A multidisciplinary approach to describe protists: redescription of Chattonidium setense Villenueve,...

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J. Eukaryot. Microbiol., 50(3), 2003 pp. 175–189q 2003 by the Society of Protozoologists

A Multidisciplinary Approach to Describe Protists: Redescriptions ofNovistrombidium testaceum Anigstein 1914 and Strombidium inclinatum

Montagnes, Taylor, and Lynn 1990 (Ciliophora, Oligotrichia)LETIZIA MODEO,a GIULIO PETRONI,a GIOVANNA ROSATIa and DAVID J. S. MONTAGNESb

aDipartimento di Etologia, Ecologia ed Evoluzione, Universita di Pisa, Pisa, 56126, Italy, andbSchool of Biological Sciences, University of Liverpool, Liverpool, L69 7ZL, UK

ABSTRACT. We combined behavioral, morphological (live, stained, scanning and transmission electron-microscope), and moleculardata to redescribe two common, intertidal oligotrich ciliates, Novistrombidium testaceum and Strombidium inclinatum. Both specieswere collected from a rocky shore near Leghorn, Ligurian Sea. A literature review revealed four morphotypes of Novistrombidiumtestaceum that differ in subtle ways, including oral morphology. These differences may be diagnostic, but we do not consider themsufficient to distinguish different taxa. Although other studies have synonymised Strombidium inclinatum and S. sulcatum, based onoral structures, size, and nuclear structure, there are morphological distinctions between them. In particular, the present study supportsa lack of anterior protuberance in both live and preserved S. inclinatum, while S. sulcatum possesses a protuberance. The 18S rDNAmolecular data, in accordance with morphological and ultrastructural observations, indicate that the Strombidiida (Oligotrichia) constitutea well-supported clade. The separation of the genera within this clade, even between Novistrombidium and Strombidium, remainsunresolved, and the analysis of more species is required. Finally, we recommend that when possible, ecologists, morphological taxon-omists, and molecular biologists combine their expertise to provide comprehensive taxonomic descriptions.

Key Words. Ciliate, electron microscopy, marine, oligotrich, phylogeny, protargol, 18S rDNA, taxonomy.

OVER the last 10 to 20 years there have been substantialefforts to improve existing descriptions of both fresh-

water and marine oligotrich ciliates (e.g. Montagnes and Lynn1991; Petz and Foissner 1992; Song, Wang, and Warren 2000).These works provide data that substantially improve the rela-tively simple descriptions made over the last two centuries. Inthis work we have continued this effort, by providing data ontwo common, intertidal species, Novistrombidium testaceumand Strombidium inclinatum. Furthermore, we have taken thisopportunity to combine behavioral, morphological (live,stained, scanning and transmission electron-microscope), andmolecular data. Thus, we have taken advantage of the majortechniques of this century to improve the description of thesespecies.

MATERIALS AND METHODS

Live observations. Ciliates were viewed using direct inter-ference contrast microscopy with a Leitz microscope at 400–1,2503 magnification. For each species, at least 15 individualswere observed. To examine the phototactic behaviour, ciliateswere placed for 15 min in a 9-cm Petri dish, completely dark-ened except for a 1.5-cm hole through which light passed (300mmol photons m22 s21).

Fixation, staining, and electron microscopy. Ciliates wereprotargol-stained (Montagnes and Lynn 1987). Ciliates wereviewed using bright field and direct interference contrast mi-croscopy with a Zeiss Axiovert microscope at 200–1,600 3magnification. Images were captured with a 35-mm camera ora video camera (JVC model KY-F55B, 3-CCD, 750 horizontal3 480 vertical line resolution), attached to a Pentium II PCwith image analysis software (Scion image for windows, Scion,MD, USA).

For scanning electron microscopy, samples of Novistrombi-dium testaceum were fixed for 20 min at 20 8C in a 1:1 mixtureof 2% OsO4 and a saturated solution of HgCl2 both initiallydissolved in 33 ppt seawater, while samples of Strombidiuminclinatum were fixed for 20 min at 20 8C in 2% OsO4 in 33ppt seawater. All the specimens were then processed as de-scribed by Rosati et al. (1999). For transmission electron mi-croscopy, both species were fixed for 30 min at 20 8C in a 1:1combination of 2% OsO4 in distilled water and 5% glutaralde-

Corresponding Author: L. Modeo—Telephone number: 139-050-500840; FAX number: 139-050-24653; E-mail: [email protected]

hyde in 0.2 M cacodylate buffer, pH 7.4. Specimens were thenethanol-dehydrated, transferred to 100% acetone, and embed-ded in an Epon-araldite mixture. Thin sections were placed oncopper grids and stained with uranyl acetate and lead citrate.

Measurements. Examination of protargol-stained cells fol-lowed the recommendations of Montagnes and Lynn (1991).Meristic and morphometric data are presented as means (range).Unless otherwise indicated, measurements are from protargol-stained specimens. Morphometrics were made using the imageanalysis system described above; . 30 longitudinally orientedspecimens were examined. Counts of the ventral polykinetidsmay be variable due to the orientation of cells, which may ob-scure the smaller polykinetids in the cytostomal region. Fur-thermore, the dimensions of cells are based on osmium tetrox-ide and/or mercuric chloride fixed, and protargol-stained ma-terial, which may shrink cells (Jerome, Montagnes, and Taylor1993).

18S rDNA amplification and sequencing. For each species; 50 starved specimens were picked with a micropipette andwashed repeatedly in sterilized 33 ppt seawater to completelyremove food contaminants. Specimens were then placed in a0.2-ml PCR cap, harvested by centrifugation, fixed and washedwith 70% ethanol to minimize salt concentration, and let dry.Caps were stored at 220 8C. PCR mix was added directly intothe cap at the moment of use.

The small subunit ribosomal RNA (SSrRNA) genes were am-plified in a Gene Amp PCR system 2400 (Applied Biosystems)using the universal eukaryotic primer forward 18F6 Euk [59-AA(CT) CTG GTT GAT (CT)(CT)T GCC AG-39] for S. incli-natum, or 18S F9 [59-CTG GTT GAT CCT GCC AG-39] (Med-lin et al. 1988) for N. testaceum, and reverse 18S R1513 Hypo[59-TGA TCC TTC (CT)GC AGG TTC-39] (Petroni et al.2002). PCR products were purified and directly sequenced inboth directions by MWG-BIOTECH AG. To minimize the pos-sibility of amplification errors, high-fidelity Taqs were alwaysused (Expand High Fidelity PCR System, Roche Diagnostics;TaKaRa Ex Taq, TaKaRa Biomedicals), and the sequences oftwo amplicons were compared. Internal and external primersfor sequencing were derived from Sogin’s Laboratory Internethome page: http://www.mbl.edu/labs/Sogin/Pages/Methods.html and modified according to the characteristics of Spirotri-chea (Petroni et al. 2002).

Sequence availability and phylogenetic analyses. The ac-cession numbers of the nucleotide sequences determined in this

176 J. EUKARYOT. MICROBIOL., VOL. 50, NO. 3, MAY–JUNE 2003

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Fig. 1. Scanning electron (a, b, c) and direct interference contrast (d, inset) micrographs of Novistrombidium testaceum (strain S1). a, c.Ventral views: arrows, the two ends of the girdle. b. Dorsal view. d. Ventral view. Inset. Antero-ventral view: arrowhead indicates the oral cavity.APZ, anterior polykinetid zone; CC, cytoplasmic collar; G, girdle; L, oral lip; T, trichite groups; VPZ, ventral polykinetid zone. a, b, c, d, inset,bars 5 10 mm.

study, deposited in the EMBL Nucleotide Sequence Database,are: Novistrombidium testaceum strain S1 AJ488910; Strombi-dium inclinatum strain S3 AJ488911. The other sequences usedin phylogenetic reconstruction are available from GenBank/EMBL databases under the following accession numbers: As-pidisca steini AF305625 (Chen and Song 2002); Diophrys ap-pendiculata AY004773 (Chen and Song 2001); Euplotes aedi-culatus M14590, X03949 (Sogin et al. 1986), AJ305253 (Pe-troni et al. 2002); E. charon AJ305249 (Petroni et al. 2002); E.crassus AJ310492 (Bernhard et al. 2001), AJ305239,AJ305240, AJ305255 (Petroni et al. 2002), AY007437–40 (Ril-ey and Katz 2001); E. harpa AJ305252 (Petroni et al. 2002);E. eurystomus AJ310491 (Bernhard et al. 2001); E. magnicir-ratus AJ305250 (Petroni et al. 2002); E. minuta AJ310490(Bernhard et al. 2001), AJ305244–46 (Petroni et al. 2002); E.muscicola AJ305254 (Petroni et al. 2002); E. octocarinatusAJ310489 (Bernhard et al. 2001); E. parkei AJ305247 (Petroniet al. 2002); E. raikovi AJ305251 (Petroni et al. 2002); E. rar-iseta AJ305248 (Petroni et al. 2002); E. vannus AJ305241–43(Petroni et al. 2002); Euplotidium arenarium Y19166 (Petroniet al. 2000); Eutintinnus pectinis AF399169–71 (Snoeyenbos-West et al. 2002); Favella ehrenbergii AF399159–64 (Snoe-yenbos-West et al. 2002); Halteria grandinella AF194410(Shin et al. 2000), AY007441–44 (Riley and Katz 2001),AF164137 (Prescott et al., unpubl.); Holosticha multistylataAJ277876 (Shin et al. 2000); Laboea strobila AF399151–54(Snoeyenbos-West et al. 2002); Metacylis angulata AF399143–46 (Snoeyenbos-West et al. 2002); Onychodromus quadricor-nutus X53485 (Schlegel, Elwood, and Sogin 1991); O. grandisAJ310486 (Bernhard et al. 2001); Oxytricha granuliferaX53486 (Schlegel, Elwood, and Sogin 1991); Paraurostylaweissei AJ310485 (Bernhard et al. 2001); Pattersoniella viti-phila AJ310495 (Bernhard et al. 2001); Phacodinium metchn-icoffi AJ277877 (Shin et al. 2000); Protocruzia sp.1 X65153(Hammerschmidt et al. 1996); Protocruzia sp. 2 AF194409(Shin et al. 2000); Steinia sphagnicola AJ310494 (Bernhard etal. 2001); Sterkiella nova (previously Oxytricha nova Foissnerand Berger 1999) X03948, M14601 (Elwood, Olsen, and Sogin1985); Strobilidium sp. AF399122–27 (Snoeyenbos-West et al.2002); Strombidinopsis sp. AF399132–35 (Snoeyenbos-West etal. 2002); Strombidium purpureum U97112 (Hirt et al., un-publ.); Strombidium sp. AF399115–17 (Snoeyenbos-West et al.2002); Stylonychia lemnae AJ310496–7 (Bernhard et al. 2001);S. mytilus AJ310498–9 (Bernhard et al. 2001); Tetmemena pus-tulata (previously Stylonychia pustulata; Eigner 1997, 1999)M14600, X03947 (Elwood, Olsen, and Sogin 1985); Tintinnop-sis tubulosoides AF399108–11 (Snoeyenbos-West et al. 2002);Uronychia transfuga AF260120 (Chen and Song 2001).

Sequences were aligned using a sequence editor and aligner(ARB program package, Ludwig and Strunk 1997). The auto-mated alignment was then corrected by hand to optimize thebase-pairing scheme of the rRNA secondary structure (Van dePeer et al. 2000). Four filters were constructed to selectivelyremove the more variable positions before performing the phy-logenetic analysis: filter Cili50 retains only positions conservedin at least 50% of ciliates within a selection of 100 almost full-length ciliate sequences representative of all groups; filters Spi-ro50 and Spiro40 retain only positions conserved, respectively,in at least 50% and 40% of the Spirotrichea analyzed in the

present work; filter EukTermini retains all positions comprisedwithin the primers used for initial amplification. Gap positionswere also counted as characters in the filter construction. Phy-logenetic analyses were performed using the DNAPARS pro-gram for maximum parsimony (Felsenstein 1989) and theFastDNAml program for maximum likelihood (Felsenstein1981; Olsen et al. 1994) from the ARB package (Ludwig andStrunk 1997). For each reconstruction method, the four filterswere used. Eight trees were thus obtained, and their topologieswere compared to recognize stable nodes (Ludwig et al. 1998).Maximum parsimony tree stability was also warranted by boot-strap analysis. Interaktiv parsimony was used to add Strombi-dium sp. partial sequences AF399115–17 (Snoeyenbos-West etal. 2002) to maximum parsimony tree without altering thebranching order (Ludwig and Strunk 1997).

RESULTS

Novistrombidium testaceum Anigstein 1914(Oligotrichia, Strombidiidae)

(Fig. 1, 2, 3 (a–g))

Gross and fine structure. Size: 38 (range 30–47) mm long,40 (30–50) mm wide (in vivo 35–80 long, 30–70 mm wide).Shape: roughly obconical, posterior rounded to conical, anteriorend slopes obliquely from upper right to lower left. Large ven-tral oral groove occupies ; 30% of the ventral surface of theapical hemisphere. Pronounced oral lip on right of oral groove(Fig. 1a, c). Cytoplasmic collar (visible in SEM specimens, Fig.1c) surrounds dorsal perimeter of oral groove. Oral ciliature:bases of anterior polykinetids (APk) of anterior polykinetedzone (APZ) follow the oblique slope of apical end; starting fromright side of ventral surface at an upper level, APk continuearound dorsal surface and end on left side of ventral surface,below level where they started; displacement between APk oneither side of oral groove is ; 14 mm (SEM measurements).Ventral polykinetid zone (VPZ) composed of ventral polyki-netids (VPk), continuous with APZ. APZ comprised of 15 (14–17) APk, 3–4 mm apart. APk composed of 3 rows of ciliatedkinetosomes; maximum length of APk cilia 19 (16–20) mm.VPZ comprised of 10 (7–12) VPk, 1.6 (1.1–1.9) mm apart, end-ing underneath oral lip. VPk composed of 3 rows of ciliatedkinetosomes. In serial section the rows appear equally long.Paroral kinety extends beneath oral lip, along right border oforal cavity; composed of ciliated dikinetids. A tuft of cilia (vis-ible in TEM preparations) occurs in terminal part of oral region;it is not clear if it is formed by VPKs close together or isdistinct from both VPK and paroral kinety (Fig. 3e). Somaticciliature: a girdle kinety encircles cell, showing pronounced dis-placement of its ends; starting from right side of the ventralsurface, 19 (16–21) mm from the posterior, girdle kinety ex-tends around to dorsal surface and continues obliquely towardposterior, ending ; 14 mm below the origin (SEM measure-ments). Girdle kinety composed of 49 (41–56) dikinetids,spaced ; 1 mm apart, with one kinetosome bearing ; 1.5 mmlong cilia. Kinetosomes of girdle dikinetids originate 0.3 mmbelow cortex (Fig. 3d). Ventral kinety ; 19 mm long, extendsfrom the cell posterior towards base of oral groove; it forms acharacteristic left hook on posterior ventral surface. Ventral ki-nety lies in a groove and is composed of 27 (25–30) dikinetids,

177MODEO ET AL.—MULTIDISCIPLINARY APPROACH TO DESCRIBE PROTISTS


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Fig. 2. Schematic drawings of Novistrombidium testaceum morphotypes. a–d. N. testaceum (strain S1, this study). a. Ventral view indicatingthe oral ciliature and the displacement of the two girdle kinety ends (arrows). b. Ventral view. c. Anterior view. d. Shapes of the nuclei. e. N.testaceum from Anigstein (1914). f. N. testaceum from Kahl (1932). g. N. testaceum from Song and Bradbury (1998). APk of APZ, polykinetidsof anterior polykinetid zone; Bs, black shapes; Ma, macronucleus; Mu, mucocyst-like structures; Pa, paroral kinety; T, trichite groups; Vk, ventralkinety; VPk of VPZ, polykinetids of ventral polykinetid zone. a–f, bars 5 10 mm. g, bar 5 20 mm.

spaced ; 0.75 mm apart; only one kinetosome appears ciliated;cilia are ; 1 mm long. Perilemma: envelops cell and somaticciliature; extends enveloping the three ciliary rows of each APkup to ; 70% of their length (visible in SEM preparations).Cortex: irregular thickenings of dense material (Fig. 3a) dis-tributed over cell surface between the plasmalemma and cellmembrane. Clear zones, likely alveolar spaces, delimited byinternal membrane, supported by a microtubular layer (Fig. 3b).Polygonal platelets occur, over most of cell, from just belowouter margin of APZ to posterior. Platelets of polysaccharidicnature (Modeo et al. 2001), 100–120 nm thick, interconnectedby thin fibrous laminae variable in thickness and width (Fig.3b). Where platelets occur, microtubular layer separates alve-olar membrane system from platelets (Fig. 3b). Nuclei: mac-ronucleus typically C-shaped, often divided into two parts orconstricted to appear as two parts, but shape can vary (Fig. 2d).Macronucleus lies beneath the APZ and above level of girdle;59 (40–86) mm long, 3–6 mm wide. Micronucleus spheroid;typically centrally located between macronuclear segments;sometimes displaced laterally, 1.5–4 mm in diam. Extrusomes:trichite insertions follow oblique pattern of outer margin ofAPZ, above upper margin of cortical platelets, not closely as-sociated with the girdle kinety. Some (3–4) trichite groups mayinsert near ventral kinety. Extruded trichites appeared as rodswith a head (in protargol preparations). Trichites 14–18 mmlong, ; 0.45 mm wide; occur in ; 14 groups of 8–10 trichites.At cortical end, trichite groups surrounded by short curved tu-bules (composed of polysaccharidic substances, Modeo et al.2001) and encircled by microtubules; outside the perimeter de-limited by microtubules, many mitochondria occur (Fig. 3g).Under the cortex, each trichite covered by dense material form-ing a ‘‘cap’’ (Fig. 3f). Trichite structure (Fig. 3f, g), membrane-bound with three distinct compartments: external amorphouselectron-dense layer (EL), 0.03 mm thick; layer consisting ofconcentric sheets (CSL), 0.13 mm thick; lumen (L), 0.15 mm indiam. (for details, see Modeo et al. 2001). Mucocyst-like struc-tures occur just below and between APk, composed of 15 (14–17) spherical bodies 1.8 mm in diam. (Fig. 3c). Other spheroidor rod-shaped bodies of unknown nature (1.5 mm, 1.0–2.2) oc-cur primarily below but sometimes beside or above macronu-cleus (darkly staining with protargol); these structures alternatewith trichite groups.

Live observations and behavior. Cells usually transparent.Swimming behavior similar to that described by Fenchel andJonsson (1988) for Strombidium sulcatum, redescribed by Mon-tagnes, Taylor, and Lynn (1990) as S. inclinatum: ciliates rotateabout their central axis, swimming through the water in helicesclockwise or anti-clockwise alternatively, but this movement isaccompanied by to and fro oscillations, conferring to the wholeswimming path a waving appearance. Sudden tumbles occurirregularly during movement. There was no phototactic re-sponse.

SSU rRNA gene sequences. The almost complete 18SrRNA gene sequence of Novistrombidium testaceum (strain S1)was determined through the direct sequencing of PCR productsand deposited in EMBL database (Accession numberAJ488910). Novistrombidium testaceum 18S rDNA is 1733 bplong (excluding primers) and has a GC content of 48%. When

gaps in the alignment are not considered as characters, N. tes-taceum 18S rDNA shares a 94.8% similarity with S. inclinatumand a 94.0% similarity with S. purpureum, the only otherStrombidium species whose 18S rDNA included in our phylo-genetic analyses (see discussion) (Fig. 4, 5). The similarityshared with Laboea strobila, the only other strombidiid ana-lyzed to date, is 90.9%.

Time and locality of isolation of type material. Ciliateswere isolated in Autumn 1997 from pools on a rocky shorenear Leghorn (Ligurian Sea) 438 309 N, 108 209 E: water tem-perature 19 8C, salinity 33 ppt. The rocks form a platform onwhich pools (50–100 cm diam., 5–10 cm deep) may be inter-connected and linked to the open sea by channels at high tide(tidal amplitude, 20–30 cm) or when waves occur. The bottomof pools was covered by medium-coarse sand.

Deposition of type material. A slide of protargol-stainedNovistrombidium testaceum (dated) representing the hapanto-type resides in the collections of the Natural History Museum,London, UK, with registration numbers 2003:1:4:1.

Strombidium inclinatum Montagnes, Taylor, and Lynn 1990(Oligotrichia, Strombidiidae)

(Fig. 3 (h–n), 6, 7)

Gross and fine structure. Size: 26 (range 14–32) mm long,20 (15–28) mm wide (in vivo 32–46 long, 25–37 mm wide).Shape: roughly obconical, posterior rounded to bluntly-pointed,anterior end slopes obliquely from upper right to lower left. Anapical protrusion is not evident in either fixed or live specimens(Fig. 6a, e, g). A non-prominent lip, on right of oral groove, isvisible in SEM preparations (Fig. 6d). Oral ciliature: bases ofanterior polykinetids (APk) of anterior polykinetid zone (APZ)follow slanted anterior end, starting from right side of ventralsurface at an upper level, APZ continues around dorsal surfaceand ends on left side of ventral surface, below level where itstarted; displacement between ends of APZ on either side oforal groove is ; 5 mm. Ventral polykinetid zone (VPZ) com-posed of ventral polykinetids (VPk), continuous with APZ. APZcomprised of 14 (12–16) APk, 2–4 mm apart. APk composedof 3 rows of ciliated kinetosomes; maximum length of APk cilia15 (12–17) mm. VPZ comprised of 8 (7–10) VPk, 0.9 (0.7–1.2)mm apart; proximal 2–4 sit in oral vestibule. VPk composed of3 rows of ciliated kinetosomes, two equally long plus innermostshorter one of 5–6 kinetosomes (Fig. 3l). Paroral kinety locatedwithin oral cavity, under oral lip, on right side, composed ofdikinetids. Somatic ciliature: equatorial girdle kinety complete-ly encircles cell, no break observed, ; 3 mm below the VPZ,13 (10–16) mm above posterior end of cell. Girdle kinety com-posed of 37 (32–44) dikinetids spaced ; 1 mm apart, inserted0.3 mm into cortex, with both kinetosomes ciliated (Fig 3j, k).One ; 0.7 mm-long cilium per dikinetid, emerging from cell,slightly bent with respect to kinetosome (Fig. 3k); granularbody (; 0.3 mm thick) within axoneme of emergent cilium (Fig.3k, inset). Bulges occur between emerging cilia (Fig. 6b),caused by second dikinetid cilium, which is sharply bent, lyingclose to alveolar system under perilemma (Fig. 3j, k). Ventralkinety ; 4 mm long, lies in ; 7 mm-long groove, extendingupward from cell posterior; composed of 7 (5–9) dikinetids,


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Fig. 3. Transmission electron micrographs of Novistrombidium testaceum (strain S1, this study) (a–g) and Strombidium inclinatum (h–n). Thecortex of N. testaceum (a, b): arrow, microtubular layer; arrowhead, the lamina joining the platelets. c. Kinetosomes of an anterior polykinetid ofN. testaceum. d. Girdle kinety cilium of N. testaceum. e. Proximal region of the oral cavity of N. testaceum. Longitudinal (f) and cross sections(g) of trichites of N. testaceum: arrows, the microtubular layer surrounding the trichite group. The cortex of S. inclinatum (h, i): arrow, microtubularlayer. j. Girdle dikinetid cilium of S. inclinatum trapped under perilemma. k. Emergent girdle dikinetid cilium of S. inclinatum: arrows, cilium;inset, granular capping body. l. Proximal part of the oral cavity of S. inclinatum (note the innermost row of ventral polykinetids considerablyshorter than others). Longitudinal (m) and cross-sections (n) of trichites of S. inclinatum: arrow, microtubular layer. CSL, concentric sheet layer;CT, curved tubules; DT, dense thickenings; EC, electron-dense cap; EL, external layer; ER, electron-dense rings ; L, lumen; LS, lens-shapedstructure; Mi, mitochondrium; Mu, mucocyst-like structure; P, polysaccharidic platelets; Pa, paroral kinety; Tc, tuft of cilia; VPk, ventral poly-kinetids. a, b, bars 5 0.3 mm; c, bar 5 0.5 mm; d, bar 5 0.3 mm; e, bar 5 2 mm; f, g, bars 5 1 mm; h, i, bars 5 0.3 mm; j–n, bars 5 0.5 mm.

spaced ; 0.5 mm apart; one kinetosome ciliated, bearing ; 0.8mm-long cilium. Perilemma: as determined by observing manyultrathin sections from different cells at different levels; it en-velops the whole cell including cilia of girdle, ventral kinety,and proximal region of APk cilia. Cortex: lens-shaped struc-tures, composed of opaque, homogeneous material, occur be-tween perilemma and cell membrane (Fig. 3h). External alve-olar membrane wrinkled (Fig. 3i). Polygonal polysaccharidicplatelets (100–120 nm thick) occur under cell surface, posteriorto girdle kinety (Modeo et al. 2001). Microtubular layer under-lies cortical membranes; in posterior it separates cortical mem-branes from platelets (Fig. 3h, i). Thin laminae connect plate-lets, but are not polysaccharidic (Modeo et al. 2001). Nuclei:macronucleus, spheroid to ovoid, located slightly left of cellcentral axis, half way between poles, 11 (8–16) mm long, 8 (4–12) mm wide. Spheroid micronucleus located near posterior orleft-lateral edge of macronucleus; 3.8 (1.2–6.4) mm in diam.Extrusomes: trichites insert above girdle kinety, grouped in ;3 mm-thick band, in regular longitudinal rows of 6–8 trichites.Trichites, 9–12 mm long, 0.35 mm wide; form funnel-like com-plex within cell, extending from insertion towards, but notreaching, posterior. Trichite structure has been illustrated in de-tail elsewhere (Modeo et al. 2001). They are membrane-boundwith three distinct compartments: external layer (EL) 0.08 mmthick; concentric sheets layer (CSL) ; 0.04 mm thick; lumen(L) 0.11 mm in diam. Lumen with electron-dense ‘‘rings’’ (ER),0.02 mm high, occurring at a distance from each other thatbecomes smaller closer to cortical end of structure (Fig. 3m).Trichites covered by ‘‘cap’’ of dense material at cortical end;caps covering trichites of the same row (6–8 trichites) are con-tiguous, forming a layer under pellicle. Microtubule sheetsemerge from caps, paralleling trichites along ; 20% of theirlength (Fig. 3m). Short curved tubules occur in cytoplasm sur-rounding trichites (Fig. 3m, n). Cells may contain 10 (4–20)food vacuoles 1–5 mm in diameter.

Live observations and behavior. Cells often light orange-colored, especially at posterior, due to the presence of vacuoleslikely containing carotenoid material retained from digestedprey or secondary metabolites. Swimming behavior typical ofstrombidiids (Fenchel and Jonsson 1988): ciliates rotate abouttheir central axis swimming in helices with a narrow pitch anda clockwise rotation. This movement is not accompanied by thewaving oscillations described for Novistrombidium testaceum.The swimming proceeds along a straight line and is interruptedby frequent sharp tumbles. The ciliate is positively phototactic,accumulating in illuminated areas.

SSU rRNA gene sequences. The almost complete 18SrRNA gene sequences of Strombidium inclinatum (strain S3)was determined through the direct sequencing of PCR productsand deposited in the EMBL database (Accession numberAJ488911). Strombidium inclinatum 18S rDNA is 1734 bp long(excluding primers) and has a GC content of 47%. When gapsin the alignment are not counted as characters, S. inclinatum

18S rDNA shares a 94.8% similarity with Novistrombidium tes-taceum (strain S1) and a 93.2% similarity with S. purpureum,the only other Strombidium species whose 18S rDNA is in-cluded in our phylogenetic analyses (see discussion) (Fig. 4, 5).The similarity shared with Laboea strobila, the only otherstrombidiid analyzed to date is 90.4%.

Time and locality of isolation of type material. Ciliateswere collected in Autumn 1997. They were isolated from thesame tidal pools on a rocky shore near Leghorn (Ligurian Sea)where specimens of Novistrombidium testaceum were found(see above).

Deposition of material. A slide of protargol-stained Strom-bidium inclinatum from this study (dated) resides in the collec-tions of the Natural History Museum, London, UK, with reg-istration numbers 2003:1:4:2.

Phylogenetic analysis. In all trees, the four strombidiidsStrombidium inclinatum, S. purpureum, Novistrombidium tes-taceum, and Laboea strobila, form a stable, highly supported,monophyletic clade (Oligotrichia).

In most phylogenetic reconstructions with both maximumlikelihood and maximum parsimony alghorithms (Fig. 4, 5), L.strobila branches basally to the other strombidiid species, witha statistical support below 70%. In all maximum likelihoodtrees N. testaceum branches together the two Strombidium spe-cies as a sister taxon of S. purpureum (Fig. 4) whereas in max-imum parsimony trees the two Strombidium species form aclade separate from N. testaceum, although never supported bybootstrap percentages above 50%.

With the present dataset of sequences, both the Strombidiida(Oligotrichia), and Choreotrichia are well-supported clades;they also join together in a well-supported group. However, theHalteriida do not cluster with Strombidiida; they fall withinStichotrichia.

DISCUSSION

Both species examined in this study have been described, butthere are controversies associated with their status. Thus, wehave added a substantial amount of new information on thesetaxa at a number of levels. Below we first assess the descrip-tions of the taxa, comparing our data to those in the literature.We then examine the Strombidiidae phylogenetically in thecontext of a larger data set, using molecular data.

Novistrombidium testaceum. Anigstein (1914) made a de-tailed description of Strombidium testaceum (Fig. 2e), whichhas now been designated the type of the genus NovistrombidiumSong and Bradbury 1998. Strombidium obliquum Kahl 1932(Fig. 2f) was also synonymised with Novistrombidium testa-ceum and a third morphotype (Fig. 2g), identified from marineenclosures in China (Song and Bradbury 1998). The diagnosisof Novistrombidium is: Strombidiidae with incomplete girdlekinety around the equatorial area that is conspicuously openwith a large ventral gap, through which ventral kinety extends(Song and Bradbury 1998). Although Novistrombidium may


Fig. 4. Maximum likelihood tree inferred using filter Cili50. The sequences of the species here analysed are represented in bold as are thenames for subclass groupings. Evolutionary distance is represented by the length of the horizontal components. The scale bar corresponds to 10changes per 100 positions. Species comprised in the same genus are grouped. * indicates consensus sequences used.

represent a distinct lineage containing other species (Agatha,S., pers. commun.), the use of relative terms in the diagnosismakes it imprecise. Also, our molecular analysis (Fig. 4, 5)suggests that Novistrombidium might not be a distinct lineage.Furthermore, the position and structure of the somatic kineties,used to diagnose Novistrombidium, are highly variable in theStrombidiidae, and these features may have arisen more thanonce. Finally, there was no ventral kinety observed in the orig-inal population of the type species of this genus, although de-tailed observations were provided for the girdle kinety and theposterior region of the cell (Anigstein 1914); there was also noventral kinety noted by Kahl (1932). If Novistrombidium tes-taceum described by Anigstein (1914) actually lacks a ventral

kinety, then it lacks a diagnostic feature of the genus (sensuSong and Bradbury 1998), creating a serious conundrum. Basedon the points raised above, a revised diagnosis of Novistrom-bidium, using morphological features, is now being considered(Agatha, S., unpubl. data). We further suggest that, after moremolecular data are available, a rigorous reassessment of a num-ber of the recently created genera of Strombidiids is needed(e.g. Krainer 1991, Song and Bradbury 1998, Song, Wang, andWarren 2000). We also propose that, given the above concerns,in the immediate future, taxonomists should take a more con-servative approach and refrain from creating new genera withinthe Strombidiidae; this approach will prevent the creation ofspurious lineages and will reduce the alienation of ecologists,


Fig. 5. Maximum parsimony tree inferred using filter Cili50. Note that Strombidium sp. partial sequences were independently added to theparsimony tree without altering its branching order (using interaktiv parsimony, see text), and have not been included in the phylogenetic analysis.The sequences of the species here analysed are represented in bold as are the names for subclass groupings. The numbers at the bifurcationsrepresent the bootstrap values out of 100 resampling of the data set. Evolutionary distance is calculated by interaktiv parsimony and representedby the length of the horizontal components. The scale bar corresponds to 10 changes per 100 positions. Species comprised in the same genus aregrouped. * indicates consensus sequences used.

who often find the idiosyncrasies of taxonomic debate to becounterproductive. At present, however, until the Strombidiidaehave been reassessed, we support maintaining the genus Nov-istrombidium.

We recognize four distinct morphotypes of Novistrombidiumtestaceum: N. testaceum, sensu Anigstein (1914); N. testaceum,as described above; N. testaceum, sensu Song and Bradbury(1998); N. testaceum, sensu Kahl (1932) (Table 1). The fourmorphotypes of N. testaceum are similar in size, habitat, APkand VPk number, trichite arrangement, nuclear structure, andposition of the girdle kinety. However, they differ in subtleways (Table 1, Fig. 2). We speculate that all four morphotypes

have a ventral kinety, although both Anigstein (1914) and Kahl(1932) did not observe one in live/fixed specimens; such struc-tures may be difficult to see without modern optics or stainingtechniques. However, the four morphotypes also differ in theiroral structures (Table 1, Fig. 2): N. testaceum (sensu Song andBradbury 1998) lacks an oral lip, collar, or anterior bump-likeprotuberance, and thus we question its identification as N. tes-taceum; however, as no type material exists for this descriptionwe cannot confirm the absence of these oral structures. In con-trast, the other three morphotypes have some form of oral pro-jection, of varying size and position; these minor differencesmay be diagnostic, but at this time, we do not consider them


Fig. 6. Scanning electron (a–d), Feulgen stained (e), and direct interference contrast (f, g) micrographs of Strombidium inclinatum. a. Dorsalview. b. Posterior view. c. Ventral view. d. Cilia of oral region; note the small oral lip (L). e. Whole cell. f. A dividing live-cell. g. A live cellviewed with direct interference microscopy. APZ, anterior polykinetid zone; G, girdle kinety; Ma, macronucleus; T, trichites; VPZ, ventralpolykinetid zone. a–c, bars 5 5 mm; d, bar 5 1 mm; e–g, bars 5 10 mm.


Table 1. Meristics and morphometrics distinguishing the morphotypes of Novistrombidium testaceum. All measurements are in mm, and numbersseparated by – are ranges.

Character

N. testaceumsensu Anigstein

(1914)N. testaceum

sensu this study

N. testaceumsensu Song andBradbury (1998)

N. testaceumsensu Kahl (1932)

Somatic length (live)Somatic width (live)Length:width ratio

70–8450–60;1.3

35–8030–70;1.0

50–7055–75;1.0

50—;1.0

Apical structure anterior protuberance filledwith granules

relatively large collar absent relatively smallcollar

Peristomial lip present (relatively small) present (relativelylarge)

absent absent

Ventral kinety not observed relatively distinct relatively distinct not observed

sufficient to distinguish different taxa. As deposited type ma-terial is lacking for the genus, we provide our hapantotype (seeabove) as a type for the genus and species.

Strombidium inclinatum. Montagnes, Taylor, and Lynn(1990) described S. inclinatum (Fig. 7d) and specifically distin-guished it from S. sulcatum (Fig. 7b, c). Subsequently, Song,Wang, and Warren (2000) redescribed S. sulcatum (Fig. 7e, f),using a population from China, and synonymized S. inclinatumwith S. sulcatum. We suggest that this synonymy may be im-prudent; our reasons follow. There are a number of morpho-metric differences between the characterized taxa (Table 2).These differences include cell size; length of anterior polyki-netids cilia; trichite length; the presence of an anterior bump-like protuberance, and macronuclear size, position, and shape.Note that the macronuclear shape of S. sulcatum was reassessedby us (Table 2, Fig. 7c), using original silver stains made byFaure-Fremiet (marked CC. viii. 53; Universite Paris Sud,France). Moreover, rather than examining specimens from thetype location, Song, Wang, and Warren (2000) based their syn-onymy of taxa isolated from the North Sea (S. inclinatum) andNorth Atlantic (S. sulcatum) on a species isolated from the Yel-low Sea (S. sulcatum sensu Song, Wang, and Warren 2000)(Table 2). Although protists may be ubiquitous (Finlay et al.1999), endemism likely occurs (Foissner 1999), and it thus maybe inappropriate to make synonymies based on specimens takenfrom such distinct and distant locations.

A main morphometric component of the description of S.inclinatum (Montagnes, Taylor, and Lynn 1990) and the sub-sequent synonymy of S. inclinatum and S. sulcatum (sensuSong, Wang, and Warren 2000) was the presence/absence ofan anterior bump-like protuberance. Faure-Fremiet (1912) andFaure-Fremiet and Ganier (1970) indicated that S. sulcatumpossesses an anterior protuberance in both live and preserved/silver-nitrate stained material (Fig. 7b). We have subsequentlyexamined the original slides of Faure-Fremiet (marked CC. viii.53, vi 68) used by Faure-Fremiet and Ganier (1970) and supportthe presence of an anterior bump-like protuberance.

Montagnes, Taylor, and Lynn (1990) observed live speci-mens of S. inclinatum and did not remark on the presence ofan anterior protuberance in these samples. Furthermore, theydid state that there was no protuberance in fixed/protargol-stained material (Fig. 7d). Following these observations, thepresent study has indicated a lack of protuberance in both liveand preserved S. inclinatum (Fig. 6c, e, g, Fig. 7a). Strombidiuminclinatum does possess a small oral lip, running along the rightside of the oral groove (Fig. 6d, see also Fig. 1 in Fenchel andJonsson 1988), but this is not similar to the anterior bump-likeprotuberance mentioned above. In contrast, Song, Wang, andWarren (2000) suggested that in their population of Strombi-dium from China (Fig. 7e, f), the protuberance disappeared

when cells were preserved and protargol-stained; although atone point they stated that the protuberance ‘‘is usually unde-tectable’’ and at another point that it ‘‘becomes completely un-detectable’’.

Thus, S. sulcatum (sensu Faure-Fremiet 1912; Faure-Fremietand Ganier 1970) has an anterior bump-like protuberance (invivo and preserved), S. sulcatum (sensu Song, Wang, and War-ren 2000) only has a protuberance in vivo, and S. inclinatumlacks a protuberance. Coupled with the other differences, es-pecially the difference in cell size and nuclear shape (Table 2),there appears to be a clear morphological distinction betweenS. sulcatum (sensu Faure-Fremiet 1912; Faure-Fremiet andGanier 1970) and S. inclinatum. We have also collected spec-imens of S. inclinatum from marine enclosures on the Isle ofMan (Irish Sea) (unpubl. data) that also lack an anterior pro-tuberance and in all other respects match the description of S.inclinatum; thus, S. inclinatum appears to occur in a number ofAtlantic habitats.

Although there are gross morphological differences betweenS. inclinatum and S. sulcatum, there are a number of ultrastruc-tural similarities (cf. Faure-Fremiet and Ganier 1970; Modeo etal. 2001). For instance, the trichite structure of the two speciesis almost identical. Furthermore, both species have a similar,but not identical, structure of the dikinetids of the girdle kinety;they both have lens-shaped structures occuring between the per-ilemma and the cell membrane, and show a granular body with-in the axoneme of the emergent cilium of each girdle dikinetid.These similarities lead us to suggest that S. inclinatum and S.sulcatum are closely related.

One of the strengths of the present study is that it combinesbehavioral, live, stained, SEM, TEM, and molecular data todescribe the studied species. The molecular data are an obviousmeans to assess the diversity of the strombidiids; we anticipatethat the only means by which this S. inclinatum-S. sulcatumquestion and the distinction between morphotypes of Novis-trombidium testaceum (see above) will be unequivocally solvedis by using molecular techniques. With this approach the close-ly related and often confused Euplotes crassus and E. vannusspecies have recently been separated (Petroni et al. 2002). Inthis case, species-specific oligonucleotidic probes were also de-signed for a rapid identification by means of in situ hybridiza-tion of these morphologically similar species (Petroni et al.2003). The molecular characterization of other strombidiid spe-cies, and the design of species-specific oligonucleotide probesfor their rapid recognition is clearly a challenge for future work.

Phylogenetic analysis. According to the molecular analysisof 18S rDNA, the Strombidiida constitute a well-supportedclade (Oligotrichia). These results based on molecular data arein accordance with those inferred by morphological and ultra-structural studies. Indeed, peculiar features such as trichites and


Table 2. Meristics and morphometrics of Strombidium sulcatum and S. inclinatum. Values are means or single values; all measurements arein mm, and numbers separated by – are ranges. All the measurements are after protargol staining except for those indicated by * which areobserved and/or supported by examination of original material, and those indicated by ** which are measured from figure. Abbreviations are asin text.

StudyS. inclinatum(this study)

S. inclinatum(Montagnes, Taylor,

and Lynn 1990)

S. sulcatum(Faure-Fremiet 1912;

Faure-Fremiet andGanier 1970)

S. sulcatum(Song, Wang, and

Warren 2000)

Habitat marine, tide poolssemi-planktonic

marine, semiplanktonic marine marine, maricultureponds

Location Ligurian Sea, Leghorn(Italy)

Northern Sea, Limfjor-den (Denmark)

Ligurian Sea, Ville-franche sur Mer;Roscoff; Endoume(France)

Qingdao (China)

Somatic length 14–32 12.5–30 30–50* 30–47Somatic width 15–28 12.5–21 25–30* 24–41Apical protrusion absent absent present* present in vivo, ab-

sent after fixationAPK number 12–16 12–15 12–14* 13–16Length of APK cilia 12–17 15 20 ;20–25**VPK number 7–10 7–9 7–8* 7–9Length of VPK cilia 3.2–6.7 4–7 3–8* —Macronuclear shape/position spheroid to ovoid/left

lateralellipsoid/left lateral compressed spheroid*/

centralspheroid/central

Macronucleus maximum width 4–12 6–12.5 ;12* (diameters ;123 6)

10–15**

Girdle kinety equatorial equatorial equatorial-subequa-torial*

equatorial

Dikinetids in the girdle 32–44 — ;50 ;50Ventral kinety length ;4 ;6** 10–12 ;8Dikinetids in ventral kinety 5–9 7–9 7–8* 7–8Trichites length 9–12 15–16 7–8 (TEM) 12–15

←

Fig. 7. Schematic drawings of protargol- stained and living Strombidium inclinatum and Strombidium sulcatum. a. S. inclinatum ventral view,from this study. b. S. sulcatum from Faure-Fremiet and Ganier (1970). c. Nuclear shape of S. sulcatum reassessed by us (Table 2), using originalsilver stains made by Faure-Fremiet (marked CC. viii. 53; Universite Paris Sud, France). d. S. inclinatum from Montagnes, Taylor, and Lynn(1990). e, f. S. sulcatum from Song, Wang, and Warren (2000). APk of APZ, polykinetids of anterior polykinetid zone; Gk, girdle kinety; Ma,macronucleus; Mi, micronucleus; Pa, paroral kinety; T, trichites; Vk, ventral kinety; VPk of VPZ, polykinetids of ventral polykinetid zone. a, b,bars 5 10 mm; d, bar 5 30 mm; e, bar 5 25 mm; f, bar 5 20 mm.

polysaccharidic platelets are present in Novistrombidium testa-ceum and Strombidium inclinatum as well as in the other stud-ied Strombidiida (Faure-Fremiet 1969; Maeda and Carey 1985;Montagnes, Taylor, and Lynn 1990).

However, phylogenetic relationship within the Oligotrichiaare not unambiguously resolved. Our data show molecular dis-tances always greater than 5% between the Strombidiida spe-cies so far characterized. Furthermore, the genus Laboea ap-pears to branch basally to other Strombidiida but with a branch-ing pattern that is not highly supported; it is noteworthy thatthis genus was synonimized with Strombidium by some authors(Kahl 1932; Maeda and Carey 1985). The analysis of morespecies will likely support the separation of the two genera.

The separation between the genera Strombidium and Novis-trombidium remains questionable due to the different topologiesof maximum likelihood and parsimony trees (Fig. 4, 5). More-over the lack of a rigorous morphological description of theStrombidium purpureum strain, to which the never officiallypublished sequence U97112 by Hirt et al. belongs, renders amorphological comparison impossible.

There are other Strombidium sequences in database, namelyStrombidium sp. sequences AF399115–17 by Snoeyenbos-Westet al. (2002). They were not included in the selection of speciesused for the calculation of maximum likelihood and maximum

parsimony trees for the following reason. The authors reportthem as partial sequences from the same species and use theirconsensus sequence for phylogenetic analysis, but the latter se-quence, in our opinion, can be considered a chimera. This hasbeen revealed by the independent addition of these three partialsequences to a reference tree using ARB interaktiv parsimony.By this method, in which the inclusion of partial sequences doesnot modify the branching order of the tree, Strombidium clonesAF399115–16 cluster within Oligotrichia, while Strombidiumclone AF399117 is closely related to Strobilidium sp. (Choreo-trichia) (Fig. 5). Moreover, sequence AF399117 weakly match-es AF399115–16 sequences in the overlapping region (data notshown).

The results here reported, in accordance with both morpho-logical (Petz and Foissner 1992) and molecular (Hoffman andPrescott 1997; Lynn and Sogin 1988; Riley and Katz 2001;Shin et al. 2000; Snoeyenbos-West et al. 2002) data so far col-lected, support the exclusion of genus Halteria, and likely theorder Halteriida, from subclass Oligotrichia. The latter taxonwould be represented only by the order Strombidiida.

Conclusion. This is one of the first studies on ciliates todiagnose taxa based on data from behavioral studies and ma-terial prepared for live, stained, SEM, TEM, and molecular ob-servations. Recently, Baumgartner, Stetter, and Foissner (2002)


redescribed Trimyema minutum using a combination of mor-phological, physiological, and molecular data. Other authors, ina set of related papers, have described the novel group of Li-tostomatea Macropodiniidae marsupial endosymbionts usingmorphological (Cameron and O’Donoghue 2001; Cameron,O’Donoghue, and Adlard 2001), ultrastructural (Cameron, andO’Donoghue 2002a, b), and molecular approaches (Cameron,Adlard, and O’Donoghue 2001). We are now at a turning pointin taxonomic studies. Molecular methodologies are well estab-lished and are clearly a new means to describe and discriminatetaxa. However, it will be many years, if ever, before moleculartechniques entirely supersede morphological taxonomic meth-ods. Especially in ecological surveys, where literally hundredsof taxa may be observed in a single study, molecular techniqueswill likely remain intractable. Thus, combining methodologies,as in this work, might provide a versatile approach. We rec-ommend that when possible, ecologists, morphological taxon-omists, and molecular biologists combine their expertise to pro-vide comprehensive taxonomic descriptions. Such multidisci-plinary works will hopefully help to unravel some of the bio-diversity questions that are being so hotly debated in the presentliterature.

ACKNOWLEDGMENTS

Thanks are given to: Anne Fleury-Aubusson (Universite Par-is Sud) for allowing the use of the original slides of Faure-Fremiet; Reinhard Kypke and Claudio Ghezzani for assistancewith figures; Simone Gabrielli for photographic assistance; Fa-brizio Erra for assistance with behavioral observations. Manythanks are given to Sabine Agatha for her guidance and con-structive criticism related to the designation of taxa.

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Received 07/26/02, 02/11/03; accepted 02/11/03

A multidisciplinary approach to describe protists: redescription of Chattonidium setense Villenueve,...

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