Redescription of Pseudovorticella cylindrica (Dons, 1915) nov. comb. and Zoothamnium hiketes Precht, 1935, two poorly defined marine peritrichs (Ciliophora: Peritrichia) from the north China Sea

The living morphology, silverline system and infraciliature of two marine peritrichous ciliates, Pseudovorticella cylindrica (Dons, 1915) nov. comb. (formerly Vorticella cylindrica) and Zoothamnium hiketes Precht, 1935, collected in Qingdao, China, were investigated. Based on the Qingdao populations, both species are redefined. Pseudovorticella cylindrica is characterized by: cell inverted bell‐shaped, about 45×50 µm in vivo; macronucleus J‐shaped; one apically positioned contractile vacuole; pellicle smooth with inconspicuous, closely spaced striations; the number of transverse silverlines from peristome to aboral ciliary wreath 33–40, from aboral ciliary wreath to scopula 14–19; the inner row of peniculus 3 conspicuously short. An updated and supplementary description of Zoothamnium hiketes is also supplied: size of zooid in vivo 70×35 µm on average, elongate in shape with inconspicuously double‐layered peristomial lip; macronucleus C‐formed; one large contractile vacuole apically located; colony dichotomously branched; the number of striations from peristome to aboral ciliary wreath 89–109, from aboral ciliary wreath to scopula 35–43; rows in peniculus 3 parallel to each other.


Introduction
In recent years, it has been proposed that a minimum requirement for an acceptable species-level description in peritrichous ciliates is that it should be based on observations of the organisms both in vivo and following silver impregnation (Foissner 1984).
Among the solitary peritrichs, members of the genus Pseudovorticella and Zoothamnium, which have plastic body shape, variable size and highly contractile nature, are difficult to identify to species level. This problem is compounded by the large number of taxa described, and a lack of information on the infraciliature and the silverline system based on silver impregnation (Kahl 1935;Stiller 1971;Warren 1986Warren , 1987Foissner et al. 1992).
During faunistic surveys of marine ciliates in coastal waters of north China, two less common peritrich species were isolated and investigated. After comparison with known congeners, one of them is believed to be a poorly described Pseudovorticella which, for many decades, has been known as Vorticella cylindrica Dons, 1915, and another one is identified as Zoothamnium hiketes.

Materials and methods
Pseudovorticella cylindrica (Dons, 1915) nov. comb. was discovered in a shrimpbreeding pond on the coast of Qingdao (Tsingtao, 36u089N, 120u439E), China in April 2004 with salinity ca 30 and pH 8.1. Zoothamnium hiketes was collected in June 2003 from an abalone-farming pond near Qingdao with water temperature 22uC and salinity ca 28.
Ciliates were studied in vivo using a differential interference contrast microscope. The infraciliature was revealed with protargol impregnation according to Wilbert (1975), while the silverline system was impregnated by the Chatton-Lwoff silver nitrate method according to Song and Wilbert (1995). Drawings of stained specimens were performed at 12506 with the aid of a camera lucida. Terminology is mainly according to Corliss (1979), Foissner et al. (1985 and Warren (1986Warren ( , 1987.

Voucher slides
Silver nitrate and protargol-prepared materials are deposited as voucher slides in the collection of the Laboratory of Protozoology, Ocean University of China with the following registration numbers: Pseudovorticella cylindrica, 04040801-2; Zoothamnium hiketes, 03061901-2.

Subclass PERITRICHIA Stein, 1859
Order SESSILIDA Kahl, 1933Family VORTICELLIDAE Ehrenberg, 1938Genus Pseudovorticella Foissner and Schiffmann, 1974 Pseudovorticella cylindrica (Dons, 1915) nov. comb. (Figures 1-18; Table I) Synonym Vorticella cylindrica Dons, 1915. Since no ciliature information was previously available for this organism, here we supply an improved diagnosis and detailed data on the infraciliature as well as on the morphology of living cells, based on the Qingdao population.

Description
Cell constant in size and shape, about 40-50645-50 mm in vivo, usually with a slight constriction in the aboral region. Maximum width of cell at peristomial area with thin and relatively rigid peristomial lip (PL;Figures 1,5,9). Peristomial disc (PD) flat and only slightly elevated when cell fully extended (Figures 1, 9). Pellicle generally smooth, while a finely reticulate pattern of striations can be observed only under high magnification (10006) (Figure 1).
Individuals often closely grouped together thus forming pseudocolonies of up to 30 zooids. Telotroch (swarmer) was not observed.
Infraciliature as shown in Figures 6, 12, 15, 17, 18. Haplokinety (H) and polykinety (Po) describing about 1.5 turns around peristomial disc before entering vestibulum, where they make a further turn. Similar to that of other congeners, polykinety forms three peniculi in lower half of vestibulum, each consisting of three rows. The posterior ends of peniculus 1 (P1) and P3 terminate at the same level whereas P2 situates between and terminates conspicuously above P1 and P3 (Figures 6, 15). The inner row of P3 is characteristically displaced relative to the other two: very short, about one-third length of the outer two rows which converge with P1. The haplokinety passing around the vestibulum on the opposite wall to the peniculi. Germinal kinety (G) consisting of zig-zag structure of kinetosomes and extending to the upper one-third of the vestibulum, where it passes immediately beyond the haplokinety ( Figure 6). The epistomial membrane is short, located near the opening of the infundibulum (Figures 6, 12, arrow). Aboral ciliary wreath encircles cell in posterior region and seems to be composed of a Z-rowed structure ( Figure 14, double arrowhead; Figure 17, arrowheads).

Comparison
Pseudovorticella cylindrica was originally described by Dons (1915) (Figure 3) under the name of Vorticella cylindrica. Its infraciliature remained unknown though it was redescribed in the last decade by Song (1991a) (Figure 4). The Qingdao population resembles the previous descriptions in all aspects, e.g. body shape, size, number of contractile vacuoles, as well as marine habitat, hence we are confident that the identification is correct. A summary of comparisons between P. cylindrica and similar Pseudovorticella and Vorticella spp. is given in Table II.
Morphologically, Pseudovorticella patellina (Mü ller, 1776) Song and Warren, 2000 ( Figure 39) should be closely related to P. cylindrica. Nevertheless, the former differs from P. cylindrica in its larger size (55-110 versus 40-50 mm), the number of transverse silverlines between the aboral ciliary wreath and the anterior end (19-22 versus 33-40), number of contractile vacuoles (two versus one), and the extreme outwards extension of the peristomial lip. In addition, the structure of peniculus 3 is completely different (see Song and Warren 2000).
Pseudovorticella punctata (Dons, 1918) Warren, 1987) is most similar to P. cylindrica, especially in terms of body size and the habitat. However, P. punctata can be clearly distinguished from the latter by the appearance of the peristomial lip (considerably thick versus thin), the lower position of the contractile vacuole (below the vestibulum versus apically located), and having significantly fewer transverse lines (about 30 versus 52) (Warren 1987).
Considering the general morphology, further comparisons should be also made with some Vorticella species. Vorticella campanula Ehrenberg, 1831 (Figures 40, 41) corresponds very well in vivo with Pseudovorticella cylindrica. In the past 80 years, P. cylindrica was repeatedly considered to be a marine variety of V. campanula (Noland and Finley 1931;Warren 1987). In 1992, a thorough redescription of V. campanula using modern techniques was given by Foissner et al. (1992). Thus, we have the opportunity to compare these two forms. Vorticella campanula can be separated from P. cylindrica by size (50-160 versus 40-50 mm), the habitat (freshwater versus marine), Vorticella-type silverline system (versus Pseudovorticella pattern), and dissimilarities considering the infraciliature (Foissner et al. 1992).
Vorticella nebulifera Mü ller, 1786 also bears some resemblance to Pseudovorticella cylindrica, particularly with respect to the smooth pellicle and cell size. However, the two taxa can be clearly separated by the position of the contractile vacuole (lower ventral versus apically dorsal located) (Figure 42), different pattern of silverline system, and the body shape (slender in V. nebulifera) (Song 1991a). With reference to body shape and the habitat, another similar form, Vorticella marina Greeff, 1870 whose silverline system and infraciliature remain unclear, should also be compared with P. cylindrica. The former differs from the latter, even at the in vivo level, in the lower position of contractile vacuole ( Figure 44) and appearance of pellicle (distinctly striated versus finely striated) (Song 1991a).

Genus Zoothamnium Bory de St. Vincent, 1826
Zoothamnium hiketes Precht, 1935 (Figures 19-38; Table I) Zoothamnium hiketes was originally described by Precht (1935) from the North Sea, Germany. No further studies have been carried out since then and hence neither the infraciliature nor silverline system is clear. Based on the Qingdao population and previous studies, an updated diagnosis and a detailed redescription are presented here.

Description of Qingdao population
Zooids usually elongated vase-shaped, in vivo about 60-80630-40 mm, and only slightly constricted below the thick peristomial lip, which is inconspicuously double-layered; large peristomial disc highly elevated (Figures 19, 29). Pellicle smooth when observed at low magnification, fine striations recognizable under high magnification (6400 or higher) (Figure 31).
Colony dichotomously branched with zooids located regularly in pairs (Figures 23, 27), consisting of up to 100 zooids in a large colony (Figure 34). Stalk up to 400 mm long, diameter about 12 mm in main stalk and 6-8 mm in accessory branches. Surface of stalk smooth and often covered with rod-shaped bacteria (about 1.2-1.8 mm in length) (Figures 21, 28, arrowheads). Spasmoneme about 2 mm in diameter, without visible mitochondria (thecoplasmic granules).

Comparison
Zoothamnium hiketes Precht, 1935 was originally found on the seta of Gammarus species as epizoons, and the brief description concerned only the morphology of live cells (Precht 1935;Figure 22a, b). Since then, no redescriptions have been made. In the absence of data concerning the infraciliature and silverline system, we identified our organism mainly on the basis of its body shape, appearance of the thick peristomial lip (inconspicuous doublelayered), position of contractile vacuole, branching form, and marine habitat. The only difference is the size of the zooid. According to the original description, the zooid of Z. hiketes is smaller, about 40-55 mm in length, while in the Qingdao population the length is about 60-80 mm. Since the size of the organism, to the authors' knowledge, is usually a population-dependent feature in most cases, we consider this difference as an intra-species variation.
Morphologically, Zoothamnium cienkowskii Wrzesniowski, 1877 ( Figure 45; Table III) is similar to Z. hiketes in vivo. Unfortunately, neither the infraciliature nor silverline system of the former has been described. However, Z. cienkowskii can be distinguished at least by the appearance of the stalk (transversely wrinkled at accessory branch versus smooth) and differentiation of the zooids (having enlarged zooids versus all zooids about equal size) (Kahl 1935). Zoothamnium affine sensu Song, 1991 (Figures 46, 47) whose infraciliature also remains unknown, matches in some aspects Z. hiketes, e.g. in body size, in branching form and in some other morphometric features (Table III). However, the former can be identified by having fewer silverlines between oral area and aboral ciliary wreath (75-81 versus 89-109) and a cross-striated stalk (versus smooth) (Song 1991b).
Zoothamnium duplicatum Kahl, 1933 (Figures 50, 51; Table III) also exhibits a similar size and dichotomous branching pattern, but differs from Z. hiketes by the following features: (1) significantly fewer silverlines between peristomial area and aboral ciliary wreath (48-53 versus 89-109); (2) the form of P3 (outer two rows close set, with their upper halves separated widely from the inner one versus three rows parallel to each other) (Ji et al., 2005).