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A new investigation of the marine ciliate, Trachelostyla pediculiformis (Cohn, 1866) Borror, 1972 (Ciliophora, Hypotrichida), with establishment of a new genus, Spirotrachelostyla nov. gen.
ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 42 (2006) 63–73 www.elsevier.de/ejop
A new investigation of the marine ciliate, Trachelostyla pediculiformis (Cohn, 1866) Borror, 1972 (Ciliophora, Hypotrichida), with establishment of a new genus, Spirotrachelostyla nov. gen. Jun Gonga,b, Weibo Songa,b,, Lifang Lia, Chen Shaoa, Zigui Chena a
Laboratory of Protozoology, Ocean University of China, Qingdao 266003, P.R. China College of Life Science, South China Normal University, Guangzhou 510631, P.R. China
b
Received 28 August 2005; received in revised form 25 October 2005; accepted 3 December 2005
Abstract The morphology and infraciliature of the type species of the ciliate genus Trachelostyla, T. pediculiformis (Cohn, 1866) Borror, 1972, collected from the Bohai Sea near Tianjin, north China, were investigated using live observation and protargol impregnation. The Chinese population corresponds well with the original description, as follows: size about 80–150 20–30 mm in vivo; body dorsoventrally flattened and non-spiral; outline elongate, with anterior region conspicuously narrowed while posterior not; on average 14 macronuclear nodules, two micronuclei and 42 adoral membranelles; 16–24 cirri in left and 21–31 in right marginal row. In total 18 frontoventral-transverse cirri: constantly 11 cirri in frontal area and two ventral ones together with five enlarged transverse cirri (TC). Three inconspicuous caudal cirri (CC); six dorsal kineties with prominent cilia. The literature on T. pediculiformis is critically reviewed, showing that the descriptions so far do not agree very well. To clarify the taxonomic status of this species, the population from the Chinese coast of the Bohai Sea is designated as a neotype. The small subunit rRNA gene was sequenced for the neotype and the molecular phylogenetic tree basically supports the establishment of the family Trachelostylidae Small & Lynn, 1985. Based on the data obtained, the genus Trachelostyla Borror, 1972 is redefined thus: dorsoventrally flattened trachelostylids, body non-spirally twisted and elongate, with peristomal region conspicuously narrowed; ciliature generally in 11:2:5:3 pattern, i.e. 11 cirri in frontal region, two ventral cirri located anterior to five TC, three CC present; one left and one right row of marginal cirri not confluent posteriorly. A new genus Spirotrachelostyla nov. gen. is proposed to include the taxa which were previously assigned to Trachelostyla but characterized by spirally twisted, spindle-like body shape and a variable number of cirri in the peristomal area. Three new combinations are suggested: Spirotrachelostyla spiralis (Dragesco & Dragesco-Kerne´is, 1986) nov. comb. [basionym: Trachelostyla spiralis Dragesco & Dragesco-Kerne´is, 1986], S. tani (Hu & Song, 2002) nov. comb. [basionym: T. tani Hu & Song, 2002] and S. simplex (Kahl, 1932) nov. comb. [basionym: Stichotricha simplex Kahl, 1932]. r 2006 Elsevier GmbH. All rights reserved. Keywords: Spirotrachelostyla nov. gen.; New combinations; Redescription; Small unit ribosomal RNA gene; Trachelostyla pediculiformis; Neotypification
Corresponding author. Laboratory of Protozoology, Ocean University of China, Qingdao 266003, P.R. China.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2005.12.001
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Introduction Species of the genus Trachelostyla, known for their fragile, flexible and non-contractile body, have been found frequently in marine or terrestrial environments worldwide (Biernacka 1963; Borror 1963; Carey 1992; Jones 1974; Kahl 1932; Kattar 1970; Lepsi 1962; Maeda and Carey 1984). Trachelostyla was established by Kahl (1932) with two species, namely T. pediculiformis (Cohn, 1866) and T. caudata Kahl, 1932, but he did not fix either as the type. As indicated by Aescht (2001), it was Borror (1972) who fixed T. pediculiformis as the type species and thus made the genus valid according to ICZN (1999). However, the infraciliature of T. pediculiformis has never been sufficiently investigated (Aescht 2001; Berger 1999), making characterization of this genus difficult, though several morphotypes were added to Trachelostyla in recent decades (Buitkamp and Wilbert 1974; Dragesco and Dragesco-Kerne´is 1986; Hu and Song 2002). In the summer of 2003, ciliate communities in littoral sediments in the Bohai Bay, north China were taxonomically examined and a Trachelostyla was isolated. Detailed observations and investigations have demonstrated that it was a population of T. pediculiformis. The present paper gives a morphological description and a phylogenetic analysis based on the SSrRNA gene sequence of the Chinese population. In addition, some taxonomical problems of the genus Trachelostyla have been critically reconsidered.
vitiphila AJ310495, Steinia sphagnicola AJ310494 (Bernhard et al. 2001); Cyrtohymena citrina AY498653, Gonostomum namibiense AY498655, Hemiurosoma terricola AY498651, Onychodromopsis flexilis AY498652, Orthoamphisiella breviseries AY498654 (Foissner et al. 2004); Aspidisca steini AF305625, Euplotes vannus AY004772 (Chen and Song 2002); Euplotes charon AF492705, Gastrostyla steinei AF164133, Pleurotricha lanceolota AF164128, Stylonychia lemnae AF164124, Stylonychia mytilus AJ310499, Oxytricha ferruginea AF370027, Oxytricha longa AF164125; Loxodes magnus L31519 (Hirt et al. 1995) was used as the outgroup species. The SSrRNA gene sequences were aligned using a computer assisted procedure, Clustal W, ver. 1.80 (Thompson et al. 1994), and refined by considering the conservation of primary structures. PHYLIP package, version 3.57c (Felsenstein 1995) was used to calculate the sequence similarity and evolutionary distances between pairs of nucleotide sequences using the Kimura (1980) two-parameter model. Distance–matrix trees were then constructed using the Fitch and Margoliash (1967) least-squares (LS) method. The distance data were bootstrap resampled 1000 times (Felsenstein 1985).
Results and discussion Trachelostyla pediculiformis (Cohn, 1866) Borror, 1972 (Tables 1 and 2 and Figs. 1–29)
Material and methods Trachelostyla pediculiformis was collected from the top 5 cm of sandy littoral sediments near Tianjin, Bohai Sea, China (391100 N; 1171100 E) in August 2003. The water temperature was 18 1C. A raw culture was set up at room temperature (20 1C) and maintained in Petri dishes with boiled seawater of salinity 30%. Living cells were observed by differential interference contrast microscopy. The infraciliature was revealed by the protargol impregnation method of Wilbert (1975). Living individuals were examined and measured at 1000 magnification; counts, measurements and drawings of stained specimens were performed at 1250 with the aid of a camera lucida. Terminology is mainly according to Corliss (1979) and Berger (1999). Genomic DNA extraction, polymerase chain reaction (PCR) amplification of SSrRNA gene, cloning and sequencing were performed according to Shang et al. (2003). The nucleotide sequences used in this paper are available from the GenBank/EMBL databases under the following accession numbers: Gonostomum strenuum AJ310493, Laurentiella strenua AJ310487, Pattersoniella
1866 Stichochaeta pediculiformis – Cohn, Z. wiss. Zool. 16, 285; Pl. XV, Fig. 38a and b (Fig. 30A; original description, no type material available). 1883 Gonostomum pediculiforme (Cohn, 1866) – Maupas, Arch. Zool. Exp. ge´n. (Se´r 2) 1, 550; Pl. XXIV, Figs. 8–13 (Fig. 30B; combination with Gonostomum and detailed redescription). 1888 Stichochaeta corsica – Gourret and Roeser, Arch. Biol. 8, 187; Pl. XIV, Fig. 6 (Fig. 30C; synonym and redescription). 1928 Gonostomum pediculiforme (Cohn, 1866) – Kahl, Arch. Hydrobiol. 19, 210; Fig. 44h (Fig. 30D; synonym and redescription). 1932 Trachelostyla pediculiformis (Cohn, 1866) – Kahl, Tierwelt Dtl. 25, 596; p. 580, Fig. 106 parts 5, 6 and 11. (Fig. 30E; redescription). 1933 Trachelostyla pediculiformis (Cohn, 1866) – Kahl, Tierwelt N.- u. Ostsee 23, 112; Fig. 17.25 (Fig. 30F; guide to marine ciliates). 1963 Trachelostyla pediculiformis (Cohn, 1866) – Borror, Arch. Protistenkd. 106, 511; Fig. 116 (Fig. 30G; the first study using silver staining methods; no voucher material available).
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Table 1.
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Morphometric characterization of the Tianjin population of Trachelostyla pediculiformis
Character
Min
Max
Mean
SD
CV
n
Body length Body width Buccal field, length Adoral membranelles, number Frontoventral cirri, numbera Pretransverse ventral cirri, number Transverse cirri, number Left marginal cirri, number Right marginal cirri, number Caudal cirri, number Macronuclear nodules, number Macronuclei, diameter Micronuclei, number Dorsal kineties, number
80 20 40 36 11 2 5 16 21 3 9 4 2 6
128 48 64 49 11 2 5 24 31 3 17 8 2 6
99.7 30.6 49.3 41.8 11 2 5 19.4 25.6 3 14.4 4.9 2 6
13.0 7.5 6.7 3.3 0 0 0 2.3 2.5 0 1.9 1.4 0 0
13.0 24.5 13.6 7.9 0 0 0 11.9 9.8 0 13.2 28.6 0 0
26 26 26 25 26 26 21 21 21 19 27 26 26 26
Data from protargol impregnated specimens; measurements in mm. (CV ¼ coefficient of variation in %, Max ¼ maximum, Mean ¼ arithmetic mean, Min ¼ minimum, n ¼ number of individuals examined, SD ¼ standard deviation). a Counting all the cirri in frontal area, i.e. anterior to the level of cytostome.
Table 2.
Comparison among populations of Trachelostyla pediculiformis
Data source
Body length (mm)
Cirri in frontal area, number
Pretransverse ventral cirri, number
Transverse cirri, number
Caudal cirri, number
Dorsal kineties, number
Macronuclei, number
Cohn (1866) Maupas (1883) Kahl (1928) Kahl (1932) Biernacka (1963) Borror (1963) Kattar (1970) Jones (1974) Maeda and Carey (1984) Present work
100 100–200 150–200 100–250 150 135 150 110 136–196
— 11 10 8–9 5 11–12 11 11 10
— 2 0 0 0 2 2 2 0
— 5 5 5 4 5 5 5 5–6
3? 3 3 0 — 0 0 0 0
— — — — 3 3 2 — 5
— 14–20 16 11 12 16–64 18 26–30 11
80–150
11
2
5
3
6
9–17
—, data not available.
1963 Trachelostyla pediculiformis (Cohn, 1866) – Biernacka, Pol. Arch. Hydrobiol. 11, 51; Fig. 100 (Fig. 30H; redescription). 1970 Trachelostyla pediculiformis (Cohn, 1866) – Kattar, Bol. Zool. Biol. Mar. (Sa˜o Paulo) N. S. 27, 188; Fig. 33 (Fig. 30I; redescription). 1972 Trachelostyla pediculiformis (Cohn, 1866) – Borror, J. Protozool. 19, 15; Fig. 43 (Fig. 30J; combining author; revision of hypotrichs). 1974 Trachelostyla pediculiformis (Cohn, 1866) Kahl, 1932 – Jones, Univ. South Alabama Monogr. 1, 42; Pl. XXIX, Fig. 6 (Fig. 30K; redescription). 1984 Trachelostyla pediculiformis (Cohn, 1866) Kahl, 1932 – Maeda and Carey, Bull. Br. Mus.
Nat. Hist. (Zool.) 47, 5; Fig. 1 (Fig. 30L; redescription of the species and revision of the genus). 1992 Trachelostyla pediculiformis (Kahl, 1930–5) Maeda & Carey, 1984 – Carey, Marine Interstitial Ciliates, p. 186, Fig. 737 (guide; description identical with Maeda and Carey 1984, the illustration is a redrawing of Fig. 30L). All the morphological data as well as the ecological features of the Tianjin population correspond perfectly well with both the original description by Cohn (1866) and the redescription by Maupas (1883), so the identification of our organism is beyond doubt.
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Figs. 1–9. Trachelostyla pediculiformis from life (1, 2, 7) and after protargol impregnation (3–6, 8, 9). Neotype population from Bohai Sea. 1. Ventral view of a typical specimen. 2. Left view. 3, 4. Infraciliature from ventral (3) and dorsal (4) views of the same individual, noting that the entire left-most dorsal kinety and the posterior half of right-most dorsal kinety can be observed from the ventral view. 5. Ventral view of a middle divider, showing the oral and cirral primordia (arrows) for both proter and opisthe and the dorsal kinety anlagen (arrowheads). 6. Detailed infraciliature of oral region, showing the 3–4 enlarged adoral membranelles (arrow) and the zig-zag fibril structure (arrowheads) along adoral zone of membranelles. 7. Dorsal view showing endoplasmic granules and 3 dorsal bristles. 8. Nuclear apparatus of an early divider, showing the two micronuclei (arrows) and macronuclear replication bands (arrowheads). 9. Ventral view of an early divider, with simultaneously formed oral primordia for the proter (arrow) and opisthe (arrowhead). AZM, adoral zone of membranelles; BC, buccal cirrus; CC, caudal cirri; DK, dorsal kineties; EM, endoral membrane; FC, frontal cirri; FVC, frontoventral cirri; LMR, left marginal row; Ma, macronuclei; PM, paroral membrane; PVC, postoral ventral cirri; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri. Bars ¼ 40 mm.
Description of the morphology based on the Chinese population Size 80–150 20–30 mm, mostly 120 25 mm in vivo; body shape rather constant and bipartite, trunk having elongate ellipsoidal outline, but with a narrow neck-like constriction at anterior 1/5 of body length. Left and right body margins almost straight and parallel to each other, posterior end bluntly rounded (Figs. 1, 10–12). Dorsoventrally flattened, width:thickness ratio about
2:1, ventral side flat, dorsal often vaulted in mid-body (Figs. 2 and 15). Cell flexible but non-contractile. Pellicle thin, no cortical granules recognizable. Endoplasm colorless and opaque, typically packed with 2–3-mmsized granules (Figs. 7 and 16). Nine to 17 macronuclear nodules (Ma) arranged in a ring in trunk region, each nodule ovoid to ellipsoid (about 5 4 mm after impregnation), with small nucleoli; two micronuclei, each 2–3 mm across, one anterior and one posterior, located
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Figs. 10–16. Neotype population of Trachelostyla pediculiformis from life. 10, 12. Ventral view of two typical specimens, arrows mark the anterior adoral membranelles and arrowheads indicate the prominent transverse cirri. 11. A small individual. 13. Anterior portion of cell. 14. Ventral view, to show the stiff dorsal cilia. 15. Left side view, arrowhead marks the transverse cirri. 16. Endoplasmic granules. AZM, adoral zone of membranelles; DK, bristles of dorsal kinety. Bars in Figs. 10–13, 15 ¼ 30 mm, in Figs. 14 and 16 ¼ 15 mm.
inside macronuclear ring (Figs. 4, 20, 28 and 29). No contractile vacuole observed. Feeding on bacteria digested in 5–10-mm-sized food vacuoles. Movement rapid, sometimes jerky on microscope slide and between sediment particles, body showing great flexibility. Adoral zone occupies about 40% of body length in life, composed of about 40 membranelles that form two groups of different appearance: the apical three to five membranelles strong and about 15–20 mm long, extended radially; the others conspicuously shorter, densely arranged along the left margin of buccal field (Figs. 1, 10, 12 and 13). Bases of longest membranelles about 3 mm in length (Figs. 3, 6, 20, 28 and 29). A zig-zag structure (arrowheads in Figs. 6 and 28), probably composed of fibers, always positioned along adoral zone of membranelles (AZM). Undulating membranes inconspicuous, arranged approximately in parallel, slightly curved; paroral about 13 mm and endoral 6 mm long (Fig. 6). Right marginal row (RMR) composed of about 26 cirri, begins at anterior 1/5 of body length and ends near the rightmost transverse cirrus. Left marginal row (LMR) composed of about 19 cirri terminating subcaudally (Figs. 3, 6, 19 and 24; Table 1). Frontal–ventral–transverse cirri (FVT-cirri) in a typical 8:5:5 pattern. Consistently 11 cirri in frontal area, which are homologous with 8 frontal and 3 postoral ventral cirri in other typical oxytrichids: three anterior-most (FC), four posterior frontal cirri (FVC), one buccal cirrus (BC) and three ‘‘ventral cirri’’ (PVC). Two pretransverse ventral cirri (PTVC) anterior of transverse cirri (TC) (Figs. 3, 18 and 27). Five TC highly
developed, arranged in a J-shaped row. Three inconspicuous caudal cirri (CC), of which the right two are often arranged close to each other on right of body midline (Figs. 3, 21, 22 and 26). Cilia of marginal, caudal, frontal and ventral cirri about 5–7 mm, while TC about 12–15 mm long. Dorsal kineties (DK) composed of six rows of stiff and conspicuous bristles 7–8 mm in length, extend whole body length (Figs. 1, 4, 14 and 23). Notes on morphogenesis Two individuals at early stages of morphogenesis were observed in protargol preparations. Some basic features can be recognized as follows: (1) FVTprimordia in both dividers (arrow and arrowhead in Fig. 9) develop de novo on the surface of the cell (Figs. 5 and 25, arrows); (2) replication bands of macronuclear nodules (Fig. 8, arrowheads) appear in early stage of morphogenesis; (3) the dorsal kinety anlagen originate from the old structure (Fig. 5, arrowheads). Comparison of Chinese population with Trachelostyla pediculiformis populations This species was originally described by Cohn (1866) under the name Stichochaeta pediculiformis and later was transferred by Kahl (1932) into the genus Trachelostyla (see Maeda and Carey (1984) for review and introduction for comment on nomenclature). Cohn (1866) presented some living information such as body size, shape, behavior, and appearance of cytoplasm and adoral zone. More importantly, he mentioned that this
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Figs. 17–29. Neotype population of Trachelostyla pediculiformis after protargol impregnation. 17, 28. Ventral views of infraciliature, showing anterior portion of cells; arrow in Fig. 17 marks the enlarged distal adoral membranelles and arrowheads in Fig. 28 indicate the zig-zag fibril structure along adoral zone of membranelles. 18. Focus on the 11 cirri (arrows) on anterior portion of cell. 19. Showing the three frontal cirri (arrows), anterior ends of left marginal row (double-arrowheads) and right marginal row (arrowhead). 20, 29. Focus on nuclear apparatus, arrows in Fig. 20 indicate micronuclei. 21, 22. Posterior ends of cells, arrowheads mark the caudal cirri. 23. Dorsal view, to note the dorsal kineties (arrows). 24, 27. Posterior portion of ventral side, arrows in Fig. 24 mark marginal rows and arrows in Fig. 27 indicate the two pretransverse ventral cirri. 25. Mid-portion of a middle divider; arrows mark the separated oral primordia for proter and opisthe. 26. Focus on the three caudal cirri (arrows) and dorsal cilia (arrowheads). AZM, adoral zone of membranelles; Ma, macronuclei; TC, transverse cirri.
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Fig. 30. Trachelostyla pediculiformis (A, after Cohn 1866; B, from Maupas 1883; C, after Gourret and Roeser 1888, from Kahl 1932; D, from Kahl 1928; E, from Kahl 1932; F, from Kahl 1933; G, from Borror 1963; H, from Biernacka 1963; I, from Kattar 1970; J, from Borror, 1972; K, from Jones 1974; L, from Maeda and Carey 1984), T. rostrata (M, from Small and Lynn 1985, called ‘‘T. pediculiformis’’; R, from Lepsi 1962), T. caudata (N, from Kahl 1932), Spirotrachelostyla simplex (Kahl, 1932) nov. comb. (O, from Kahl 1932, originally Stichotricha simplex Kahl 1932, transferred to Trachelostyla by Borror in 1972), S. spiralis (Dragesco & Dragesco-Kerne´is, 1986 nov. comb. (P, Q, from Dragesco and Dragesco-Kerne´is 1986, formerly T. spiralis) and S. tani (Hu & Song, 2002) nov. comb. (S–U, from Hu and Song 2002; formerly T. tani).
organism had three CC, though he was not convinced about the number. Nearly 20 years later, Maupas (1883) redescribed this species in great detail. Some diagnostic features were given as follows: cell length 100–200 mm, outline elongate; body highly flexible but non-contractile; cytoplasm granulated, grayish and opaque; about 14–20 macronuclear nodules and two micronuclei; left and right marginal cirri rows not confluent at the posterior end of body; 11 cirri in frontal area, two PTVC, five TC and three CC (Fig. 30B). There have been at least 10 redescriptions of this organism since 1883 (see list of synonyms). These redescriptions are basically in accordance with Cohn (1866) and Maupas (1883) in features including body size and shape, flexibility and cytoplasmic characters (Figs. 30A–L). Nevertheless, disagreements remain, especially in the following five aspects (Table 2): (1) The number of macronuclear nodules. Cohn (1866) did not see the nuclear apparatus, but illustrated
in his Fig. 38b (not shown in the present paper) two bright patches, which he interpreted as contractile vacuoles. However, he did not see contraction. We know that the macronuclear nodules sometimes occur as bright areas, especially when the cells are packed with various inclusions. Thus, one cannot exclude the possibility that Cohn’s type population had indeed only two macronuclear nodules. Another possibility is due to Maupas (1883), who first explicitly stated his population has about 14–20 macronuclear nodules. This record basically agrees with most of the subsequent redescriptions by Kahl (1928, 1932), Biernacka (1963), Borror (1963), Kattar (1970), Jones (1974) and Maeda and Carey (1984), and hence, in our opinion, is virtually authoritative in identification of this species. Considering the majority rule and more consistency with present classification system, we prefer the latter possibility. Stichochaeta corsica Gourret and Roeser, 1888 was described with a
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(2)
(3)
(4)
(5)
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single macronucleus, which has to be interpreted as a mis-observation. Caudal cirri. Cohn (1866) did not conclusively determine the number of CC, but later Maupas (1883) confirmed the presence of three CC in this species. Gourret and Roeser (1888) and Kahl (1928) also mentioned that there are several long and fine cirri at the posterior end of cells (Fig. 30C and D). In contrast to his previous description in 1928, Kahl (1932) suggested that the three filose cirri in the caudal area, which Maupas (1883) described as CC, were in fact dorsal cilia which could be seen from the ventral side. This interpretation was accepted by subsequent authors (Biernacka 1963; Borror 1963; Kattar 1970; Jones 1974; Maeda and Carey 1984). The present study revealed that this organism consistently has three CC (Figs. 3, 21, 22 and 26). Hence, we believe that Kahl’s conclusion is due to a misinterpretation, i.e. he overlooked these fine cilia. In fact, it is virtually impossible to distinguish the thin CC from the long stiff dorsal cilia in live specimens. The number of the FVT cirri. Cohn (1866) did not clearly describe the cirral pattern and number of cirri. Maupas (1883) observed 11 cirri in the frontal area and 2 ventral cirri anterior of the TC, which agrees with Borror (1963), Kattar (1970), Jones (1974) and our observations, but contrary to Kahl (1928, 1932), Biernacka (1963) and Maeda and Carey (1984), who recorded that there were fewer cirri (5–10) in the frontal area and no ventral cirri anterior to the transverse ones. We suppose that 11 frontal-ventral cirri in the frontal area should be the typical pattern of this taxon, which is equivalent to eight frontal (including the single buccal one) and three postoral ventral cirri in other typical oxytrichids (Berger 1999), that is, three postoral ventral cirri have moved to the frontal area in this organism. The strongly granulated and opaque cytoplasm of T. pediculiformis may lead to undercounting of the cirri in living observations. A similar misinterpretation could also have been made by Borror (1963), who recorded 12 frontal-ventral cirri from Chatton–Lwoff impregnated specimens. The numbers of TC recorded are always 5 in our population, but variation (from 4 to 6), as noted by previous authors, indicates that this character might be population-dependent (Table 2). Dorsal kineties. The number of DK in most Oxytrichids (s.l.) is highly consistent (Berger 1999). This ciliature was recorded as 3 rows by Biernacka (1963) and Borror (1963), 2 rows by Kattar (1970) and 5 rows by Maeda and Carey (1984). However, we observed consistently 6 rows in the Chinese population (Tables 1 and 2). Cortical granules. The Chinese population lacks cortical granules, while Cohn (1866) described and
illustrated ‘‘geko¨rnte Linien’’ (granulated lines) along the cirral rows and dorsal bristle rows. Such ‘‘granulated rows’’ are possibly cortical granules, which often occur along the cirral and bristle rows. However, since the body of this species is rather fragile, opaque and highly granulated, detailed observation of living cells at high magnification is usually not easy. It is known that cortical granules of ciliates are only well recognized with modern microscopic techniques, there is reasonable doubt whether Cohn could truly tell the cortical granules apart from endoplasmic granules. This difference hence should not be over-interpreted.
Comparison with related species The morphotype from Chesapeake Bay, USA, illustrated under the name ‘‘Trachelostyla pediculiformis’’ by Small and Lynn (1985) and Lynn and Small (2002), has the following features: body elongate and nonspiral, two macronuclear nodules, 12 cirri in frontal area, two ventral and five TC (Fig. 30M). In our opinion, this form should not be named T. pediculiformis because of the presence of only two macronuclear nodules (vs. many macronuclear nodules). Up to now, the only species confirmed as possessing two macronuclei is T. rostrata Lepsi, 1962 (Fig. 30R). It is therefore reasonable to suggest that Small and Lynn’s illustrations probably represent a population of T. rostrata. Morphologically, T. caudata resembles T. pediculiformis in terms of body size (150–220 mm), having many macronuclear nodules and long dorsal cilia, but differs in its characteristic tail shape (conspicuously narrowed vs. almost equal in width relative to body trunk) (Kahl 1932).
Neotypification of Trachelostyla pediculiformis As mentioned above, the original description of T. pediculiformis is seriously incomplete and there are some discrepancies between subsequent redescriptions (Gourret and Roeser 1888; Biernacka 1963; Borror 1963; Carey 1992; Maeda and Carey 1984; Jones 1974; Kattar 1970). There is no type material available for these morphotypes, thus it seems wise to redefine this species and to designate a neotype (ICZN 1999; Foissner et al. 2002). According to Article 75.3 of the ICZN (1999), the designation has to comply with the following particulars: (1) the taxonomic status of the present species is somewhat unclear because the original description and subsequent redescriptions do not agree in some important features (see above for details); (2) for differentiation of T. pediculiformis from other related
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nominal species, see above; (3) the neotype is clearly described (see above) and illustrated (Figs. 3 and 4), so that the recognition of the specimen designated is ensured; (4) it is generally known that no type material is available from species described by Cohn (1866), Maupas (1883) and Kahl (1928, 1932, 1933), and furthermore, there is no indication that Biernacka (1963), Borror (1963), Kattar (1970), Jones (1974) and Maeda and Carey (1984) made permanent preparations of the present species; (5) there is strong evidence that the neotype is consistent with T. pediculiformis as originally described by Cohn (1866); (6) Cohn (1866) did not fix the type locality in the original description because his aquarium contained seawater from two different sites: Helgoland, Germany, and Dorsetshire, UK, consequently a description of the type locality, that is, the sample site of the neotype population, is given in the section material and methods; (7) the slide containing a protargol-impregnated neotype specimen is deposited in the Natural History Museum, UK with the registration number 2005:3:24:15. Systematic position of Trachelostyla pediculiformis inferred from SSrRNA gene sequence The length of the SSrRNA gene of the Chinese population was determined to be 1769 nucleotides (GenBank accession No. DQ057346). The LS tree constructed from SSrRNA gene sequences is shown as Fig. 31, which gives similar topology to that shown in the phylogenetic studies of stichotrichines by Foissner et al. (2004). Trachelostyla pediculiformis clusters with two species of the genus Gonostomum Sterki, 1878 and Orthoamphisiella breviseries Foissner et al., 2002, with weak bootstrap value of 55%. The branching pattern basically confirms the establishment of the family Trachelostylidae Small & Lynn, 1985, which includes both Trachelostyla and Gonostomum, and is morphologically distinguished from the family Oxytrichidae Ehrenberg, 1838 by the following features: (1) three postoral ventral cirri are positioned anteriorly; (2) undulating membranes are short relative to the length of adoral zone; and (3) the gonostomoid adoral zone (Small and Lynn 1985; Berger 1999; Lynn and Small 2002). Orthoamphisiella breviseries, which was recognized by Foissner et al. (2002) as a member of the family Orthoamphisiellidae Eigner, 1997, is clustered within the Trachelostya–Gonostomum-clade, the relationship with Gonostomum namibiense showing 59% bootstrap support; it is interesting that Orthoamphisiella has a gonostomoid oral zone, few frontoventral cirri, and anterior displaced postoral cirri (Foissner et al. 2002; Foissner et al. 2004). Reconsideration on the genus Trachelostyla Although 14 nominal species have been assigned to the genus Trachelostyla, only six of them have been
Fig. 31. A least-squares tree inferred from the nucleotide sequences of complete SSrRNA gene. Numbers at nodes represent bootstrap values (in %) out of 1000 replicates: the number is from the distance–matrix based least-squares method. Asterisks indicate bootstrap values less than 50%. The new sequence is highlighted in boldface.
recognized and widely accepted: T. pediculiformis (Cohn, 1866), T. caudata Kahl, 1932, T. simplex (Kahl, 1932) Borror, 1972, T. spiralis Dragesco & DragescoKerne´is, 1986, T. rostrata Lepsi, 1962 and T. tani Hu & Song, 2002 (for reviews see Berger 1999, 2001; Borror 1972; Buitkamp 1977; Hu and Song 2002; Fig. 30). Among these, two basic types or groups can be recognized according to the general appearance and ciliary configuration: one group is characterized by: (1) Oxytricha-like, dorsoventrally flattened but non-spiral body shape; and (2) possessing a typical pattern of frontoventral cirri (i.e. in 11+2 mode) as in T. pediculiformis. The second group has a spirally twisted body shape with conspicuously pointed cell ends and a variable number of frontoventral cirri (hence no typical pattern, usually several anterior ventral cirri present). In addition, organisms in the latter group seem to prefer to live in a tube-like lorica and hence are mostly sessile forms, though not all of them have been confirmed to have this life-style. The pointed and spirally twisted body shape, which is rare in oxytrichids and trachelostylids, represents evidently a response to a loricadwelling life-style (living in a tube which is either made by the organism itself or constructed from environmental fragments). Based on these differences, we believe that these two groups should be separated at the generic level and hence suggest the present genus
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should be split into two by redefining the genus Trachelostyla and establishing a new genus Spirotrachelostyla nov. gen.
Trachelostyla Borror, 1972 Redefined diagnosis Dorsoventrally flattened trachelostylids, body nonspirally twisted and elongate, with peristomal region conspicuously narrowed; ciliature generally in 11:2:5:3 pattern, i.e.11 cirri in frontal region; two ventral cirri located anterior to five TC; three CC present. One left and one right row of marginal cirri not confluent posteriorly. Species assignable At least two morphospecies, i.e. the type species T. pediculiformis (Cohn, 1866) and T. rostrata Lepsi, 1962 should clearly be included in this genus. The third one, T. caudata Kahl, 1932 could be a valid member of this genus on the basis of its general appearance (Fig. 30N), though its infraciliature remains undescribed.
Spirotrachelostyla nov. gen. Diagnosis: Spirally twisted trachelostylids with spindle-shaped body; about 13 cirri scattered on anterior peristomal region; two PTVC present or absent; usually five TC; one left and one right row of marginal cirri not confluent posteriorly; CC present. Generally sessile forms in a lorica. Etymology: Spirotrachelostyla is a composite of the Greek noun hespeira (spiral, helix) and the genus-group noun Trachelostyla. It alludes to the spirally twisted body and, like Trachelostyla, has feminine gender. Type species: T. spiralis Dragesco & DragescoKerne´is, 1986. Species assignable: Spirotrachelostyla spiralis (Dragesco & Dragesco-Kerne´is, 1986) nov. comb. [basionym: T. spiralis Dragesco & Dragesco-Kerne´is, 1986] (Figs. 30P and Q), S. tani (Hu & Song, 2002) nov. comb. [basionym: T. tani Hu & Song, 2002] (Figs. 30S–U) and S. simplex (Kahl, 1932) nov. comb. [basionym: Stichotricha simplex Kahl, 1932] (Fig. 30O). Comparison with related genera: Considering the spirally twisted and spindle-like body shape and the relative length of the AZM to body length (40–50%), three genera, namely Stichotricha Perty, 1849, Chaetospira Lachmann, 1856 and Urostrongylum Kahl, 1932, are rather similar to the new genus. However, both Stichotricha and Chaetospira have 2 ventral cirral rows (vs. a few ventral cirri, never in rows, or even absent in Spirotrachelostyla) and no TC (vs. present in Spirotrachelostyla), thus these taxa can be clearly separated (Foissner 1980; Song and Wilbert 1989; Foissner et al.
1991; Hu and Song 2001). Urostrongylum differs from Spirotrachelostyla nov. gen. in having a single row of ventral cirri (vs. several not in rows or even absent), strongly pointed posterior end (vs. slightly tailed and bluntly rounded) and absence of TC (vs. present) (Borror 1972).
Acknowledgments This work was supported by ‘‘The National Science Foundation of China’’ (Projects No. 30430090 and 40376045). The authors are grateful to helpful comments on the manuscript provided by reviewers.
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PROTISTOLOGY European Journal of Protistology 42 (2006) 63–73 www.elsevier.de/ejop
A new investigation of the marine ciliate, Trachelostyla pediculiformis (Cohn, 1866) Borror, 1972 (Ciliophora, Hypotrichida), with establishment of a new genus, Spirotrachelostyla nov. gen. Jun Gonga,b, Weibo Songa,b,, Lifang Lia, Chen Shaoa, Zigui Chena a
Laboratory of Protozoology, Ocean University of China, Qingdao 266003, P.R. China College of Life Science, South China Normal University, Guangzhou 510631, P.R. China
b
Received 28 August 2005; received in revised form 25 October 2005; accepted 3 December 2005
Abstract The morphology and infraciliature of the type species of the ciliate genus Trachelostyla, T. pediculiformis (Cohn, 1866) Borror, 1972, collected from the Bohai Sea near Tianjin, north China, were investigated using live observation and protargol impregnation. The Chinese population corresponds well with the original description, as follows: size about 80–150 20–30 mm in vivo; body dorsoventrally flattened and non-spiral; outline elongate, with anterior region conspicuously narrowed while posterior not; on average 14 macronuclear nodules, two micronuclei and 42 adoral membranelles; 16–24 cirri in left and 21–31 in right marginal row. In total 18 frontoventral-transverse cirri: constantly 11 cirri in frontal area and two ventral ones together with five enlarged transverse cirri (TC). Three inconspicuous caudal cirri (CC); six dorsal kineties with prominent cilia. The literature on T. pediculiformis is critically reviewed, showing that the descriptions so far do not agree very well. To clarify the taxonomic status of this species, the population from the Chinese coast of the Bohai Sea is designated as a neotype. The small subunit rRNA gene was sequenced for the neotype and the molecular phylogenetic tree basically supports the establishment of the family Trachelostylidae Small & Lynn, 1985. Based on the data obtained, the genus Trachelostyla Borror, 1972 is redefined thus: dorsoventrally flattened trachelostylids, body non-spirally twisted and elongate, with peristomal region conspicuously narrowed; ciliature generally in 11:2:5:3 pattern, i.e. 11 cirri in frontal region, two ventral cirri located anterior to five TC, three CC present; one left and one right row of marginal cirri not confluent posteriorly. A new genus Spirotrachelostyla nov. gen. is proposed to include the taxa which were previously assigned to Trachelostyla but characterized by spirally twisted, spindle-like body shape and a variable number of cirri in the peristomal area. Three new combinations are suggested: Spirotrachelostyla spiralis (Dragesco & Dragesco-Kerne´is, 1986) nov. comb. [basionym: Trachelostyla spiralis Dragesco & Dragesco-Kerne´is, 1986], S. tani (Hu & Song, 2002) nov. comb. [basionym: T. tani Hu & Song, 2002] and S. simplex (Kahl, 1932) nov. comb. [basionym: Stichotricha simplex Kahl, 1932]. r 2006 Elsevier GmbH. All rights reserved. Keywords: Spirotrachelostyla nov. gen.; New combinations; Redescription; Small unit ribosomal RNA gene; Trachelostyla pediculiformis; Neotypification
Corresponding author. Laboratory of Protozoology, Ocean University of China, Qingdao 266003, P.R. China.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2005.12.001
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Introduction Species of the genus Trachelostyla, known for their fragile, flexible and non-contractile body, have been found frequently in marine or terrestrial environments worldwide (Biernacka 1963; Borror 1963; Carey 1992; Jones 1974; Kahl 1932; Kattar 1970; Lepsi 1962; Maeda and Carey 1984). Trachelostyla was established by Kahl (1932) with two species, namely T. pediculiformis (Cohn, 1866) and T. caudata Kahl, 1932, but he did not fix either as the type. As indicated by Aescht (2001), it was Borror (1972) who fixed T. pediculiformis as the type species and thus made the genus valid according to ICZN (1999). However, the infraciliature of T. pediculiformis has never been sufficiently investigated (Aescht 2001; Berger 1999), making characterization of this genus difficult, though several morphotypes were added to Trachelostyla in recent decades (Buitkamp and Wilbert 1974; Dragesco and Dragesco-Kerne´is 1986; Hu and Song 2002). In the summer of 2003, ciliate communities in littoral sediments in the Bohai Bay, north China were taxonomically examined and a Trachelostyla was isolated. Detailed observations and investigations have demonstrated that it was a population of T. pediculiformis. The present paper gives a morphological description and a phylogenetic analysis based on the SSrRNA gene sequence of the Chinese population. In addition, some taxonomical problems of the genus Trachelostyla have been critically reconsidered.
vitiphila AJ310495, Steinia sphagnicola AJ310494 (Bernhard et al. 2001); Cyrtohymena citrina AY498653, Gonostomum namibiense AY498655, Hemiurosoma terricola AY498651, Onychodromopsis flexilis AY498652, Orthoamphisiella breviseries AY498654 (Foissner et al. 2004); Aspidisca steini AF305625, Euplotes vannus AY004772 (Chen and Song 2002); Euplotes charon AF492705, Gastrostyla steinei AF164133, Pleurotricha lanceolota AF164128, Stylonychia lemnae AF164124, Stylonychia mytilus AJ310499, Oxytricha ferruginea AF370027, Oxytricha longa AF164125; Loxodes magnus L31519 (Hirt et al. 1995) was used as the outgroup species. The SSrRNA gene sequences were aligned using a computer assisted procedure, Clustal W, ver. 1.80 (Thompson et al. 1994), and refined by considering the conservation of primary structures. PHYLIP package, version 3.57c (Felsenstein 1995) was used to calculate the sequence similarity and evolutionary distances between pairs of nucleotide sequences using the Kimura (1980) two-parameter model. Distance–matrix trees were then constructed using the Fitch and Margoliash (1967) least-squares (LS) method. The distance data were bootstrap resampled 1000 times (Felsenstein 1985).
Results and discussion Trachelostyla pediculiformis (Cohn, 1866) Borror, 1972 (Tables 1 and 2 and Figs. 1–29)
Material and methods Trachelostyla pediculiformis was collected from the top 5 cm of sandy littoral sediments near Tianjin, Bohai Sea, China (391100 N; 1171100 E) in August 2003. The water temperature was 18 1C. A raw culture was set up at room temperature (20 1C) and maintained in Petri dishes with boiled seawater of salinity 30%. Living cells were observed by differential interference contrast microscopy. The infraciliature was revealed by the protargol impregnation method of Wilbert (1975). Living individuals were examined and measured at 1000 magnification; counts, measurements and drawings of stained specimens were performed at 1250 with the aid of a camera lucida. Terminology is mainly according to Corliss (1979) and Berger (1999). Genomic DNA extraction, polymerase chain reaction (PCR) amplification of SSrRNA gene, cloning and sequencing were performed according to Shang et al. (2003). The nucleotide sequences used in this paper are available from the GenBank/EMBL databases under the following accession numbers: Gonostomum strenuum AJ310493, Laurentiella strenua AJ310487, Pattersoniella
1866 Stichochaeta pediculiformis – Cohn, Z. wiss. Zool. 16, 285; Pl. XV, Fig. 38a and b (Fig. 30A; original description, no type material available). 1883 Gonostomum pediculiforme (Cohn, 1866) – Maupas, Arch. Zool. Exp. ge´n. (Se´r 2) 1, 550; Pl. XXIV, Figs. 8–13 (Fig. 30B; combination with Gonostomum and detailed redescription). 1888 Stichochaeta corsica – Gourret and Roeser, Arch. Biol. 8, 187; Pl. XIV, Fig. 6 (Fig. 30C; synonym and redescription). 1928 Gonostomum pediculiforme (Cohn, 1866) – Kahl, Arch. Hydrobiol. 19, 210; Fig. 44h (Fig. 30D; synonym and redescription). 1932 Trachelostyla pediculiformis (Cohn, 1866) – Kahl, Tierwelt Dtl. 25, 596; p. 580, Fig. 106 parts 5, 6 and 11. (Fig. 30E; redescription). 1933 Trachelostyla pediculiformis (Cohn, 1866) – Kahl, Tierwelt N.- u. Ostsee 23, 112; Fig. 17.25 (Fig. 30F; guide to marine ciliates). 1963 Trachelostyla pediculiformis (Cohn, 1866) – Borror, Arch. Protistenkd. 106, 511; Fig. 116 (Fig. 30G; the first study using silver staining methods; no voucher material available).
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Table 1.
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Morphometric characterization of the Tianjin population of Trachelostyla pediculiformis
Character
Min
Max
Mean
SD
CV
n
Body length Body width Buccal field, length Adoral membranelles, number Frontoventral cirri, numbera Pretransverse ventral cirri, number Transverse cirri, number Left marginal cirri, number Right marginal cirri, number Caudal cirri, number Macronuclear nodules, number Macronuclei, diameter Micronuclei, number Dorsal kineties, number
80 20 40 36 11 2 5 16 21 3 9 4 2 6
128 48 64 49 11 2 5 24 31 3 17 8 2 6
99.7 30.6 49.3 41.8 11 2 5 19.4 25.6 3 14.4 4.9 2 6
13.0 7.5 6.7 3.3 0 0 0 2.3 2.5 0 1.9 1.4 0 0
13.0 24.5 13.6 7.9 0 0 0 11.9 9.8 0 13.2 28.6 0 0
26 26 26 25 26 26 21 21 21 19 27 26 26 26
Data from protargol impregnated specimens; measurements in mm. (CV ¼ coefficient of variation in %, Max ¼ maximum, Mean ¼ arithmetic mean, Min ¼ minimum, n ¼ number of individuals examined, SD ¼ standard deviation). a Counting all the cirri in frontal area, i.e. anterior to the level of cytostome.
Table 2.
Comparison among populations of Trachelostyla pediculiformis
Data source
Body length (mm)
Cirri in frontal area, number
Pretransverse ventral cirri, number
Transverse cirri, number
Caudal cirri, number
Dorsal kineties, number
Macronuclei, number
Cohn (1866) Maupas (1883) Kahl (1928) Kahl (1932) Biernacka (1963) Borror (1963) Kattar (1970) Jones (1974) Maeda and Carey (1984) Present work
100 100–200 150–200 100–250 150 135 150 110 136–196
— 11 10 8–9 5 11–12 11 11 10
— 2 0 0 0 2 2 2 0
— 5 5 5 4 5 5 5 5–6
3? 3 3 0 — 0 0 0 0
— — — — 3 3 2 — 5
— 14–20 16 11 12 16–64 18 26–30 11
80–150
11
2
5
3
6
9–17
—, data not available.
1963 Trachelostyla pediculiformis (Cohn, 1866) – Biernacka, Pol. Arch. Hydrobiol. 11, 51; Fig. 100 (Fig. 30H; redescription). 1970 Trachelostyla pediculiformis (Cohn, 1866) – Kattar, Bol. Zool. Biol. Mar. (Sa˜o Paulo) N. S. 27, 188; Fig. 33 (Fig. 30I; redescription). 1972 Trachelostyla pediculiformis (Cohn, 1866) – Borror, J. Protozool. 19, 15; Fig. 43 (Fig. 30J; combining author; revision of hypotrichs). 1974 Trachelostyla pediculiformis (Cohn, 1866) Kahl, 1932 – Jones, Univ. South Alabama Monogr. 1, 42; Pl. XXIX, Fig. 6 (Fig. 30K; redescription). 1984 Trachelostyla pediculiformis (Cohn, 1866) Kahl, 1932 – Maeda and Carey, Bull. Br. Mus.
Nat. Hist. (Zool.) 47, 5; Fig. 1 (Fig. 30L; redescription of the species and revision of the genus). 1992 Trachelostyla pediculiformis (Kahl, 1930–5) Maeda & Carey, 1984 – Carey, Marine Interstitial Ciliates, p. 186, Fig. 737 (guide; description identical with Maeda and Carey 1984, the illustration is a redrawing of Fig. 30L). All the morphological data as well as the ecological features of the Tianjin population correspond perfectly well with both the original description by Cohn (1866) and the redescription by Maupas (1883), so the identification of our organism is beyond doubt.
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Figs. 1–9. Trachelostyla pediculiformis from life (1, 2, 7) and after protargol impregnation (3–6, 8, 9). Neotype population from Bohai Sea. 1. Ventral view of a typical specimen. 2. Left view. 3, 4. Infraciliature from ventral (3) and dorsal (4) views of the same individual, noting that the entire left-most dorsal kinety and the posterior half of right-most dorsal kinety can be observed from the ventral view. 5. Ventral view of a middle divider, showing the oral and cirral primordia (arrows) for both proter and opisthe and the dorsal kinety anlagen (arrowheads). 6. Detailed infraciliature of oral region, showing the 3–4 enlarged adoral membranelles (arrow) and the zig-zag fibril structure (arrowheads) along adoral zone of membranelles. 7. Dorsal view showing endoplasmic granules and 3 dorsal bristles. 8. Nuclear apparatus of an early divider, showing the two micronuclei (arrows) and macronuclear replication bands (arrowheads). 9. Ventral view of an early divider, with simultaneously formed oral primordia for the proter (arrow) and opisthe (arrowhead). AZM, adoral zone of membranelles; BC, buccal cirrus; CC, caudal cirri; DK, dorsal kineties; EM, endoral membrane; FC, frontal cirri; FVC, frontoventral cirri; LMR, left marginal row; Ma, macronuclei; PM, paroral membrane; PVC, postoral ventral cirri; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri. Bars ¼ 40 mm.
Description of the morphology based on the Chinese population Size 80–150 20–30 mm, mostly 120 25 mm in vivo; body shape rather constant and bipartite, trunk having elongate ellipsoidal outline, but with a narrow neck-like constriction at anterior 1/5 of body length. Left and right body margins almost straight and parallel to each other, posterior end bluntly rounded (Figs. 1, 10–12). Dorsoventrally flattened, width:thickness ratio about
2:1, ventral side flat, dorsal often vaulted in mid-body (Figs. 2 and 15). Cell flexible but non-contractile. Pellicle thin, no cortical granules recognizable. Endoplasm colorless and opaque, typically packed with 2–3-mmsized granules (Figs. 7 and 16). Nine to 17 macronuclear nodules (Ma) arranged in a ring in trunk region, each nodule ovoid to ellipsoid (about 5 4 mm after impregnation), with small nucleoli; two micronuclei, each 2–3 mm across, one anterior and one posterior, located
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Figs. 10–16. Neotype population of Trachelostyla pediculiformis from life. 10, 12. Ventral view of two typical specimens, arrows mark the anterior adoral membranelles and arrowheads indicate the prominent transverse cirri. 11. A small individual. 13. Anterior portion of cell. 14. Ventral view, to show the stiff dorsal cilia. 15. Left side view, arrowhead marks the transverse cirri. 16. Endoplasmic granules. AZM, adoral zone of membranelles; DK, bristles of dorsal kinety. Bars in Figs. 10–13, 15 ¼ 30 mm, in Figs. 14 and 16 ¼ 15 mm.
inside macronuclear ring (Figs. 4, 20, 28 and 29). No contractile vacuole observed. Feeding on bacteria digested in 5–10-mm-sized food vacuoles. Movement rapid, sometimes jerky on microscope slide and between sediment particles, body showing great flexibility. Adoral zone occupies about 40% of body length in life, composed of about 40 membranelles that form two groups of different appearance: the apical three to five membranelles strong and about 15–20 mm long, extended radially; the others conspicuously shorter, densely arranged along the left margin of buccal field (Figs. 1, 10, 12 and 13). Bases of longest membranelles about 3 mm in length (Figs. 3, 6, 20, 28 and 29). A zig-zag structure (arrowheads in Figs. 6 and 28), probably composed of fibers, always positioned along adoral zone of membranelles (AZM). Undulating membranes inconspicuous, arranged approximately in parallel, slightly curved; paroral about 13 mm and endoral 6 mm long (Fig. 6). Right marginal row (RMR) composed of about 26 cirri, begins at anterior 1/5 of body length and ends near the rightmost transverse cirrus. Left marginal row (LMR) composed of about 19 cirri terminating subcaudally (Figs. 3, 6, 19 and 24; Table 1). Frontal–ventral–transverse cirri (FVT-cirri) in a typical 8:5:5 pattern. Consistently 11 cirri in frontal area, which are homologous with 8 frontal and 3 postoral ventral cirri in other typical oxytrichids: three anterior-most (FC), four posterior frontal cirri (FVC), one buccal cirrus (BC) and three ‘‘ventral cirri’’ (PVC). Two pretransverse ventral cirri (PTVC) anterior of transverse cirri (TC) (Figs. 3, 18 and 27). Five TC highly
developed, arranged in a J-shaped row. Three inconspicuous caudal cirri (CC), of which the right two are often arranged close to each other on right of body midline (Figs. 3, 21, 22 and 26). Cilia of marginal, caudal, frontal and ventral cirri about 5–7 mm, while TC about 12–15 mm long. Dorsal kineties (DK) composed of six rows of stiff and conspicuous bristles 7–8 mm in length, extend whole body length (Figs. 1, 4, 14 and 23). Notes on morphogenesis Two individuals at early stages of morphogenesis were observed in protargol preparations. Some basic features can be recognized as follows: (1) FVTprimordia in both dividers (arrow and arrowhead in Fig. 9) develop de novo on the surface of the cell (Figs. 5 and 25, arrows); (2) replication bands of macronuclear nodules (Fig. 8, arrowheads) appear in early stage of morphogenesis; (3) the dorsal kinety anlagen originate from the old structure (Fig. 5, arrowheads). Comparison of Chinese population with Trachelostyla pediculiformis populations This species was originally described by Cohn (1866) under the name Stichochaeta pediculiformis and later was transferred by Kahl (1932) into the genus Trachelostyla (see Maeda and Carey (1984) for review and introduction for comment on nomenclature). Cohn (1866) presented some living information such as body size, shape, behavior, and appearance of cytoplasm and adoral zone. More importantly, he mentioned that this
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Figs. 17–29. Neotype population of Trachelostyla pediculiformis after protargol impregnation. 17, 28. Ventral views of infraciliature, showing anterior portion of cells; arrow in Fig. 17 marks the enlarged distal adoral membranelles and arrowheads in Fig. 28 indicate the zig-zag fibril structure along adoral zone of membranelles. 18. Focus on the 11 cirri (arrows) on anterior portion of cell. 19. Showing the three frontal cirri (arrows), anterior ends of left marginal row (double-arrowheads) and right marginal row (arrowhead). 20, 29. Focus on nuclear apparatus, arrows in Fig. 20 indicate micronuclei. 21, 22. Posterior ends of cells, arrowheads mark the caudal cirri. 23. Dorsal view, to note the dorsal kineties (arrows). 24, 27. Posterior portion of ventral side, arrows in Fig. 24 mark marginal rows and arrows in Fig. 27 indicate the two pretransverse ventral cirri. 25. Mid-portion of a middle divider; arrows mark the separated oral primordia for proter and opisthe. 26. Focus on the three caudal cirri (arrows) and dorsal cilia (arrowheads). AZM, adoral zone of membranelles; Ma, macronuclei; TC, transverse cirri.
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Fig. 30. Trachelostyla pediculiformis (A, after Cohn 1866; B, from Maupas 1883; C, after Gourret and Roeser 1888, from Kahl 1932; D, from Kahl 1928; E, from Kahl 1932; F, from Kahl 1933; G, from Borror 1963; H, from Biernacka 1963; I, from Kattar 1970; J, from Borror, 1972; K, from Jones 1974; L, from Maeda and Carey 1984), T. rostrata (M, from Small and Lynn 1985, called ‘‘T. pediculiformis’’; R, from Lepsi 1962), T. caudata (N, from Kahl 1932), Spirotrachelostyla simplex (Kahl, 1932) nov. comb. (O, from Kahl 1932, originally Stichotricha simplex Kahl 1932, transferred to Trachelostyla by Borror in 1972), S. spiralis (Dragesco & Dragesco-Kerne´is, 1986 nov. comb. (P, Q, from Dragesco and Dragesco-Kerne´is 1986, formerly T. spiralis) and S. tani (Hu & Song, 2002) nov. comb. (S–U, from Hu and Song 2002; formerly T. tani).
organism had three CC, though he was not convinced about the number. Nearly 20 years later, Maupas (1883) redescribed this species in great detail. Some diagnostic features were given as follows: cell length 100–200 mm, outline elongate; body highly flexible but non-contractile; cytoplasm granulated, grayish and opaque; about 14–20 macronuclear nodules and two micronuclei; left and right marginal cirri rows not confluent at the posterior end of body; 11 cirri in frontal area, two PTVC, five TC and three CC (Fig. 30B). There have been at least 10 redescriptions of this organism since 1883 (see list of synonyms). These redescriptions are basically in accordance with Cohn (1866) and Maupas (1883) in features including body size and shape, flexibility and cytoplasmic characters (Figs. 30A–L). Nevertheless, disagreements remain, especially in the following five aspects (Table 2): (1) The number of macronuclear nodules. Cohn (1866) did not see the nuclear apparatus, but illustrated
in his Fig. 38b (not shown in the present paper) two bright patches, which he interpreted as contractile vacuoles. However, he did not see contraction. We know that the macronuclear nodules sometimes occur as bright areas, especially when the cells are packed with various inclusions. Thus, one cannot exclude the possibility that Cohn’s type population had indeed only two macronuclear nodules. Another possibility is due to Maupas (1883), who first explicitly stated his population has about 14–20 macronuclear nodules. This record basically agrees with most of the subsequent redescriptions by Kahl (1928, 1932), Biernacka (1963), Borror (1963), Kattar (1970), Jones (1974) and Maeda and Carey (1984), and hence, in our opinion, is virtually authoritative in identification of this species. Considering the majority rule and more consistency with present classification system, we prefer the latter possibility. Stichochaeta corsica Gourret and Roeser, 1888 was described with a
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(2)
(3)
(4)
(5)
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single macronucleus, which has to be interpreted as a mis-observation. Caudal cirri. Cohn (1866) did not conclusively determine the number of CC, but later Maupas (1883) confirmed the presence of three CC in this species. Gourret and Roeser (1888) and Kahl (1928) also mentioned that there are several long and fine cirri at the posterior end of cells (Fig. 30C and D). In contrast to his previous description in 1928, Kahl (1932) suggested that the three filose cirri in the caudal area, which Maupas (1883) described as CC, were in fact dorsal cilia which could be seen from the ventral side. This interpretation was accepted by subsequent authors (Biernacka 1963; Borror 1963; Kattar 1970; Jones 1974; Maeda and Carey 1984). The present study revealed that this organism consistently has three CC (Figs. 3, 21, 22 and 26). Hence, we believe that Kahl’s conclusion is due to a misinterpretation, i.e. he overlooked these fine cilia. In fact, it is virtually impossible to distinguish the thin CC from the long stiff dorsal cilia in live specimens. The number of the FVT cirri. Cohn (1866) did not clearly describe the cirral pattern and number of cirri. Maupas (1883) observed 11 cirri in the frontal area and 2 ventral cirri anterior of the TC, which agrees with Borror (1963), Kattar (1970), Jones (1974) and our observations, but contrary to Kahl (1928, 1932), Biernacka (1963) and Maeda and Carey (1984), who recorded that there were fewer cirri (5–10) in the frontal area and no ventral cirri anterior to the transverse ones. We suppose that 11 frontal-ventral cirri in the frontal area should be the typical pattern of this taxon, which is equivalent to eight frontal (including the single buccal one) and three postoral ventral cirri in other typical oxytrichids (Berger 1999), that is, three postoral ventral cirri have moved to the frontal area in this organism. The strongly granulated and opaque cytoplasm of T. pediculiformis may lead to undercounting of the cirri in living observations. A similar misinterpretation could also have been made by Borror (1963), who recorded 12 frontal-ventral cirri from Chatton–Lwoff impregnated specimens. The numbers of TC recorded are always 5 in our population, but variation (from 4 to 6), as noted by previous authors, indicates that this character might be population-dependent (Table 2). Dorsal kineties. The number of DK in most Oxytrichids (s.l.) is highly consistent (Berger 1999). This ciliature was recorded as 3 rows by Biernacka (1963) and Borror (1963), 2 rows by Kattar (1970) and 5 rows by Maeda and Carey (1984). However, we observed consistently 6 rows in the Chinese population (Tables 1 and 2). Cortical granules. The Chinese population lacks cortical granules, while Cohn (1866) described and
illustrated ‘‘geko¨rnte Linien’’ (granulated lines) along the cirral rows and dorsal bristle rows. Such ‘‘granulated rows’’ are possibly cortical granules, which often occur along the cirral and bristle rows. However, since the body of this species is rather fragile, opaque and highly granulated, detailed observation of living cells at high magnification is usually not easy. It is known that cortical granules of ciliates are only well recognized with modern microscopic techniques, there is reasonable doubt whether Cohn could truly tell the cortical granules apart from endoplasmic granules. This difference hence should not be over-interpreted.
Comparison with related species The morphotype from Chesapeake Bay, USA, illustrated under the name ‘‘Trachelostyla pediculiformis’’ by Small and Lynn (1985) and Lynn and Small (2002), has the following features: body elongate and nonspiral, two macronuclear nodules, 12 cirri in frontal area, two ventral and five TC (Fig. 30M). In our opinion, this form should not be named T. pediculiformis because of the presence of only two macronuclear nodules (vs. many macronuclear nodules). Up to now, the only species confirmed as possessing two macronuclei is T. rostrata Lepsi, 1962 (Fig. 30R). It is therefore reasonable to suggest that Small and Lynn’s illustrations probably represent a population of T. rostrata. Morphologically, T. caudata resembles T. pediculiformis in terms of body size (150–220 mm), having many macronuclear nodules and long dorsal cilia, but differs in its characteristic tail shape (conspicuously narrowed vs. almost equal in width relative to body trunk) (Kahl 1932).
Neotypification of Trachelostyla pediculiformis As mentioned above, the original description of T. pediculiformis is seriously incomplete and there are some discrepancies between subsequent redescriptions (Gourret and Roeser 1888; Biernacka 1963; Borror 1963; Carey 1992; Maeda and Carey 1984; Jones 1974; Kattar 1970). There is no type material available for these morphotypes, thus it seems wise to redefine this species and to designate a neotype (ICZN 1999; Foissner et al. 2002). According to Article 75.3 of the ICZN (1999), the designation has to comply with the following particulars: (1) the taxonomic status of the present species is somewhat unclear because the original description and subsequent redescriptions do not agree in some important features (see above for details); (2) for differentiation of T. pediculiformis from other related
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nominal species, see above; (3) the neotype is clearly described (see above) and illustrated (Figs. 3 and 4), so that the recognition of the specimen designated is ensured; (4) it is generally known that no type material is available from species described by Cohn (1866), Maupas (1883) and Kahl (1928, 1932, 1933), and furthermore, there is no indication that Biernacka (1963), Borror (1963), Kattar (1970), Jones (1974) and Maeda and Carey (1984) made permanent preparations of the present species; (5) there is strong evidence that the neotype is consistent with T. pediculiformis as originally described by Cohn (1866); (6) Cohn (1866) did not fix the type locality in the original description because his aquarium contained seawater from two different sites: Helgoland, Germany, and Dorsetshire, UK, consequently a description of the type locality, that is, the sample site of the neotype population, is given in the section material and methods; (7) the slide containing a protargol-impregnated neotype specimen is deposited in the Natural History Museum, UK with the registration number 2005:3:24:15. Systematic position of Trachelostyla pediculiformis inferred from SSrRNA gene sequence The length of the SSrRNA gene of the Chinese population was determined to be 1769 nucleotides (GenBank accession No. DQ057346). The LS tree constructed from SSrRNA gene sequences is shown as Fig. 31, which gives similar topology to that shown in the phylogenetic studies of stichotrichines by Foissner et al. (2004). Trachelostyla pediculiformis clusters with two species of the genus Gonostomum Sterki, 1878 and Orthoamphisiella breviseries Foissner et al., 2002, with weak bootstrap value of 55%. The branching pattern basically confirms the establishment of the family Trachelostylidae Small & Lynn, 1985, which includes both Trachelostyla and Gonostomum, and is morphologically distinguished from the family Oxytrichidae Ehrenberg, 1838 by the following features: (1) three postoral ventral cirri are positioned anteriorly; (2) undulating membranes are short relative to the length of adoral zone; and (3) the gonostomoid adoral zone (Small and Lynn 1985; Berger 1999; Lynn and Small 2002). Orthoamphisiella breviseries, which was recognized by Foissner et al. (2002) as a member of the family Orthoamphisiellidae Eigner, 1997, is clustered within the Trachelostya–Gonostomum-clade, the relationship with Gonostomum namibiense showing 59% bootstrap support; it is interesting that Orthoamphisiella has a gonostomoid oral zone, few frontoventral cirri, and anterior displaced postoral cirri (Foissner et al. 2002; Foissner et al. 2004). Reconsideration on the genus Trachelostyla Although 14 nominal species have been assigned to the genus Trachelostyla, only six of them have been
Fig. 31. A least-squares tree inferred from the nucleotide sequences of complete SSrRNA gene. Numbers at nodes represent bootstrap values (in %) out of 1000 replicates: the number is from the distance–matrix based least-squares method. Asterisks indicate bootstrap values less than 50%. The new sequence is highlighted in boldface.
recognized and widely accepted: T. pediculiformis (Cohn, 1866), T. caudata Kahl, 1932, T. simplex (Kahl, 1932) Borror, 1972, T. spiralis Dragesco & DragescoKerne´is, 1986, T. rostrata Lepsi, 1962 and T. tani Hu & Song, 2002 (for reviews see Berger 1999, 2001; Borror 1972; Buitkamp 1977; Hu and Song 2002; Fig. 30). Among these, two basic types or groups can be recognized according to the general appearance and ciliary configuration: one group is characterized by: (1) Oxytricha-like, dorsoventrally flattened but non-spiral body shape; and (2) possessing a typical pattern of frontoventral cirri (i.e. in 11+2 mode) as in T. pediculiformis. The second group has a spirally twisted body shape with conspicuously pointed cell ends and a variable number of frontoventral cirri (hence no typical pattern, usually several anterior ventral cirri present). In addition, organisms in the latter group seem to prefer to live in a tube-like lorica and hence are mostly sessile forms, though not all of them have been confirmed to have this life-style. The pointed and spirally twisted body shape, which is rare in oxytrichids and trachelostylids, represents evidently a response to a loricadwelling life-style (living in a tube which is either made by the organism itself or constructed from environmental fragments). Based on these differences, we believe that these two groups should be separated at the generic level and hence suggest the present genus
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should be split into two by redefining the genus Trachelostyla and establishing a new genus Spirotrachelostyla nov. gen.
Trachelostyla Borror, 1972 Redefined diagnosis Dorsoventrally flattened trachelostylids, body nonspirally twisted and elongate, with peristomal region conspicuously narrowed; ciliature generally in 11:2:5:3 pattern, i.e.11 cirri in frontal region; two ventral cirri located anterior to five TC; three CC present. One left and one right row of marginal cirri not confluent posteriorly. Species assignable At least two morphospecies, i.e. the type species T. pediculiformis (Cohn, 1866) and T. rostrata Lepsi, 1962 should clearly be included in this genus. The third one, T. caudata Kahl, 1932 could be a valid member of this genus on the basis of its general appearance (Fig. 30N), though its infraciliature remains undescribed.
Spirotrachelostyla nov. gen. Diagnosis: Spirally twisted trachelostylids with spindle-shaped body; about 13 cirri scattered on anterior peristomal region; two PTVC present or absent; usually five TC; one left and one right row of marginal cirri not confluent posteriorly; CC present. Generally sessile forms in a lorica. Etymology: Spirotrachelostyla is a composite of the Greek noun hespeira (spiral, helix) and the genus-group noun Trachelostyla. It alludes to the spirally twisted body and, like Trachelostyla, has feminine gender. Type species: T. spiralis Dragesco & DragescoKerne´is, 1986. Species assignable: Spirotrachelostyla spiralis (Dragesco & Dragesco-Kerne´is, 1986) nov. comb. [basionym: T. spiralis Dragesco & Dragesco-Kerne´is, 1986] (Figs. 30P and Q), S. tani (Hu & Song, 2002) nov. comb. [basionym: T. tani Hu & Song, 2002] (Figs. 30S–U) and S. simplex (Kahl, 1932) nov. comb. [basionym: Stichotricha simplex Kahl, 1932] (Fig. 30O). Comparison with related genera: Considering the spirally twisted and spindle-like body shape and the relative length of the AZM to body length (40–50%), three genera, namely Stichotricha Perty, 1849, Chaetospira Lachmann, 1856 and Urostrongylum Kahl, 1932, are rather similar to the new genus. However, both Stichotricha and Chaetospira have 2 ventral cirral rows (vs. a few ventral cirri, never in rows, or even absent in Spirotrachelostyla) and no TC (vs. present in Spirotrachelostyla), thus these taxa can be clearly separated (Foissner 1980; Song and Wilbert 1989; Foissner et al.
1991; Hu and Song 2001). Urostrongylum differs from Spirotrachelostyla nov. gen. in having a single row of ventral cirri (vs. several not in rows or even absent), strongly pointed posterior end (vs. slightly tailed and bluntly rounded) and absence of TC (vs. present) (Borror 1972).
Acknowledgments This work was supported by ‘‘The National Science Foundation of China’’ (Projects No. 30430090 and 40376045). The authors are grateful to helpful comments on the manuscript provided by reviewers.
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