THE stichotrichous family Amphisiellidae is typically characterized

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1 J. Eukaryot. Microbiol., 54(4), 2007 pp r 2007 The Author(s) Journal compilation r 2007 by the International Society of Protistologists DOI: /j x Morphological Redescription and Neotypification of the Marine Ciliate, Amphisiella marioni Gourret & Roeser, 1888 (Ciliophora: Hypotrichida), a Poorly Known Form Misidentified for a Long Time JIQIU LI a, XIAOFENG LIN a, CHEN SHAO b, JUN GONG a, XIAOZHONG HU b and WEIBO SONG a,b a Laboratory of Protozoology, College of Life Science, South China Normal University, Guangzhou , China, and b Laboratory of Protozoology, KLM, Ocean University of China, Qingdao , China ABSTRACT. The well-known marine hypotrichous ciliate, Amphisiella marioni Gourret & Roeser, 1888 has been repeatedly misidentified for over a century, which has led to great confusion in the species identification. Based on a population collected from mariculture water in Qingdao (Tsingtao), north China, the morphology and infraciliature were investigated using live observations and the protargol impregnation method. The Chinese population corresponds perfectly with the original description by Gourret and Roeser (1888). As the type species of Amphisiella, its morphological characteristics include: an elongated body about mm long in vivo; with grouped cortical granules arranged in irregular rows throughout the whole body; a contractile vacuole located slightly behind mid-body; adoral zone of membranelles slightly bipartite in structure with the anterior part ventrally located; amphisiellid median cirral row (ACR) extending to the level of the transverse cirri (TC); about eight frontal cirri left of the ACR; closely spaced marginal rows, displaced inwards, each with about 32 cirri; six TC arranged in a V-shape; no caudal cirrus; five to six dorsal kineties; and typically two macronuclei. We conclude that the populations described by Mansfeld (1923) and Wicklow (1982), respectively, were misidentified. To clarify the taxonomic status of this species, the population from the northern Chinese coast of the Yellow Sea is designated as a neotype. Key Words. Amphisiella marioni, marine ciliate. THE stichotrichous family Amphisiellidae is typically characterized by the presence of the amphisiellid median cirral row (ACR), which arises by a unique ontogenetic process (Eigner and Foissner 1994; Jankowski 1979). Based on redefinitions given by the authors, the type genus Amphisiella has the following morphological characters: one right and one left marginal cirral row; one relatively long ACR; more than one cirrus left of the ACR; transverse cirri (TC) obliquely arranged; and caudal cirri lacking (Eigner and Foissner 1994; Foissner 1988; Lynn and Small 2002; Petz and Foissner 1996; Voss 1992). Up to now, over 10 Amphisiella morphotypes have been reported and the morphology and division morphogenesis of most of them have been carefully investigated (Berger 2004; Berger and Foissner 1989; Blatterer and Foissner 1988; Eigner and Foissner 1994; Fauré-Fremiet 1954; Fernandez-Leborans 1984; Fernandez-Leborans and Novillo 1992; Foissner 1982, 1988; Gourret and Roeser 1888; Hu, Warren, and Suzuki 2004; Petz and Foissner 1996). In the spring of 2004, we found a stichotrichous ciliate in a mariculture water system, Qingdao, China. Subsequent observation demonstrated it to be conspecific with Amphisiella marioni Gourret & Roeser, 1888 (Borror 1972, 1979; Kahl 1932; Wicklow 1982). This gave us the opportunity to clarify the confusion in species identification and re-investigate its ciliary pattern and other aspects of its infraciliature for the first time since the original description. Corresponding Author: X. Lin, Laboratory of Protozoology, College of Life Science, South China Normal University, Guangzhou , China xlin@scnu.edu.cn 364 MATERIALS AND METHODS Samples were collected on 21 April 2004 from the discharge outlet of a mariculturing water system in Qingdao (Tsingtao, N; E), China. The water temperature was about 25 1C, salinity 31%, and ph about 7.9. Raw cultures were established at room temperature in the laboratory using Petri dishes filled with seawater from the sample site. Some rice grains were added to support microbial growth. This species was found in small numbers after several days, and attempts at pure culture failed. The living ciliates were isolated and examined using brightfield and differential interference contrast microscopy. The protargol silver staining method (Wilbert 1975) was used to reveal the infraciliature. All drawings were made at a magnification of 1,250X with the aid of a camera lucida (Nikon 80i, Japan). Measurements were carried out with an ocular micrometer (Nikon 80i). Terminology is mainly according to Berger (2004). RESULTS Redescription of Amphisiella marioni Gourret & Roeser, 1888 (Table 1, Fig. 1 23). Morphology and infraciliature of the Qingdao population. Cells were mostly mm long (Fig. 1, 9, 10). The body was elongate, with a length to width ratio 4 5:1. The anterior end was broadly rounded (Fig. 9 14, 17), while the posterior end was slightly tapered (Fig. 11, 12), dorsoventrally cell was flattened about 2:1. The ventral side was flat with three conspicuous grooves along marginal rows and amphisiellid median ciliary row (Fig. 1, 9). Body was flexible and only slightly contractile (Fig. 2, 3, 11 14). Buccal field was dominant, the adoral zone occupied about 30% 40% of cell length, of which the bases of membranelles were more or less bipartite and the membranelles in distal portion located characteristically on ventral side (Fig. 1 4, 7, 17). In addition, the lengths of cilia in two parts of adoral zone of membranelles (AZM) seemed not equally long: cilia in the distal part were clearly longer than those in the proximal portion (Fig. 1 3, 20). Cytoplasm was colorless to grayish, often filled with many shining globules (3 5 mm in diam.). Pellicle was comparatively thick, with dot-like cortical granules (about 0.5 mm in diam.), which were dark red in color and basically irregularly grouped in longitudinal lines distributed throughout whole body (Fig. 5, 6, 22, 23). Mitochondria-like structures, about mm in size, were very dense and positioned just beneath cell surface; they were conspicuous even at low magnifications (Fig. 2, 6, 20 23). Single contractile vacuole, about 10 mm in diam., located slightly behind mid-body near left margin (Fig. 1, 2, 10). Usually two large ellipsoid macronuclear nodules centrally located, about 14 8 mm in size, contained many spherical chromatin granules; they were usually detectable in vivo under differential interference contrast microscopy (Fig. 2, 4, 8, 19). Micronuclei were not observed. Locomotion was moderately quick. Individuals crawled on the bottom of a Petri dish or on debris, with short and frequent pauses and then changed their direction.

2 LI ET AL. TAXONOMIC STUDY OF AMPHISIELLA MARIONI 365 Table 1. Morphometrical data of the Qingdao population of Amphisiella marioni. Character Min. Max. Mean SD CV n Body length Body width Length of buccal field Number of adoral membranelles Number of frontal cirri Number of amphisiellid median cirri Number of transverse cirri Number of left marginal cirri Number of right marginal cirri Number of dorsal kineties Number of macronuclei Macronuclei length Macronuclei width All data are based on protargol-impregnated specimens. Measurements in mm. Including the buccal cirrus. CV, coefficient of variation in %; Max., maximum; Mean, arithmetic mean; Min., minimum; n, number of specimens investigated; SD, standard deviation. Bipartite AZM contained (mean 31) membranelles, with no distinct gap between the two portions (Fig. 3, 7, 20, arrows): distal part consisted of 8 10 membranelles, with cilia ca mm long; proximal part composed of membranelles, with cilia about 8 mm long (Fig. 7, 17 20). Undulating membranes were straight and parallel, anteriorly terminated near buccal cirrus (BC); paroral membrane consisted of a row of zig-zag kinetosomes; endoral membrane was single-rowed (Fig. 7). With seven to nine frontal cirri (FC, including BC), usually about five located anteriorly, and mostly three located in posterior area and longitudinally arranged as a short row (Fig. 7, 18, 19). ACR composed of (mean 36) densely spaced cirri, commencing near distal end of AZM and extending distinctly sigmoidally close to the TC. Bases of cirri in central portion of ACR were slightly longer than those at both ends (Fig. 7, 19). Marginal rows were conspicuously away from cell margins, hence near to each other, with and cirri in the right and the left marginal row, respectively (Fig. 1 3, 7, 9, 19); characteristically, the right row (RMC) extended anteriorly to about 30% of cell length (Fig. 7, 19). Invariably six TC arranged in V-shape, terminally located; cirri were about 10 mm long in life and thus extended distinctly projecting beyond posterior (Fig. 7, 19). Dorsal cilia loosely arranged, bristle-like, about 3 mm long, mostly in six rows, which extended over entire length of body (Fig. 8). Caudal cirri are lacking. DISCUSSION Comparison of Chinese population with other populations. The original description of A. marioni by Gourret and Roeser (1888) is rather brief, and no information was given on body size or the number of macronucleus. Fortunately, the illustration was excellent: (i) the adoral membranelles on the anterior margin are located on ventral side and appear to separate into two parts with different lengths of cilia although there is no conspicuous gap between them; (ii) the distal end of AZM extends to about 25% of buccal length on the right body margin; (iii) the ACR extends nearly to the posterior end of the body; (iv) the two marginal cirral rows are arranged quite close to each other; and (v) the low-positioned contractile vacuole (recorded in its original description) (Fig. 24). All these diagnostic characters and the general appearance demonstrate that our Qingdao population should be conspecific with it. Mansfeld (1923) reported a new species that also named as A. marioni (Fig. 25). However, this is very likely a misidentification because it is obviously different from the original description of A. marioni in the following points: (i) the orientation of the AZM structure, of which the distal end extends strongly to about 50% of buccal length on the right body margin (vs. only 25% in A. marioni); (ii) the general appearance of AZM, which is of clearly a uniform structure (vs. bipartite in A. marioni); and (iii) both the marginal cirri and those in the amphisiellid median (the ventral row) are more widely spaced. In addition, the TC is 7 (vs. 6) in number and seem much more conspicuous in Mansfeld s isolate. Wicklow (1982) gave a description of both morphology and morphogenesis of A. marioni, which was subsequently accepted by several authors (Berger 2004; Eigner and Foissner 1994; Petz and Foissner 1996). However, based on the comparison at the infraciliature level (Fig. 26), we are convinced that this is also a misidentification. It can be clearly separated from A. marioni sensu Gourret & Roeser as follows: (i) conspicuously narrow buccal cavity (vs. prominent in A. marioni); (ii) the posterior end of ACR conspicuously shortened (vs. extending nearly to the posterior end); (iii) with a short fragment of the ciliary row right of ACR (vs. absent); and (iv) two marginal rows far away from each other and hence located on the cell margin (vs. adjacent to the ACR in A. marioni). In addition, the bases of membranelles in the distal portion of AZM seem to be longer than those in the latter. Comparison with related species. Considering the living morphology, the infraciliature, and especially the morphometric data, Amphisiella annulata ((Kahl, 1928) Borror, 1972 (Fig ) must be extremely similar to A. marioni. The former is a common form and its morphology and morphogenesis have been investigated in detail (Berger 2004; Hu et al. 2004). According to the data available, it can be distinguished from A. marioni by its broadly rounded posterior end (vs. tapered in the latter), by the dorsally located collar membranelles (vs. ventrally located in A. marioni), by the uniform structure of its AZM in vivo (vs. bipartite in the latter), by the presence of pre-tc (vs. absent), by more membranelles, dorsal kineties, and cirri in the amphisiellid median row (Table 2), by differently arranged cortical granules (Table 2), and by the absence of mitochondria-like granules (vs. presence in A. marioni). Further, the A. annulata populations described by Kahl (1928, 1932) and Berger (2004) have several ringshaped structures in the cytoplasm making the identification of this species usually rather easy. Another marine form, Amphisiella capitata (Perejaslawzewa 1886) Borror, 1972 (Fig. 27) could be also compared with A. marioni. However, the infraciliature remains undescribed, and hence the identity of this species is uncertain. According to the original description and illustration, it differs from A. marioni in the strongly curved distal end of AZM (extending to 50% of buccal length vs. to 25% of buccal length) and the location of contractile vacuole (at anterior 30% of body length vs. slightly below the equator). Neotypification. As mentioned above, the original description of A. marioni Gourret & Roeser, 1888 was rather incomplete and both subsequent isolates of Mansfeld (1923) and Wicklow (1982) have been considered to be misidentifications (see above for details). Thus, it seems wise to designate a neotype (ICZN 1999; Foissner, Agatha, and Berger 2002) with silver-impregnated specimens. According to Article 75.3 of the ICZN (1999), the designation has to be accompanied by the publication of some particulars: (i) The taxonomic status of the present species is somewhat unclear because the original description and two

3 366 J. EUKARYOT. MICROBIOL., VOL. 54, NO. 4, JULY AUGUST 2007 Fig Amphisiella marioni from life (1 6) and after protargol impregnation (7, 8). 1. Ventral view of a typical individual. 2, 3. Ventral views, to show the shape variants; arrows in Fig. 2 mark the mitochondria-like granules, note the AZM have a bipartite trend (arrow in Fig. 3) and membranelles in anterior part of AZM located on ventral side. 4. Lateral view; arrow points to the ventrally located adoral membranelles. 5. Dorsal view, to show the distribution of dark-red cortical granules. 6. Showing the small cortical granules and the large mitochondria-like granules. 7, 8. Infraciliature of ventral (7) and dorsal (8) sides of the neotype specimen; arrow in Fig. 7 marks the bipartite AZM. ACR, amphisiellid median cirral row; AZM, adoral zone of membranelles; BC, buccal cirri; CV, contractile vacuole; DK, dorsal kinety; EM, endoral membrane; FC, frontal cirri; LMC, left marginal cirri; Ma, macronuclei; PM, paroral membrane; RMC, right marginal cirri; TC, transverse cirri. Scale bar 5 50 mm.

4 LI ET AL. TAXONOMIC STUDY OF AMPHISIELLA MARIONI 367 Fig Photomicrographs of the morphology and infraciliature of Amphisiella marioni from life (9 17, 20 23) and after protargol impregnation (18, 19). 9, 10. General appearance of ventral sides, note the allocation of amphisiellid median cirral row and marginal rows; arrow in Fig. 10 marks the contractile vacuole Curved specimens; arrows in Fig. 11, 12 point to the tapered posterior end, which is typical for free (undisturbed) individuals. 15, 16. Laterally views; arrow in Fig. 16 points to the projecting anterior collar. 17. Anterior part of body, note the collar adoral membranelles ventrally located. 18. Infraciliature of the buccal field. 19. Infraciliature of ventral side. 20. Ventral view under differential interference contrast microscopy; arrow marks the bipartite adoral zone of membranelles, arrowheads point to mitochondria-like granules To show the distribution of cortical granules and mitochondria-like granules (arrowheads). Scale bar 5 50 mm.

5 368 J. EUKARYOT. MICROBIOL., VOL. 54, NO. 4, JULY AUGUST 2007 Fig Related species or populations. 24. Amphisiella marioni (from Gourret & Roeser 1888). 25. A. marioni (from Mansfeld 1923). 26. A. marioni sensu Wicklow, 1982 (misidentification) (from Wicklow 1982). 27. Amphisiella capitata (from Perejaslawzewa 1886) Amphisiella annulata (from Berger 2004), morphology (28), infraciliature (29, 30) and cortical granules (31) A. annulata (from Hu et al. 2004), cortical granules (32), morphology (33) and infraciliature (34, 35).

6 LI ET AL. TAXONOMIC STUDY OF AMPHISIELLA MARIONI 369 Table 2. Morphological and morphometrical comparisons of Amphisiella marioni (present work) and two populations of Amphisiella annulata. Character Amphisiella marioni Amphisiella annulata A. annulata Body length in mm, after protargol impregnation Body length in mm, in vivo Number of adoral membranelles 31 (25 36) 47 (31 57) 52 (41 62) Number of amphisiellid median cirri 36 (31 45) 44 (25 54) 50 (45 61) Number of dorsal kineties Structure of AZM in vivo Bipartite according to the cilia length of membranelles Uniform as a single structure Uniform as a single structure Position of membranelles in On ventral side On dorsal side On dorsal side anterior part of AZM Pre-transverse cirri Absent Present Present Living characteristics No ring-shaped granules, but with numerous very large mitochondria-like granules With several ring-shaped granules without any conspicuous mitochondria-like granules Without ring-shaped or mitochondria-like granules Distribution of cortical granules On both ventral and dorsal sides; in irregular rows, not around dorsal cilia Only on dorsal side; grouped along dorsal kineties and around cilia Only on dorsal side; grouped along dorsal kineties and around cilia Data sources Present work Berger (2004) Hu et al. (2004) AZM, adoral zone of membranelles. (ii) (iii) (iv) (v) (vi) (vii) redescriptions do not agree in important features (see above for details). For a differentiation of A. marioni from related taxa, see above. The neotype specimen is clearly described and illustrated (Fig. 7, 8), so that the recognition of the neotype designated is ensured. It is generally known that no type material is available from the species described by Gourret and Roeser (1888) and Mansfeld (1923), and furthermore, that there is no indication that Wicklow (1982) designated a neotype. There is strong evidence that the neotype is consistent with A. marioni as originally described by Gourret and Roeser (1888). For a detailed comparison, see above. Unfortunately, the neotype does not come from very near the original type locality (northern Chinese coast of the Yellow Sea vs. a harbor in Bastia, Corsica, a French island in the Mediterranean Sea). However, both sites are marine habitats, and numerous free-living ciliates especially marine ones, which live in a comparatively homogenous medium are apparently cosmopolitan (Patterson, Larsen, and Corliss 1989) so that this point should not be overinterpreted (for a thorough discussion of this problem, see Foissner et al. 2002, p. 44, and Foissner 2002). A detailed description of the new type locality, that is, the sample site of the neotype population, is given in the Materials and Methods. The neotype slide containing the protargol-impregnated specimens is deposited in the Laboratory of Protozoology, Ocean University of China, China with the Registration No. Lin Based on the Qingdao population of A. marioni, we supply here an improved diagnosis, which matches the original description by Gourret and Roeser (1888) rather well. Improved diagnosis. Marine Amphisiella with elongated body shape, size about mm in vivo; grouped cortical granules, arranged in irregular rows throughout whole body; contractile vacuole located slightly behind the equator; adoral zone with membranelles, slightly bipartite in structure with the anterior part ventrally located; about eight frontal cirri left of ACR; ACR with cirri, extending to the level of the TC; closely spaced marginal rows, with and cirri in the right and left rows, respectively; six TC arranged in V-shape; typically six dorsal kineties; and usually two macronuclear nodules. ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (Project numbers: , , and ). LITERATURE CITED Berger, H Amphisiella annulata (Kahl 1928) Borror, 1972 (Ciliophora: Hypotricha): morphology, notes on morphogenesis, review of literature, and neotypification. Acta Protozool., 43:1 14. 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(London), 18: Fernandez-Leborans, G. & Novillo, A Morphology and taxonomy of two new species of marine ciliates (Ciliophora: Spirotrichea: Stichotrichida: Amphisiellidae). Proc. Boil. Soc. Wash., 105: Foissner, W Ökologie und Taxonomie der Hypotrichida (Protozoa: Ciliophora) einiger österreichischer Böden. Arch. Protistenkd., 126: Foissner, W Gemeinsame Arten in der terricolen Ciliatenfauna (Protozoa: Ciliophora) von Australien und Afrika. Stapfia, 17: Foissner, W Neotypification of protists, especially ciliates (Protozoa, Ciliophora). Bull. Zool. Nom., 59:

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