other. Furthermore, their evolu-

tionary relationships based on their somatic ciliature are

largely supported by the SSrRNA gene sequence data.

These findings therefore indicate that the somatic ciliary

pattern is an important generic character within the family

Strobilidiidae as previously suggested (Petz & Foissner

1992; Petz et al. 1995; Agatha et al. 2005).

The genus Rimostrombidium. The original description of Ri-

mostrombidium was very brief, the main defining character

being ‘with ribbed cortex’ (Jankowski 1978). Petz & Foiss-

ner (1992) redefined the genus Strobilidium based on the

characteristic spiralling of the somatic ciliary rows in the

caudal region of the cell and transferred those strobilidiids

lacking such a caudal spiral into Rimostrombidium.

Although the ribbed cortex is not commonly found in

these species, no revised diagnosis of Rimostrombidium has

previously been proposed. Nevertheless, it is noteworthy

that the longitudinally oriented somatic kineties, which do

not extend to the posterior end of the cell, are commonly

41, 4, July 2012, pp 417–434 431


used to identify species within this genus (Petz & Foissner

1992; Agatha & Riedel-Lorje 1998; Song & Bradbury

1998; Foissner et al. 1999; Lei et al. 1999). An improved

diagnosis of Rimostrombidium is therefore here suggested.

Improved diagnosis of Rimostrombidium. Strobilidiidae with

longitudinal or slightly obliquely oriented somatic kineties

not extend to posterior pole. Oral primordium develops

left laterally or dorsally.

The genus Pelagostrobilidium. Petz et al. (1995) established

the genus Pelagostrobilidium based on its transversely

arched somatic kineties. Noting the weak curvature of kin-

ety 2, which is not transversely arched but instead spirals

along the left side of the cell in P. epacrum, Agatha et al.

(2005) improved the diagnosis of the genus Pelagostrobilidi-

um emphasizing the locations of certain kineties relative to

one another, for example, ‘kineties 3 and 4 posteriorly

shortened and abutting on curved kinety 2’, and included

some morphogenetic characters, for example, ‘oral primor-

dium develops left laterally anterior to kinety 2 and right

of kinety 3’. However, following the description of P. wil-

berti, Kuppers et al. (2006) further emended the diagnosis

of Pelagostrobilidium by supplying some additional charac-

ters such as ‘when kinety 2 is posteriorly shortened, kin-

eties 3 and 4 extend close to kinety 2; oral primordium

develops left laterally or dorsally anterior to kinety 2 and

left of kinety 3’. The locations of the somatic kineties

were, however, so strictly defined that some Pelagostrobili-

dium-like species cannot be assigned to this genus. Pelago-

strobilidium minutum sp. n., for example, is similar to other

Pelagostrobilidium spp. with K2 spiralling around the left

side of the body and the oral primordium located between

K2 and K3. Furthermore in the SSrRNA gene tree, P. min-

utum sp. n. is nested within the Pelagostrobilidium clade.

Nevertheless, its K3 and K4 are widely separated from K2

thus excluding it from Pelagostrobilidium according to the

genus diagnosis revised by Kuppers et al. (2006). This sug-

gests that the spiralled K2 should be regarded as a generic

character, whereas the locations of K3 and K4 should

probably be species level characters. Therefore, an

improved diagnosis of Pelagostrobilidium is suggested:

Improved diagnosis of Pelagostrobilidium. Strobilidiidae with

somatic kinety 2 spiralled around the left side of body. No

somatic kineties forming a spiral at posterior pole. Oral

primordium develops between kineties 2 and 3.

Remarks. Among the characters used for separating species

of Pelagostrobilidium are the locations of K3 and K4 and

the curvature of K2. Agatha & Riedel-Lorje (1998) rede-

scribed the poorly known species Rimostrombidium conicum


in which the curvature of K2 is less prominent, suggesting

that it represents an intermediate stage between Rimo-

strombidium and Pelagostrobilidium. Furthermore, in R. coni-

cum, the oral primordium develops between K2 and K3.

Therefore, this species is here transferred to the genus Pel-

agostrobilidium as P. conicum (Kahl, 1932) nov. comb [basi-

onym Rimostrombidium conicum].

Morphogenesis in choreotrichs

Morphogenesis has been documented for only four cho-

reotrich ciliates. Deroux (1974) reported the morphoge-

netic events of Strobilidium gyrans and pointed out that the

morphogenesis of the new peristome was confined to the

interior of cell by an invaginated cortical fold. Dale &

Lynn (1998) reported stomatogenesis in Strombidinopsis spi-

niferum and compared the stomatogenetic processes in

choreotrichs with those in oligotrichs. The morphogenesis

of Pelagostrobilidium neptuni was described in detail by

Agatha et al. (2005) who proposed that the position of the

new peristome in strobilidiids may depend on the shape

and length of kinety 2. Kuppers et al. (2006) described

morphogenesis in P. wilberti and proposed that the short-

ening of K3 and K4 abutting on the elongated K2 could

be a derived character in the genus Pelagostrobilidium.

In the present study, we describe for the first time sto-

matogenesis in Rimostrombidium. Compared with other

genera, stomatogenesis in Rimostrombidium most closely

resembles that in Strombidinopsis. In both cases, the new

adoral zone undergoes an anticlockwise rotation prior to

the differentiation of the polykinetids. Moreover, an oral

primordium tube is formed when the distal and proximal

portions of the adoral zone meet each other (Dale & Lynn

1998). By contrast, in Pelagostrobilidium and Strobilidium,

the anticlockwise rotation of the oral primordium takes

place concurrently with the differentiation of the polyki-

netids. In addition, an oral primordium funnel, with a

wide external portion and a narrow inner portion, is

formed rather than a tube (Deroux 1974; Petz & Foissner

1992; Agatha et al. 2005).

Considering the location of the oral primordium (OP) in

strobilidiids, in P. paraepacrum sp. n., P. minutum sp. n. and

P. wilberti, the OP develops between K2 and K3 (Kuppers

et al. 2006). This is consistent with the finding by Agatha

et al. (2005) that the location of the OP in strobilidiids is

dependent on the shape and length of K2. In other strobili-

diid species, however, the situation is different and ⁄ or vari-

able. In R. veniliae and R. brachykinetum, the OP develops

on the left-dorsal side anterior of K4 and K5 (Krainer

1995); in S. caudatum, the OP develops dorsally (Petz &

Foissner 1992); in R. orientale, the OP develops ventrally

(Song & Bradbury 1998); and in S. gyrans, the OP develops

between K1 and K2 (Deroux 1974). Therefore, the relative



positions of the OP and K2 appear to be related only in Pel-

agostrobilidium species, whereas in other strobilidiid genera,

they appear to be variable or species specific.

Classification of Lynnella

In our trees, Lynnella clusters with the choreotrichs which

together form a sister group to the oligotrichs. This result

does not concur with the previous analyses in which Lyn-

nella clusters with the oligotrichs rather than the choreo-

trichs (Liu et al. 2011). Furthermore, the possibility of a

relationship with the subclass Oligotrichia was not rejected

by AU (P = 0.088) and SH (P = 0.365) tests. However, the

support values for the Lynnella + Choreotrichia clade are

relatively weak (58% ML, 69% MP and 0.61 BI), which

indicates that the classification of Lynnella remains ambig-

uous. Considering the common characters shared with

both choreotrichs and oligotrichs, respectively, Lynnella

could be an intermediate form between these two and may

represent a new order and a new subclass of Spirotrichea.

More data are needed to confirm this possibility.

AcknowledgementsThis work was supported by the Natural Science Founda-

tion of China (project numbers: 41006098; 31071893).

Thanks are due to Mr. William Keel (Smithsonian Institu-

tion, National Museum of Natural History, USA) for

supplying the type material of Rimostrombidium veniliae

(Montagnes & Taylor 1994) Petz et al. 1995.

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