Taxonomic description of two new marine oligotrichous ciliates (Protozoa, Ciliophora)

The morphology and ciliary pattern of a new marine oligotrichous ciliate, Strombidium wilberti nov. spec., collected from the littoral area off Qingdao, North China, are described from live observations and after protargol impregnation. This new species is characterized by: about 45–60×35–50 µm in vivo with truncated obconical body shape and conspicuous distended cell surface which covers the posterior half of the cell; buccal cavity extending posteriorly to about one‐third of body length; about eight buccal and 15 collar membranelles; girdle kinety completely closed, consisting of ca 60 dikinetids; ventral kinety conspicuously long with about 30 densely packed dikinetids, which extends anteriorly to girdle area; extrusomes about 20 µm long, grouped in rows and arranged anterior to girdle kinety; single globular macronucleus. Related nominal congeners are compared and discussed. The Antarctic morphotype described by Petz et al. (1995) as Strombidium emergens (Leegaard, 1915) Kahl, 1932 is not conspecific with the original form and is hence recognized as a new member of this genus, Strombidium petzi nov. spec. The diagnosis for this new organism: large marine Strombidium ca 95–110×60–65 µm in vivo with elongate elliptical body shape and conspicuously shallow oral cavity. Girdle kinety continuous on ventral side with ca 100 dikinetids; ventral kinety extending from posterior area almost to girdle kinety, consisting of more than 20 dikinetids; four to five buccal and 14–15 collar membranelles. Macronucleus oval in shape; one subequatorial girdle of extrusomes.


Introduction
Most naked or aloricate oligotrichs such as strombidiids are highly motile ciliates and often dominant in microzooplankton communities in both marine and freshwater habitats (Dragesco 1960;Borror 1972;Fenchel and Jonsson 1988;Krainer 1991). Since many of these species are minute and fragile, they have often been investigated rather superficially, based largely or entirely on preserved or live material (Fauré-Fremiet 1924;Kahl 1932;Maeda and Carey 1985). For this reason, numerous ambiguities concerning the identification of taxa have accumulated over the last century and hence, most nominal species need to be redefined or re-investigated using not only live observations but also impregnation methods. This is especially true for the marine forms, which were relatively poorly considered as revealed in some recent studies ; Montagnes et al. 1990;Martin and Montagnes 1993;Montagnes and Taylor 1994;Petz et al. 1995;Agatha and Riedel-Lorjé 1997).
Faunistic research on plankton ciliates from coastal waters in north China seas has been carried out in recent years and various little-known or new oligotrichs have been isolated and studied (Song and Packroff 1997;Song and Bradbury 1998;Lei et al. 1999;Song et al. 2000). As a new contribution to our knowledge of these organisms, a further unknown species in the genus Strombidium is identified and described in the present paper.

Materials and methods
Sampling sites and cell culture. Samples were collected in December 1993 from several sites used for mollusc-farming in coastal regions of the Yellow Sea near Qingdao, China. Cells were cultured at room temperature in Petri dishes with the water in which they were isolated. These cultures were usually maintained for several days and then used for morphological studies.
Observations and staining. Specimens were observed in vivo with phase contrast and differential interference contrast microscopy. The infraciliature was revealed by protargol impregnation (Wilbert 1975). Counts, drawings (with help of a camera lucida) and measurements were performed at a magnification of 61250. Terminology is mainly according to Corliss (1979) and .
(Figures 1, 2; Table I) Diagnosis. Marine Strombidium, about 45-60635-50 mm in vivo with truncated obconical body shape and conspicuous distended cell surface which covers the posterior half of the cell; buccal cavity extending posteriorly to about one-third of body length; about eight buccal and 15 collar membranelles; girdle kinety completely closed, consisting of ca 60 dikinetids; ventral kinety prominently long with about 30 densely packed dikinetids, extends anteriorly to girdle area; extrusomes about 20 mm long, grouped in rows and arranged anterior to girdle kinety; single globular macronucleus.
Ecological characteristics for sampling sites. Sandy coastal area with clean water, salinity was 33, pH about 8.1, water temperature ranged from 5 to 9uC.
Type deposition. One permanent slide as holotype with protargol-impregnated specimens is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao.
Dedication. I dedicate this species to Prof. Dr Norbert Wilbert (Institute of Zoology, Bonn University, Germany), a well-respected and great German protozoologist, who has made significant contributions to ciliate taxonomy and ecology during the last 30 years.
Description. Size mostly about 50640 mm in vivo. Body shape generally invariable, broadly obconical and circular in cross-section, widest at anterior third of cell length ( Figure 1A), while in some individuals body might be slightly asymmetric when viewed dorso-ventrally ( Figure 1E). Apical protrusion or collar inconspicuous in vivo, usually undetectable after fixation ( Figure 1F, H). Buccal cavity relatively shallow and inconspicuous, extending posteriorly to about 25-30% of cell length ( Figure 1A, E). Ventral groove conspicuous and extending from girdle region to posterior cell end, containing long ventral kinety ( Figure 1F). Cilia in collar membranelles about 25-30 mm long, stretching anteriorly when swimming, cilia in buccal membranelles ca 10 mm in length ( Figure 1A). Perilemma remarkable, always as conspicuously distended 'lorica-like' structure covering the posterior half of live cell ( Figure 1A, E), but only about 40% after protargol impregnation where the anterior cell portion is swollen ( Figure 1F). No cortical platelets recognizable either in vivo or in silvered specimens. Extrusome girdle conspicuous, extrusomes about 20 mm long, bar-like and regularly grouped (each group with about six extrusomes), which are closely spaced anterior to the girdle kinety ( Figure 2C). Outer ends of extrusomes arranged in oblique rows (anchored obliquely beneath pellicle), which make cell surface granulated and slightly punctated ( Figure 1B).
Cytoplasm basically colourless to slightly greyish, containing numerous light-reflecting, lipid-like granules (ca 2-5 mm in diameter) and food vacuoles containing diatoms, flagellates etc., which often render cells rather opaque or even completely dark when observed at lower magnifications ( Figure 1E). Macronucleus about 15-20 mm across, spherical in shape and centrally located, usually contains many large roundish nucleoli as demonstrated after impregnation ( Figure 1F); no micronucleus recognized.
Locomotion very fast and changing direction frequently, never stops swimming ( Figure  1C). This organism seems fragile for it readily bursts especially when a cover glass is used for observing under the microscope.

Comparison with related species
As in many other oligotrichs, species identification within the genus Strombidium is problematic. This is largely because these small organisms share many characters among congeners with respect to their infraciliature and living features. This problem is usually exacerbated as many descriptions are based only on live observations or preserved material, in which many characters are not recognizable.
To date, approximately 15 Strombidium species with similar cell shape and size inhabiting marine biotopes have been reported, of which about 12 have been described using silverimpregnation methods Montagnes et al. 1988Montagnes et al. , 1990Montagnes and Lynn 1991;Lynn and Gilron 1993;Martin and Montagnes 1993;Petz et al. 1995;Lei et al. 1996;Song and Packroff 1997;Song et al. 2000) (Table I).
As accepted by most taxonomists today (Krainer 1991;Petz et al. 1995;Agatha and Riedel-Lorjé 1997;Lei et al. 1999;Song et al. 2000), the following criteria, which are observed either in vivo or from silvered specimens, are important in species separation in the genus Strombidium: (1) the buccal and somatic ciliature, especially the length and position of the ventral kinety (e.g. fragment-like near the caudal pole or as a long row positioned ventrally/ laterally) and the appearance of membranelles in buccal and collar zones (clearly separated or in a continuous structure, differences in length of bases etc.); (2) biometric data of ciliature (the number of kinetosomes in girdle and ventral kineties, membranelles in collar and buccal zones etc.); (3) length, distribution and organization (i.e. grouped or sparsely distributed) of extrusomes; (4) appearance of the cell surface (presence/absence of polygonal structure etc.); (5) length of cilia and bases of membranelles, the orientation or beating characteristics of membranelles in vivo; (6) habitat (marine or freshwater) and swimming behaviour (highly species-specific!); (7) body shape, size, length of the buccal cavity, prominence of the anterior protrusion or collar etc. (though the significance of these characters was/is often over-evaluated, in my opinion); (8) characteristics of nuclear apparatus (shape and size of macronucleus, number of micronuclei etc.) and cytoplasmic inclusions (symbionts etc.).
Compared with the well-known Strombidium sulcatum, the new species, S. wilberti, has a different pattern of ventral kinety: conspicuously long and extending across the whole postequatorial ventral portion (versus fragment-like and very short in S. sulcatum) with higher number of basal body pairs (30 versus ,10). In addition, S. wilberti is relatively large (45-60 versus about 40 mm in S. sulcatum) and has a more broadly and truncate conical body shape in life ( Figure 3H) (Fenchel and Jonsson 1988;Montagnes et al. 1990;Lei et al. 1996;Song et al. 2000).
Another similar morphotype, Strombidium inclinatum Montagnes et al., 1990, can also be easily separated from the new species by the characters of somatic ciliature mentioned above and its shorter body length (32-46 mm in vivo) ( Figure 3F; Table I) (Montagnes et al. 1990;Modeo et al. 2003).
Considering the ciliary pattern, Strombidium basimorphum Martin and Montagnes, 1993 is also similar to S. wilberti. The former, however, can be identified by: (1) the 'compressed' conical body shape (usually width.length) and (2) the longer bases of collar membranelles, i.e. about twice as long as those of the buccal membranelles, as shown in Figure 7G of the original report (Martin and Montagnes 1993) (Figure 3B; Table I). With reference to the body size, shape and general appearance from life, Strombidium crassulum (Leegaard, 1915) Kahl, 1932 should be most similar to the new organism. The infraciliature of S. crassulum was described by Petz et al. (1995) based on an Antarctic strain ( Figure 3O, P). According to this redescription, S. crassulum can be separated from the new species by its highly developed buccal membranelles (13-19 versus eight in S. wilberti) which extends to the equatorial area, and the structure of the somatic ciliature (9-15 versus about 30 dikinetids in ventral kinety and ca 115 versus ca 60 dikinetids in girdle kinety, respectively) ( Table I).
Strombidium compressum (Leegaard, 1915) Kahl, 1932 also has a similar body shape ( Figure 3G) to S. wilberti, but according to the redescription by , S. compressum differs in its short ventral kinety which is positioned in the posterior third of the cell with only several dikinetids and is considerably shorter (17-28 mm after protargol impregnation) ( Table I).
Compared to Strombidium dalum and S. epidemum, both found/described from the Isles of Shoals by , the new species described here is much larger (over 50 versus ,20 mm after impregnation) and has a conspicuously longer ventral kinety which extends from girdle kinety to posterior end of the cell (versus not observed, possibly reduced) ; Figure 3C, E; Table I).
With reference to the general infraciliature, Strombidium tressum Lynn et al., 1988 also seems to be related to S. wilberti. However, the former can be distinguished by the extremely long cilia in its collar membranelles (over 40 mm, about twice as long as body size after protargol impregnation), reduced buccal membranelles (only 'a few' in number in small buccal groove) and the conspicuous perilemma, which covers almost three-quarters of body length ( Figure 3L). In addition, S. tressum is significantly smaller (21-29 mm long after fixation) ( Table I).
Strombidium rhynchum  differs from S. wilberti in its slender body shape, highly developed perilemma which covers almost four-fifths of posterior body and reduced buccal zone of membranelles (very short and located in the inconspicuous buccal cavity) ( Figure 3K; Table I).
According to the original report and the redescription by , Strombidium acutum Leegaard, 1915 could be similar to the new species as well, but can, however, be distinguished by: (1) the highly reduced ventral kinety, which was undetectable (possibly extremely inconspicuous) as mentioned in the redescriptions, and (2) having very shallow buccal cavity ). In addition,  defined this organism possessing 10-22 buccal membranelles, which is an unusually wide range of variation ( Figure 3A; Table I).
Strombidium emergens (Leegaard, 1915) Kahl, 1932 was redescribed by  under the name Strombidium sulcatum, which was later determined as S. emergens by Montagnes et al. (1990). It differs from Strombidium wilberti in its slender body shape in vivo, smaller size (28-42 versus 44-53 mm in length after protargol impregnation) and the presence of the conspicuously shorter ventral kinety (extending anteriorly to about a quarter of cell length) ( Figure 3J; Table I).
Lynn and Gilron (1993) described a Jamaican population under the name of Strombidium inclinatum Montagnes et al., 1990, which is possibly a misidentification because it has a long ventral kinety extending anteriorly almost to the equatorial region. Since no statistical data are available about the number of kinetosomes in either girdle or ventral kineties, it is difficult to compare it with the species described here. There remains the possibility that this organism is conspecific with S. wilberti.

Establishment of a new species
Strombidium petzi nov. spec. Synonym. Strombidium emergens sensu Petz, Song andWilbert, 1995. Petz et al. (1995) described a large form from sea ice of the Weddell Sea, Antarctica as Strombidium emergens (Leegaard 1915), which was characterized by its extremely large size (in vivo 95-110660-65 mm, after protargol impregnation 90649-52 mm versus 30-40 mm in preserved material in original report) and 'reduced' buccal membranelles (only a few very short membranelles in shallow oral cavity) ( Figure 3N). Considering the combination of body size, shape and pattern of infraciliature, the Antarctic organism is clearly different from all known congeners described hitherto. Hence, it is reasonable to recognize it as a new species, Strombidium petzi nov. spec.

Diagnosis.
Large marine Strombidium about 95-110660-65 mm in vivo with elongate elliptical body shape and conspicuously shallow oral cavity. Girdle kinety continuous on ventral side with ca 100 dikinetids; ventral kinety extending from posterior area almost to girdle kinety, consisting of more than 20 dikinetids; fout to five buccal and 14-15 collar membranelles. Macronucleus oval in shape; one subequatorial girdle of extrusomes.
Type deposition. One slide as holotype with protargol-impregnated specimens is deposited in the collection of microscope slides of the Oberö sterreichische Landesmuseum, A-4040 Linz, Austria.
Dedication. This species is dedicated to Dr Wolfgang Petz (Institute of Zoology, University of Salzburg, Austria), to express my respects to his great contributions in various fields of protozoological research.