Morphology and infraciliature of two new species of marine oligotrich ciliates (Ciliophora: Oligotrichida) from China

Two new marine oligotrich ciliates, Parallelostrombidium paralatum nov. sp. and Strombidium montagnesi nov. sp., were isolated from the littoral area of Qingdao (Tsingtao), China. The morphology and infraciliature of each was studied using live observation and protargol impregnation. Based on the Qingdao population, the diagnosis for P. paralatum is given: marine Parallelostrombidium, in vivo about 70×60 µm; dorsoventrally flattened ca 2:3; cell ellipsoidal in outline with conspicuous apical protrusion; on average 27 anterior and 17 ventral membranelles; two posteriorly directed thigmotactic membranelles; macronucleus broadly ellipsoidal; extrusomes ca 10 µm long, arranged along equatorial area; girdle and ventral kineties with 71–99 and 32–42 dikinetids, respectively. Strombidium montagnesi nov. sp. is characterized thus: small marine Strombidium ∼30×20 µm in vivo with truncated conical cell shape and conspicuous equatorial ridge; hemitheca covering the posterior one‐quarter to one‐third of the cell; dorsoventrally flattened ca 2:3; about 21 anterior membranelles and six ventral membranelles; girdle kinety located in posterior one‐quarter to one‐third of the cell and has 25–27 dikinetids, while ventral kinety has 6–12 dikinetids; extrusomes ca 5 µm long, arranged along girdle kinety; single ellipsoidal macronucleus centrally positioned.


Introduction
Oligotrichs play an important role in marine planktonic microbial food webs because they serve as an important link between smaller unicellular organisms and those of higher trophic levels (Pierce and Turner 1992). Since the beginning of the 19th century, about 200 aloricate oligotrichs have been reported. Unfortunately, many descriptions were based only on live specimens or on material fixed using classical methods (Busch 1921(Busch , 1930Fauré-Fremiet 1924;Kahl 1932), leaving doubt about the validity of these species. Thus, modern techniques, in which cells are investigated in life as well as following silver impregnation, are regarded as essential for adequate species description (Song and Bradbury 1998;Song et al. 2000;Agatha 2003aAgatha , 2003bAgatha , 2004aAgatha , 2004bModeo et al. 2003;Agatha et al. 2004;Xu et al. 2005;Xu and Song 2006).
The genus Parallelostrombidium was recently established by Agatha (2004a), and is characterized thus: ''ventral kinety follows posterior portion of dextrally spiraled girdle kinety; thus, both with same orientation''. Only two species are included in this genus: P. rhyticollare (Corliss and Snyder, 1986) Agatha, 2004 andP. siculum (Montagnes andTaylor, 1994) Agatha, 2004. During surveys of the ciliate fauna in the costal regions near Qingdao, China, two oligotrich ciliates were collected. After comparison with known congeners, they are believed to be new species of Parallelostrombidium and Strombidium, respectively: Parallelostrombidium paralatum nov. sp. and Strombidium montagnesi nov. sp.

Sample collection
Parallelostrombidium paralatum was collected from shrimp-culturing waters in Jiaozhou Bay off Qingdao (Tsingtao, 36u089N, 120u439E), China on 21 April 2004, the salinity of which was ,30psu, the pH ,7.8 and the temperature ,16uC. Glass slides fixed in a slide frame served as artificial substrates and were immersed in the water until biofilm was formed (,15 days). The slides were retrieved and transferred to Petri dishes with marine water from the sampling site.
Strombidium montagnesi was collected by 20 mm mesh plankton nets on 13 April 2004 from coastal waters near Qingdao. The salinity was ,33psu, pH ,7.0 and the water temperature ,16uC.

Morphological investigations
The behaviour of the organisms was studied in the Petri dishes under a dissecting microscope. The morphology was investigated under a compound microscope equipped with a high-power oil immersion objective as well as differential interference contrast optics. The infraciliature was revealed by protargol impregnation (Wilbert 1975). Counts, drawings (with the help of a camera lucida) and measurements were performed at a magnification of 12506.
Terminology is mainly according to Agatha et al. (2005).

Etymology
The specific epithet refers to the superficial similarity in body shape between this species and Strombidium latum.

Type location
Shrimp-culturing waters in Jiaozhou Bay off Qingdao (Tsingtao, 36u089N, 120u439E), China.  Cell extremely fragile, highly sensitive to presence of cover-slip and easily bursts. Pellicle delicate with thin and transparent hemitheca that covers posterior half of cell ( Figure 1A), but no polygonal cortical platelets recognizable either in vivo or in silvered specimens. Cytoplasm colourless to greyish, containing many ingested algae (including diatoms) which often render cells opaque or dark when observed at lower magnifications ( Figure 2A). Extrusomes prominent and acicular-shaped, ca 10 mm long, evenly arranged in a single row at about the level of the hemitheca margin, but not in bundles ( Figures 1A, 2E). Neither contractile vacuole nor cytopyge were detected. Single macronucleus broadly ellipsoidal in shape and centrally located, containing several large nucleoli each ,5 mm across ( Figures 1I, 2H); no micronucleus detected.
Locomotion with two patterns: moderately fast and irregular when swimming ( Figure 1E), or very fast when crawling on debris, using its two thigmotactic membranelles for attachment with ventral side facing down ( Figure 1F).
Somatic ciliature as shown in Figures 1 and 2, consisting of one girdle kinety and one ventral kinety. Girdle kinety originates in mid-ventral region (to the left of the ventral Oral apparatus occupies anterior end of cell, consisting of a short endoral membrane on inner wall of buccal lip and a membranellar zone ( Figures 1H, I, 2I). Adoral zone of membranelles bipartite with an anterior portion of about 28 (26-30) membranelles and a ventral portion of about 17 (15-19) membranelles, all of which are composed of three kinety rows ( Figure 1D). Cilia of most anterior membranelles ca 20 mm in length, stretching anteriorly when swimming ( Figure 1A). Bases of anterior membranelles about 11-12 mm long. Anterior membranelles distinctly separated from ventral ones by the two thigmotactic membranelles, the bases of which are about 15 mm long ( Figures 1H, 2I, arrowheads). Cilia of two thigmotactic membranelles about 30-35 mm long and always directed posteriorly like two tails ( Figure 1A, B, arrowheads). Bases of ventral membranelles about 7-8 mm in length, distinctly shorter than those of the thigmotactic membranelles. Endoral membrane extending to centre of protrusion, probably composed of monokinetids ( Figure 1H). Pharyngeal fibres not detected.

Stomatogenesis
Several stages in division were found which permit the reconstruction of the main stomatogenetic processes. Stomatogenesis commences with the apokinetal development of cuneate, longitudinally orientated basal bodies in a shallow depression underneath the ventral membranelles and to the left of the anterior end of the girdle kinety ( Figure 2C, arrows; J, arrowhead). While the oral primordium elongates posteriorly, membranelles differentiate from anterior to posterior ( Figure 2F, arrowhead) and the endoral membrane originates de novo. At the same time, new basal bodies are generated by intrakinetal proliferation (Figure 1J, arrows). Simultaneously, the membranelles become ciliated. When the final number of membranelles is formed, the oral primordium moves to the left ventral side of cell. The oral primordium positions to the left of the anterior end of the girdle and ventral kinety and above the left portion of the girdle kinety ( Figure 1J).
Fauré-Fremiet (1950) described two forms under the name Strombidium sauerbreyae, despite the fact that their morphology is quite different from that of the original (Sauerbrey 1928). Considering the general morphology (namely cell size and shape, presence of the two thigmotactic membranelles, locomotion pattern etc.), S. sauerbreyae sensu Fauré-Fremiet, 1950 bears a strong resemblance to Parallelostrombidium paralatum, but it can be differentiated from the latter by (1) arrangement of extrusomes (sparsely arranged in the ventral side versus evenly arranged at about the level of hemitheca margin), and (2) much lower number of anterior membranelles (ca 17 versus 26-30) and ventral membranelles (ca 15 versus 15-19) (Fauré-Fremiet 1950).
Strombidium latum sensu Fauré-Fremiet, 1950 also has thigmotactic membranelles. However, it can be separated from Parallelostrombidium paralatum by its much larger cell size (110-170 mm versus 55-80 mm), different distribution of extrusomes (surrounding the cell versus arranging along the equatorial area) and much larger oral cavity (about twothirds of cell length versus about one-third of cell length) (Fauré-Fremiet 1950).
Strombidium latum sensu Kahl, 1932 has two to three thigmotactic membranelles, which should also be compared with Parallelostrombidium paralatum. The former can be separated from the latter by its much larger cell size (100-140 mm versus 55-80 mm), and different arrangement of extrusomes (surrounding the cell margin versus arranging along the equatorial area) (Kahl 1932).
Strombidium fourneleti Dragesco, 1960 is similar in size to Parallelostrombidium paralatum and also has two thigmotactic membranelles. However, it can be distinguished from the latter by the cell shape (globular versus ellipsoidal), the fine adoral membranelles (versus prominent and well-developed), the presence of polygonal cortical platelets (versus absent), the sparsely distributed extrusomes (versus densely arranged), and the total number of anterior and ventral membranelles (,24 versus 41-49) (Dragesco 1960).
Considering the size and presence of two thigmotactic membranelles, Strombidium faurei Dragesco, 1960 should also be compared with Parallelostrombidium paralatum. The former differs from the latter in terms of cell shape (ovoid versus ellipsoidal and dorsoventrally flattened), total number of anterior and ventral membranelles (,27 versus 41-49) and the arrangement of extrusomes (sparsely distributed on the somatic area versus densely arranged along the equatorial area) (Dragesco 1960).

Ontogenetic comparison
Only early dividers were found in Parallelostrombidium paralatum, so comparisons between Parallelostrombidium and its congeners were based on stomatogenesis information.
The position of the oral primordium of Parallelostrombidium is very similar to that of Novistrombidium, i.e. oral primordium originates above the left portion of the girdle kinety (Agatha 2003a).
Parallelostrombidium differs from Strombidium in the location of the oral primordium (anterior versus posterior portion of the girdle kinety) (Song and Wang 1996;Agatha 2003a).
Similar to Laboea and Spirotontonia, the parental oral ciliature of Parallelostrombidium does not reveal any signs of reorganization. However, the position of the oral primordium of the latter is different from that of the former (oral primordium originates anterior to the left ventral portion of the girdle kinety versus oral primordium develops posterior to the left portion of the girdle kinety) (Agatha et al. 2004).

Diagnosis
Small marine Strombidium ,30620 mm in vivo with truncated conical cell shape and conspicuous equatorial ridge; hemitheca covering the posterior one-quarter to one-third of  the cell; dorsoventrally flattened ca 2:3; about 21 anterior membranelles and six ventral membranelles; girdle kinety located in posterior one-quarter to one-third of the cell and has 25-27 dikinetids, while ventral kinety has 6-12 dikinetids; extrusomes ca 5 mm long, arranged along girdle kinety; single ellipsoidal macronucleus centrally positioned.

Dedication
We dedicate this new species to Dr David J. S. Montagnes, University of Liverpool, UK, for his great contribution to the taxonomy and ecology of planktonic ciliates.

Slide deposition
One holotype slide of protargol-impregnated specimens is deposited in the Natural History Museum  Figure 4C, arrows). Contractile vacuole not observed. Single macronucleus ,10 mm in diameter, round in shape and centrally located, contains some small nucleoli ,1 mm across and several larger nucleoli ,3 mm across ( Figures 3G, H, 4G). Micronucleus not observed.
Cell swims slowly and continuously with smooth turns, while rotating about main cell axis. When under cover-slip, cilia of anterior membranelles sometimes densely pack together forming a flame shape when cell rests ( Figure 4E).
Oral apparatus typical of genus, occupies anterior end of cell. Adoral zone of membranelles surrounds apical protrusion, divided into an anterior portion with 21-23 membranelles and a ventral portion with six to seven membranelles ( Figure 3G, H). Anterior membranelles continuous with ventral ones, each composed of three equally long basal body rows. Ventral membranelles invaginate ventrally to the right side of the cell. Cilia of anterior membranelles ,15 mm in length, projecting anteriorly when swimming. Endoral membrane on inner wall of buccal lip on right side of oral cavity, rarely recognizable in protargol-impregnated specimens, probably composed of monokinetids. Pharyngeal fibres not observed.

Stomatogenesis
In our specimens, the oral primordium originates on the left ventral side immediately posterior to the ventral membranelles. The very early stage of stomatogenesis shows only a few disordered basal bodies, which do not appear to be formed in contact with parental structure, i.e. the oral primordium develops apokinetally (Figures 3E,arrow,4F,arrowhead). The basal bodies then align almost instantly into membranelles and move deeper into the cell while the oral primordium becomes orientated longitudinally and curves. The oral primordium is always positioned anterior to the girdle kinety until its differentiation into the adoral membranelles is complete (Figures 3I, 4I).

Comparison with similar species
With respect to its small size, dorsoventrally flattened body shape (width: thickness 3:2-3:1) and the sub-equatorially located girdle kinety (positioned in posterior one-quarter to one-third of the cell), Strombidium montagnesi can be easily separated from most other congeners. Comparison should be made with two morphologically similar species.
Strombidium coronatum can be separated from S. montagnesi by its cell shape (posterior end sharply pointed versus posterior end usually broadly rounded but truncated in some individuals) and much larger cell size (ca 100 mm versus 25-30 mm) (Leegaard 1915).

Ontogenetic comparison
The process of stomatogenesis differs from that in the improved diagnosis of the genus Strombidium supplied by Agatha (2004b): ''Girdle kinety horizontal. Ventral kinety longitudinal, occasionally reduced or lacking. Oral primordium develops at or below level of girdle kinety''. These differences might indicate that the ontogenetic patterns in Strombidium are more diverse than hitherto realized. Gene sequence analysis has revealed a paraphyly of Strombidium and a considerably larger genetic variation among oligotrichs than among stichotrichs (Snoeyenbos-West et al. 2002;Modeo et al. 2003). Further investigations of strombidiid morphology and ontogenesis may well lead to a split of the genus Strombidium reflecting the situation in the phylogenetic trees inferred from gene sequence analyses (Agatha et al. 2005).