Morphologic and Molecular Data Suggest that Lynnella semiglobulosa n. g., n. sp.Represents a New Family within the Subclass Choreotrichia

(Ciliophora, Spirotrichea)

WEIWEI LIU,a ZHENZHEN YI,a XIAOFENG LINa and KHALED A. S. AL-RASHEIDb

aLaboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial

Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China, andbZoology Department, King Saud University, Riyadh 11451, Saudi Arabia

ABSTRACT. The morphology, morphogenesis, and phylogeny of an undescribed oligotrich sensu lato (s. lat.) ciliate, Lynnella semiglobu-losa n. g., n. sp., found in Daya Bay, southern China, were investigated. This species shares some features with both oligotrichs sensu strictoand choreotrichs, but most morphological and morphogenetic characters as well as the phylogenetic analysis suggest that it should be assignedinto subclass Choreotrichia temporarily. Lynnella semiglobulosa is distinguished from members of all known genera and families of thesubclass Choreotrichia by a unique combination of characteristics of the buccal and somatic ciliatures. Thus, a new family Lynnellidae n. fam.and a new genus Lynnella n. g. are proposed for it. The new family is distinguished by an open adoral zone of membranelles (AZM) in which,however, there are no ventral membranelles; the distal and proximal portions of the new adoral zone lie close to each other forming an opencircle in stomatogenesis. The new genus Lynnella is characterized by possessing two longitudinally oriented somatic kineties, one dorsal andone ventral, several proximal membranelles progressively lengthened toward the proximal end of adoral zone, and two macronuclear nodules.In phylogenetic analyses based on small subunit rRNA gene sequences, L. semiglobulosa clustered basally to all choreotrichs, but with rel-atively weak support; nevertheless, the possibility of a relationship with the subclass Oligotrichia was not rejected by the approximatelyunbiased nor Shimodaira–Hasegawa test. Based on morphological, morphogenetic, and molecular evidence of L. semiglobulosa, it is con-firmed that the open AZM should be a plesiomorphic character of oligotrichs s. lat. as suggested previously.

Key Words. Infraciliature, morphogenesis, small subunit rRNA (SSrRNA), systematics, taxonomy.

AS a large, diverse group of ciliates within the class Spiro-trichea, oligotrichs sensu lato (s. lat.) constitute an important

component of the microplankton in both marine and freshwaterhabitats (Pierce and Turner 1992). During the last several decades,a variety of morphological and morphogenetic characters havebeen employed to investigate phylogenetic relationships andtaxonomic classification of oligotrich ciliates. Some early studiesdrew attention to differences in oral structures among oligotrichss. lat. and divided them into two groups, oligotrichs sensu stricto(s. str.) with a gap in adoral zone of membranelles (AZM), andchoreotrichs with a closed AZM (Small and Lynn 1985). Petz andFoissner (1992) identified differences in formation of the oralprimordium (OP) in some oligotrichs s. str. (in an intracellulartube) and choreotrichs (in a subsurface pouch), which wereconsidered as evidences for the monophyly of each by Agatha(2004a). Based on the available morphological and ontogeneticdata, Agatha (2004a) proposed a cladistic system of classificationin which the oligotrichs s. lat. were divided into two subclasses,Halteriia and Oligotrichia, the latter of which contained twoorders, Oligotrichida and Choreotrichida.

With the development of molecular technologies, the smallsubunit rRNA (SSrRNA) gene sequence has been employed toinvestigate phylogenetic relationships of oligotrichs s. str. andchoreotrichs. Struder-Kypke and Lynn (2003) concluded from amolecular phylogenetic analysis that the family Halteriidaeshould be removed from the Oligotrichida to the subclassStichotrichia. Their results also supported the monophyly ofthe remaining oligotrichs s. str. and choreotrichs. Combiningmorphological and molecular data, Lynn (2008) proposed a newsystem of classification in which the Oligotrichia sensu Agatha(2004a) would be divided into the two subclasses Oligotrichiaand Choreotrichia within the class Spirotrichea, and he provideddiagnoses that emphasized the importance of oral structures in

characterizing these taxa. Recent years, more available SSrRNAgene sequences were used to justify and confirm this classificationof higher taxa (Agatha and Struder-Kypke 2007; Gao et al. 2009;Kim et al. 2010; Struder-Kypke and Lynn 2008; Tsai et al. 2010).

In the present study, the morphology and SSrRNA gene sequenceof an undescribed species of oligotrich s. lat. are described, and itsphylogenetic and taxonomic positions are analyzed. Results of theseanalyses confirm that this species represents a new genus and speciesof ciliate, which we name Lynnella semiglobulosa n. g., n. sp.

MATERIALS AND METHODS

Collection, observation, and identification. Plankton samplescontaining Lynnella semiglobulosa n. g., n. sp. were taken using aplankton net (mesh size 20 mm) from Daya Bay (221430N,1141320E), Guangdong Province, China, on December 20, 2007.Water temperature was 21 1C, salinity was 29%, and pH was 7.5.

After isolation, specimens were observed using bright field anddifferential interference contrast microscopy (Nikon, Tokyo,Japan). Staining with protargol was done according to the methodof Wilbert (1975) to reveal the infraciliature. Counts and mea-surements on protargol-stained cells were done at a magnificationof 1,000X, and 10 living cells were measured at magnifications of40–1,000X with an ocular micrometer. Illustrations of live spec-imens were made from free-hand drawings and photographs, andthose of protargol-stained cells were made with a camera lucida(Liu et al. 2009). Terminology is mainly according to Agatha(2004b), and systematics follows Lynn (2008). ‘‘Oligotrichs s.str.’’ is used in reference to members of only the subclass Oligotri-chia sensu Lynn (2008) and ‘‘oligotrichs s. lat.’’ is used in awider, morphotypic sense to include members of the subclassesOligotrichia and Choreotrichia.

Extraction, amplification and sequencing of DNA. Five ran-domly selected cells were isolated and repeatedly washed usingsterilized artificial seawater (salinity 30%). Then, they were trans-ferred to a 2-ml microfuge tube with the minimum possiblevolume of seawater, and genomic DNA was extracted from themaccording to methods described in Gao et al. (2009).

The universal eukaryotic primers EukA (50-AACCTGGTTGATCCTGCCAGT-30) and EukB (50-TGATCCTTCTGCAGGTT

Corresponding Author: X. Lin, Laboratory of Protozoology, Key Lab-oratory of Ecology and Environmental Science in Guangdong HigherEducation, Guangdong Provincial Key Laboratory for Healthy and SafeAquaculture, College of Life Science, South China Normal University,Guangzhou 510631, China—Telephone number: 86 20 8521 0644;e-mail: xlin@scnu.edu.cn

43

J. Eukaryot. Microbiol., 58(1), 2011 pp. 43–49r 2010 The Author(s)Journal of Eukaryotic Microbiology r 2010 International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2010.00519.x

Published bythe International Society of ProtistologistsEukaryotic Microbiology

The Journal of

CACCTAC-30) (Medlin et al. 1988) were used for the amplificationof the SSrRNA gene by polymerase chain reaction (PCR). Cyclingparameters were as follows: 5 min at 94 1C; 35 cycles of 1 min at95 1C, 2 min at 56 1C, and 2 min at 72 1C; and 15 min at 72 1C.

After confirmation of amplification by gel electrophoresis, thePCR product was purified using the TIAN gel Midi PurificationKit (Tiangen Bio. Co., Shanghai, China) and inserted into apUCm-T vector (Sangon Bio. Co., Shanghai, China). DNA fromplasmids was harvested using a QIAprep Spin Miniprep Kit (Ti-angen Bio. Co.) and sequenced (Invitrogen sequencing facility,Shanghai, China) using the RV-M and M13-20 primers.

Phylogenetic analyses. The SSrRNA gene sequences of 11stichotrich ciliates, 16 oligotrichs s. str., 29 choreotrichs, and 10other species of ciliates as an out group were obtained from theGenBank database (see Fig. 31 for accession number). An align-ment of these sequences plus the one from L. semiglobulosa wasmade using Hmmer v2.3.2 (Eddy 1998). The ends were trimmed,and ambiguously aligned sites were refined by eye using Bioedit(Hall 1999), yielding a final alignment of 1,583 characters.

MrModeltest v.2 (Nylander 2004) selected GTR1I(pinv 5 0.3963)1G (a5 0.4540) as the best model using the AICcriterion. Bayesian inference (BI) was performed with MrBayes3.1.2 (Ronquist and Huelsenbeck 2003). Four simultaneous MCMCchains were run for 2,500,000 generations, sampling every 100 gen-erations. A ‘‘burn-in’’ of 6,250 trees was discarded and remainingtrees were used to calculate posterior probabilities using a majorityrule consensus. A Maximum Likelihood (ML) tree was constructedwith the PhyML v2.4.4 (Guindon and Gascuel 2003). The reliabilityof internal branches was assessed using non-parametric bootstrapmethod with 1,000 replicates. A Maximum Parsimony (MP) anal-ysis was performed with PAUP� 4.0b10 (Swofford 2002), includingbootstrapping with 1,000 replicates.

Mesquite was used to generate a constraint tree representativeof the following hypothesis: Lynnella branches basally to the sub-class Oligotrichia. The resulting tree was compared with uncon-strained ML tree using the approximately unbiased (AU) test andShimodaira–Hasegawa (SH) test (Shimodaira 2002) as imple-mented in CONSEL package (Shimodaira and Hasegawa 2001).

RESULTS

Lynnella semiglobulosa n. sp. (Table 1 and Fig. 1–30)Morphologicaldescription. Cell size was 40–50 � 65–75mm

in vivo, with most measuring approximately 45 � 70 mm, and 50–69 � 61–83 mm after protargol impregnation (Table 1). The cellshad a compressed semiglobular to bowl-like shape in which thewidth was distinctly greater than the length (width/length up

to 3:2). The posterior end was broadly rounded, and the anteriorend was conspicuously truncated with an apical protrusion on theleft of the peristome, which was approximately 10 mm high (Fig. 1,16, arrows). Cells were asymmetrically circular when viewed froman apical perspective, with the cytostomal area slightly depressedto form a shallow concavity (Fig. 3, 17, arrows; 22). The apicalmargin of the cell decreased in height gradually from the apicalprotrusion clockwise to the oral cavity (Fig. 1, 2, 5, 6, 9, 10).

No cortical platelets were observed, and the cytoplasm wascolorless. Cells were usually dark at low magnifications becausethey were packed with food vacuoles and transparent, refringentlipid droplets measuring 1–2 mm diam. (Fig. 1, 2, 16, 18). No ext-rusomes were detected. Two ovoidal macronuclear nodules con-nected by a funiculus were located in the center of the cell andoriented horizontally with respect to the cell axis (Fig. 8, 10, 12,13, 23, 26, arrowhead; 27). Variable numbers of faintly stainedmicronuclei were closely attached to the macronuclei (Fig. 10).Neither a contractile vacuole nor a cytopyge were observed.

Cells, maintained in habitat water in a 9-cm diam. Petri dish atroom temperature, usually swam in spirals by rotating about themain cell axis without pausing, but often this pattern of motion wasinterrupted by jumps of four to five cell lengths (Fig. 4), with themembranellar cilia spreading radially, mostly perpendicularly to themain cell axis and sometimes bending aborally (Fig. 3, 5, 6, 20).

The somatic ciliature consisted of two longitudinally orientedkineties (Fig. 8, 9, 10, 23, 24, 25). A ventral kinety composed of8–15 widely spaced monokinetids began on the right side of theoral region, extended ventrally, and terminated near the posteriorend of the cell (Fig. 9, 25, arrows; 26). A dorsal kinety originatedin the anterior fourth of the cell, extended on the left dorsal side,and terminated near the posterior end of the ventral kinety (Fig. 8,10, 23, 24). There were 28–43 closely spaced dikinetids in thedorsal kinety. The posterior basal body of each dikinetid had ashort cilium, about 3mm in length in vivo.

The oral cavity was shallow and lay underneath the apical pro-trusion (Fig. 1, 2, 15). The AZM began at the apical protrusion,circled the anterior edge of the cell and finally entered trans-versely into the oral cavity (Fig. 1, 3, 7, 18, 20, arrows; 23). TheAZM consisted of approximately 65 membranelles. No ventralmembranelles were observed, but there were approximately fiveclosely spaced membranelles at the proximal end of the AZM.The lengths of these proximal membranelles gradually lengthenedfrom left to right (i.e. proximally), with the longest, terminal onebeing twice the length of membranelles outside the proximal part(Fig. 7, 9, 28, arrows). All membranelles were composed of threerows of basal bodies and were associated with rootlet fibers lo-cated between and underneath the membranelles, with the more

Table 1. Morphometric data of Lynnella semiglobulosa n. g., n. sp.

Characters Minimum Maximum Mean SD n

Length of cella 50 69 57.6 5.97 17Width of cell 61 83 69.7 7.39 17Distance from apex to cytostome 19 30 23.9 13.87 10Number of adoral membranellesb 60 71 66.6 2.38 22Number of lengthened membranelles 5 8 6.2 1.02 22Number of dikinetids in dorsal kinety 28 43 34.5 4.61 19Number of monokinetids in ventral kinety 8 15 11.0 2.40 18Length of dorsal kinety 38 57 44.8 5.42 14Length of ventral kinety 21 39 29.2 5.81 15Length of macronucleus 18 25 21.6 2.33 8Width of macronucleus 10 18 14.9 2.75 8

Data based on randomly selected specimens stained with protargol. Measurements in mm.aMost specimens in protargol preparations present their anterior-lateral (e.g. Fig. 5) or posterior-lateral side to the viewer. The length of cells was

measured from the center of the buccal area to the posterior edge of the cell. The measurements were thus larger on average than those made in vivo.bIncluding the lengthened membranelles.

44 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

posterior ones bundled closely together to form a distinct stripeunder the AZM (Fig. 11, arrow; 29, arrowheads). The endoralmembrane longitudinally located on the inner wall of the buccallip and composed of a single row of basal bodies (Fig. 9, 14, 25).No pharyngeal fibers were detected.

Morphogenesis. Several divisional stages were observed(Fig. 12, 13, 14, 26, 30), allowing description of parts of morpho-genesis. The OP originated with the apokinetal development of aC-shaped field of basal bodies on the left of the ventral kinety(Fig. 12, arrow). In middle to late stages, the opisthe’s oral polyki-netids differentiated gradually, then orientated perpendicularly tothe cell surface, and the endoral membrane originated in the centerof the OP (Fig. 13, 14, 30, double arrowheads). The new mem-branelles in the proximal part of the OP became distinctly longerthan neighboring ones, reflecting the increase in membranellelength proximally. The distal and proximal portions of the newzone are almost close to each other (Fig. 13, 14, 30). Simulta-

neously, the ventral kinety became disordered (Fig. 14, arrow-heads), and the dorsal one was lengthened by intrakinetalproliferation of basal bodies. The replication bands in both mac-ronuclear nodules were recognizable in early and middle stages ofdivision (Fig. 12, 30, arrowheads).

Phylogenetic analyses. We sampled a broad selection ofSSrRNA gene sequences from 67 species of spirotrichs for phylo-genetic analyses. All tree topologies inferred using ML, MP, andBI were basically congruent (Fig. 31). Both the oligotrichs s. str.and the choreotrichs formed monophylies (98% ML, 58% MP,1.00 BI and 100% ML, 99% MP, 1.00 BI, respectively). Lynnellasemiglobulosa n. g., n. sp. branched basally to the choreotrichs butwith relatively low support (65% ML, 79% MP, 0.61 BI). Theclade including oligotrichs s. lat. and Lynnella was moderately tostrongly supported (96% ML, 87% MP, 1.00 BI).

The subclass Oligotrichia was separated into four clades: (1) thegenus Laboea was affiliated with the family Tontoniidae, clustering

Fig. 1–14. Lynnella semiglobulosa n. g., n. sp. from life (1–6) and after staining with protargol (7–14). 1, 2. Ventral views of typical specimens; notethe apical protrusion (arrow). 3. Apical view. 4. Trace of swimming pathway. 5, 6. Dorsal views showing the anterior membranelles when flexed aborally.7, 8. Apical (7) and aboral (8) views showing the buccal apparatus, somatic kineties, and macronuclear nodules; note the lengthened membranellesinserting transversely into the oral cavity (arrow). 9, 10. Ventral (9) and dorsal (10) views of the same specimens showing the ciliary pattern; arrow marksthe lengthened proximal membranelles. 11. Detail of membranelles showing the rootlet fibers between and underneath the membranelles (arrow). 12.Ventral view of cell in early stage of division; note the replication bands in the macronucleus (arrowheads); arrow indicates the early oral primordium. 13,14. Cells in middle stages of division; note the completely differentiated lengthened membranelles (arrow); arrowheads indicate the disordered ventralkinety and double-arrowheads mark the new endoral membrane. AM, anterior membranelles; AP, apical protrusion; DK, dorsal kinety; EM, endoralmembrane; Fi, fibers; Ma, macronucleus; Mi, micronucleus; VK, ventral kinety. Scale bars in Fig. 1–3, 7–10, 12–14 5 30 mm.

45LIU ET AL.—LYNNELLA SEMIGLOBULOSA N. G., N. SP.

with Spirotontonia with full support and then with Pseudotontonia;(2) most Strombidium species except Strombidium conicum weregrouped together with variable support (74% ML, 52% MP, 1.00BI); (3) S. conicum branched basally to the Omegastrombidiumelegans and Varistrombidium kielum clade, then formed a lesssupported group (36% ML) with the cluster of Novistrombidium

sinicum, Novistrombidium orientale, and Parallelostrombidiumsp.; (4) Novistrombidium testaceum represented the basal branchof the family Strombidiidae (excluding Laboea) in the ML tree(49%).

Within the subclass Choreotrichia, the monophylies of theorders Tintinnida and Choreotrichida were not confirmed because

Fig. 15–30. Photomicrographs of Lynnella semiglobulosa n. g.,n. sp. from life (15–22) and after staining with protargol (23–30). 15, 16. Two typicalindividuals showing the apical protrusion (arrow). 17, 18. Apical (17) and aboral (18) views; note the depressed oral cavity (arrows). 19. Anterior-dorsalview showing the apical protrusion (arrow). 20. Anterior view showing the fully extended anterior membranelles and the more lengthened, ‘‘proximal’’membranelles (arrow). 21. Detail of the proximal end of the adoral zone of membranelles, showing lengthening. 22. Insertion of the lengthened mem-branelles into the oral cavity underneath the apical protrusion. 23, 27. Ventral (23) and anterior (27) views. 24. Posterior view showing the dorsal kinety.25. Detail of oral apparatus; arrows mark the widely spaced monokinetids in the ventral kinety. 26. Ventral view; arrow indicates an early oral prim-ordium and arrowhead marks the funiculus connecting the two nodules of the macronucleus. 28. Anterior view of oral field; arrows mark the lengthenedmembranelles. 29. Detail of membranelles; arrows mark the fibers between membranelles and arrowheads indicate the distinct fibrillar stripe underneaththem. 30. Ventral view of cell in middle stage of division showing the new lengthened membranelles (arrow), new endoral membrane (double arrow-heads), and the replication bands in the macronucleus (arrowheads). DK, dorsal kinety; EM, endoral membrane; Ma, macronucleus; VK, ventral kinety.Scale bars in Fig. 15–20, 23, 27, 30 5 40mm, in Fig. 21–22, 24–26, 28–29 5 10 mm.

46 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

Tintinnidium mucicola was affiliated with the Parastrombidinop-sis group, though the supports were weak (37% ML and 0.78 BI).Furthermore, choreotrichid species of the families Strobilidiidaeand Parastrombidinopsidae grouped with the tintinnids ratherthan with the family Strombidinopsidae. Most tintinnids exceptT. mucicola were grouped together with variable support (63%ML, 73% MP, 1.00 BI): a strongly supported (96% ML, 97% MP,1.00 BI) cluster comprising the genera Salpingella, Amphorellop-sis, and Steenstrupiella was basally located, and the fully sup-ported group of Eutintinnus species branched next, followed bythe clade of two Favella species (Favella panamensis and Favellacampanula), while the third species (Favella ehrenbergi)clustered with the genera Rhabdonella and Metacylis. TwoTintinnopsis clades were strongly supported (Tintinnopsisberoidea1Tintinnopsis dadayi1Tintinnopsis radix and Tin-tinnopsis tocantinensis1Tintinnopsis tubulosoides). Tintinnopsis

fimbriata and Tintinnopsis subacuta did not group with any otherTintinnopsis species.

DISCUSSION

Classification of Lynnella semiglobulosa: Oligotrichia orChoreotrichia? It is widely agreed that the oral structure hasgreat value in distinguishing higher-level taxa of oligotrichs s. lat.(Agatha 2004a; Lynn 2008; Petz and Foissner 1992; Xu, Warren,and Song 2009). In Lynn’s (2008) classification of ciliates, theoligotrichs s. lat. are divided into two subclasses, Oligotrichia andChoreotrichia. The former has a C-shaped AZM with a ventral gapand the latter has a circular AZM. In addition, the AZM of theOligotrichia (except in the family Cyrtostrombidiidae) is alwaysbipartite, with an anterior part (collar) surrounding the anteriorpole of the organism and a ventral part (lapel) inside the oral

Fig. 31. Maximum Likelihood (ML) tree inferred from small subunit rRNA gene sequences indicating the phylogenetic position of Lynnella semi-globulosa n. g., n. sp. Numbers at the nodes represent support values in the following order: ML bootstrap values, Maximum Parsimony (MP) bootstrapvalues, and Bayesian inference (BI) posterior probabilities. Nodes absent from one of the three phylogenies are indicated by a hyphen instead of a supportvalue. Scale bar indicates the number of substitutions per 10 nucleotides.

47LIU ET AL.—LYNNELLA SEMIGLOBULOSA N. G., N. SP.

cavity. The ventral membranelles usually diminish in length pro-gressively and are often separated from the anterior ones. Inchoreotrichs, by contrast, the AZM completely encircles theanterior end of the cell (except the genus Parastrombidium andParastrombidinopsis) with no typical ventral membranellar zonebut several prolonged membranelles usually extend into the oralcavity. Moreover, choreotrichs typically have two ellipsoidalmacronuclear nodules (Lynn 2008; Xu et al. 2009). In morpho-genesis, differences in the formation of OP in some oligotrichs s.str. (in an intracellular tube) and choreotrichs (in a subsurfacepouch) were considered as evidences for the monophyly of eachby Agatha (2004a). In addition, the movements of new oralpolykinetids are markedly different in the late stages of stomato-genesis in oligotrichs s. lat. and choreotrichs: in oligotrichs s. lat.,the opisthe’s oral polykinetids make a counter-clockwise circle toroll out on the cortical surface, accompanied by a movement androtation of the opisthe’s somatic kineties while in choreotrichs, thiscounter-clockwise movement of the oral polykinetids occurs belowthe cortical surface, the oral polykinetids first orientating perpen-dicularly to the cell surface, then performing a counter-clockwisetwist to splay out onto the cell surface and form the closed circle(Dale and Lynn 1998).

Lynnella semiglobulosa n. g., n. sp. has an open AZM like mostoligotrichs s. str.; however, it lacks ventral membranelles, whichwould seem to exclude it from the subclass Oligotrichia except thefamily Cyrtostrombidiidae. Cyrtostrombidiidae seem to be theonly group of oligotrichs s. str. that have no ventral membrane-lles, but they differ distinctly from L. semiglobulosa in havingcyrtos-like pharyngeal fibers that are absent in L. semiglobulosa.Cyrtostrombidiids also lack the endoral membrane and the pro-gressively lengthened membranelles in the proximal end ofthe AZM (Agatha 2004b) but these are present in L. semiglobu-losa. Furthermore, the two longitudinally oriented somatic kinetiesof L. semiglobulosa have no counterpart among the known speciesof oligotrichs s. str. Instead, most of them have one longitudinalventral kinety and one transverse or spiraled girdle kinety, bothconsisting of dikinetids (Agatha 2004a; Xu et al. 2009).

Although most choreotrichs differ from L. semiglobulosa byhaving a completely closed AZM (Lynn 2008), the two ellipsoidalmacronuclear nodules, and the longitudinally arranged somatickineties, make L. semiglobulosa similar to choreotrichs. Moreover,in the late divider of L. semiglobulosa, the opisthe’s oral polyki-netids orientate perpendicularly to the cell surface, and the distaland proximal ends of the new zone are close to each other; these areregarded as important characters that differentiate choreotrichsfrom oligotrichs in stomatogenesis (Dale and Lynn 1998).

In the phylogenetic trees, our new taxon L. semiglobulosabranches basally within the subclass Choreotrichia, though thesupport values are not high (65% ML, 79% MP, 0.61 BI) (Fig. 31),which suggests its closer relationship with choreotrichs thanoligotrichs. However, the possibility that it is associated with oli-gotrichs s. str. is not rejected (P 5 0.461 for AU test; P 5 0.444 forSH test).

In summary, L. semiglobulosa n. g., n. sp. shares some morpho-logic features with both oligotrichs s. str. and choreotrichs. How-ever, the similarities between L. semiglobulosa and choreotrichs insome morphological and stomatogenetic characters as well as itsbasal position in the choreotrich clade in phylogenetic analysis sug-gest that L. semiglobulosa seems to be more closely related with thesubclass Choreotrichia than Oligotrichia. Therefore, we assignedthis new organism to the subclass Choreotrichia temporarily;however, this needs to be tested by further investigations.

Lynnella semiglobulosa represents a new genus and a newfamily in subclass Choreotrichia. The loricate choreotrich familiescan be easily separated from L. semiglobulosa by their lorica (vs. nolorica in L. semiglobulosa) (Lynn 2008).

Compared with the aloricate choreotrichs, L. semiglobulosa canbe clearly distinguished from all other known species of thesefamilies (i.e. Leegaardiellidae, Lohmanniellidae, Strobilidiidae,and Strombidinopsidae) by having a conspicuously open AZM(vs. the AZM closed or slightly open in aloricate choreotrich fam-ilies) (Agatha and Struder-Kypke 2007). Although there is a smallto conspicuous gap present in the AZM of some genera of thefamily Strombidinopsidae (e.g. Parastrombidium and Para-strombidinopsis), L. semiglobulosa can be separated from themby the fact that the proximal end of its AZM is located in the oralcavity below the distal end (vs. proximal end of the AZM on thesame horizontal level as the distal end and only two shortermembranelles extending in the oral cavity in strombidinopsids)and having only two somatic kineties (vs. several kineties equallydistributed around body in strombidinopsids) (Kim et al. 2005;Xu et al. 2007).

Such characters in Lynnella semiglobulosa represent a morpho-logical novelty in the subclass Choreotrichia and therefore should besubstantial enough to justify the erection of not only a new genus butalso a new family, Lynnellidae n. fam. Furthermore, the phyloge-netic analyses show that other choreotrichs cluster together to theexclusion of L. semiglobulosa, to some extent corroborating theerection of the new family. However, further data are needed to testwhether this species represents a new order.

Comments on the open AZM. The open AZM occurs in allknown oligotrichs s. str. (Agatha 2004a; Agatha and Struder-Kypke2007; Lynn 2008). The open AZM was considered as a plesiomor-phic character of oligotrichs s. lat. and the closed AZM of mostchoreotrichs was an apomorphic state (Agatha 2004a; Agatha andStruder-Kypke 2007). This hypothesis is confirmed by the morpho-genetic data of oligotrichs s. lat.: an open (C-shaped) OP is presentin the early stages of stomatogenesis of oligotrichs s. str. and chore-otrichs (Lynn 2008); then along with the differentiations in thefollowing stages, the OP undergoes torsion and extension to form aC-shaped AZM in oligotrichs s. str. or a closed AZM in choreotrichs(Agatha et al. 2005; Dale and Lynn 1998; Lynn 2008; Petz 1994;Petz and Foissner 1992).

Lynnella semiglobulosa n. g., n. sp. has an obviously open AZMbut shares important morphological and morphogenetic character-istics with choreotrichs. Furthermore, it is associated basally withchoreotrichs in our phylogenetic analyses, albeit with relativelymodest support. These results suggest that as a species branchingbasally from choreotrichs, L. semiglobulosa keeps an open AZM,and thus could support the viewpoint of Agatha (2004a) that theopen AZM is a plesiomorphy of oligotrichs s. lat.

Subclass ChoreotrichiaOrder?Family Lynnellidae n. fam.Diagnosis. Adoral zone of membranelles open without differ-

entiation of ventral membranelles, the distal and proximalends of the new adoral zone closed to each other forming an opencircle in stomatogenesis; somatic kineties longitudinally oriented.

Type genus. Lynnella n. g.Etymology. The new family name is derived from the name of

its type genus Lynnella.

Genus Lynnella n. g.Diagnosis. Lynnellidae with two longitudinally oriented

somatic kineties, one on dorsal side of cell and one on ventralside; several proximal membranelles progressively lengthened to-ward proximal end of adoral zone; bipartite macronucleus.

Etymology. The generic name is dedicated to our eminentCanadian colleague, Prof. Denis Lynn, University of Guelph,Canada, in acknowledgement of his great contributions to cilia-tology; feminine gender.

Type species. Lynnella semiglobulosa n. sp.

48 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

Lynnella semiglobulosa n. sp.Diagnosis. Size in vivo 40–50 � 65–75 mm; cells semiglobular

or bowl shaped. Ventral kinety sparsely ciliated and consistingof about 10 monokinetids; dorsal kinety consisting of about 35dikinetids. Adoral zone composed of about 65 membranelles, ofwhich about five most proximal ones progressively lengthenedtoward proximal end of adoral zone. Two ellipsoidal macro-nuclear nodules oriented transversely in relation to the cell axis.

Type locality. Coastal waters off Daya Bay (221430N,1141320E), Guangdong Province, China.

Type material. One slide containing a holotype (marked in redin the slide) and a paratype (protargol preparation) has been depos-ited in the Natural History Museum, London, with registry number2010:7:14:1. Another slide containing several paratypes (protargolpreparation) has been deposited in the collection of the Laboratoryof Protozoology, South China Normal University, with registrationnumber WW07122003.

Gene sequence. A sequence of the SSrRNA gene of L. semi-globulosa has been deposited in the GenBank database with acces-sion number FJ876965.

Etymology. The Latin word ‘‘semiglobulosa’’ refers to thesemi-globular shape of the new species.

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation ofChina (projects numbers: 30870280, 30700069) and a joint grantfrom the Center of Excellence in Biodiversity, King Saud Uni-versity. Many thanks are due to Ms. Miao Miao for DNA extrac-tion and Mr. Liang Zhou, Ms. Qianqian Zhang, Ms. Feng Gao, andMs. Jie Huang for their kind help in phylogenetic analyses. Wealso thank Prof. Weibo Song, Ocean University of China, Dr. JohnC. Clamp, North Carolina Central University, and the anonymousreviewers for their constructive criticism and suggestions forimproving the language.

LITERATURE CITED

Agatha, S. 2004a. A cladistic approach for the classification of oligotrichidciliates (Ciliophora: Spirotricha). Acta Protozool., 43:201–217.

Agatha, S. 2004b. Evolution of ciliary patterns in the Oligotrichida(Ciliophora, Spirotricha) and its taxonomic implications. Zoology,107:153–168.

Agatha, S. & Struder-Kypke, M. C. 2007. Phylogeny of the order Chore-otrichida (Ciliophora, Spirotricha, Oligotrichea) as inferred from mor-phology, ultrastructure, ontogenesis, and SSrRNA gene sequences. Eur.J. Protistol., 43:37–63.

Agatha, S., Struder-Kypke, M., Beran, A. & Lynn, D. H. 2005. Pelagos-trobilidium neptuni (Montagnes and Taylor, 1994) and Strombidiumbiarmatum nov. spec. (Ciliophora, Oligotrichea): phylogenetic positioninferred from morphology, ontogenesis, and gene sequence data. Eur. J.Protistol., 41:65–83.

Dale, T. & Lynn, D. H. 1998. Stomatogenesis of the ciliate genusStrombidinopsis with an improved description of S. spiniferum andS. acuminatum. J. Eukaryot. Microbiol., 45:210–217.

Eddy, S. R. 1998. Profile hidden Markov models. Bioinformatics, 14:755–763.Gao, S., Gong, J., Lynn, D., Lin, X. & Song, W. 2009. An updated

phylogeny of oligotrich and choreotrich ciliates (Protozoa, Ciliophora,Spirotrichea) with representative taxa collected from Chinese coastalwaters. Syst. Biodiver., 7:235–242.

Guindon, S. & Gascuel, O. 2003. A simple, fast and accurate algorithmto estimate large phylogenies by maximum likelihood. Syst. Biol., 52:696–704.

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignmenteditor and analysis program for Windows 95/98/NT. Nucleic AcidsSymp. Ser., 41:95–98.

Kim, J. S., Jeong, H. J., Struder-Kypke, M. C., Lynn, D. H., Kim, S., Kim,J. H. & Lee, S. H. 2005. Parastrombidinopsis shimi n. gen., n. sp.(Ciliophora: Choreotrichia) from the coastal waters of Korea: morphol-ogy and small subunit ribosomal DNA sequence. J. Eukaryot. Micro-biol., 52:514–522.

Kim, Y., Kim, S. Y., Lee, W. & Choi, J. K. 2010. New observations on thechoreotrich ciliate Strombidinopsis acuminata Faure-Fremiet 1924, andcomparison with Strombidinopsis jeokjo Jeong et al., 2004. J. Eukaryot.Microbiol., 57:48–55.

Liu, W., Xu, D., Lin, X., Li, J., Gong, J., Al-Rasheid, K. A. S. & Song, W.2009. Novistrombidium sinicum n. sp. and Novistrombidium orientale n.sp. (Protozoa: Ciliophora): two new oligotrich ciliates from a mangrovewetland, South China. J. Eukaryot. Microbiol., 56:459–465.

Lynn, D. H. 2008. The Ciliated Protozoa. Characterization, Classification,and Guide to the Literature. 3rd ed. Springer, Dordrecht.

Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. 1988. The charac-terization of enzymatically amplified eukaryotic 16S-like rRNA-codingregions. Gene, 71:491–499.

Nylander, J. A. 2004. MrModeltest Ver.2. Distributed by the author.Department of Systematic Zoology, Evolutionary Biology Centre,Uppsala University, Sweden.

Petz, W. 1994. Morphology and morphogenesis of Strombidium kryalisnov. spec. (Ciliophora, Strombidiida) from Antarctic sea ice. Arch.Protistenkd., 144:185–195.

Petz, W. & Foissner, W. 1992. Morphology and morphogenesis ofStrobilidium caudatum (Fromentel), Meseres corlissi n. sp., Halteriagrandinella (Muller), and Strombidium rehwaldi n. sp., and a proposedphylogenetic system for oligotrich ciliates (Protozoa, Ciliophora).J. Protozool., 39:159–176.

Pierce, R. W. & Turner, J. T. 1992. Ecology of planktonic ciliates inmarine food webs. Rev. Aquat. Sci., 6:139–181.

Ronquist, F. & Huelsenbeck, J. P. 2003. MRBAYES 3: Bayesian phylo-genetic inference under mixed models. Bioinformatics, 19:1572–1574.

Shimodaira, H. 2002. An approximately unbiased test of phylogenetic treeselection. Syst. Biol., 51:492–508.

Shimodaira, H. & Hasegawa, M. 2001. CONSEL: for assessingthe confidence of phylogenetic tree selection. Bioinformatics,17:1246–1247.

Small, E. B. & Lynn, D. H. 1985. Phylum Ciliophora Doflein, 1901. In:Lee, J. J., Hutner, S. H. & Bovee, E. (ed.), An Illustrated Guide to theProtozoa. Society of Protozoologists, Allen Press, Lawrence, Kansas.p. 393–575.

Struder-Kypke, M. C. & Lynn, D. H. 2003. Sequence analyses of thesmall subunit rRNA gene confirm the paraphyly of oligotrich ciliatessensu lato and support the monophyly of the subclasses Oligotrichiaand Choreotrichia (Ciliophora, Spirotrichea). J. Zool. Lond., 260:87–97.

Struder-Kypke, M. C. & Lynn, D. H. 2008. Morphological versusmolecular data-phylogeny of tintinnid ciliates (Ciliophora, Choreotri-chia) inferred from small subunit rRNA gene sequences. Denisia,23:417–424.

Swofford, D. L. 2002. PAUP�. Phylogenetic Analysis Using Parsimony(�and Other Methods). Version 4. Sinauer Associates, Sunderland, MA.

Tsai, S., Chen, J. & Chiang, K. 2010. Spirotontonia taiwanica n. sp.(Ciliophora: Oligotrichida) from the coastal waters of NortheasternTaiwan: morphology and nuclear small subunit rDNA sequence.J. Eukaryot. Microbiol., 57:429–434.

Wilbert, N. 1975. Eine verbesserte Technik der Protargolimpragnation furCiliaten. Mikrokosmos, 64:171–179.

Xu, D., Warren, A. & Song, W. 2009. Oligotrichs. In: Song, W., Warren,A. & Hu, X. (ed.), Free-Living Ciliates in the Bohai and Yellow Seas.China. Science Press, Beijing. p. 307–351.

Xu, D., Song, W., Warren, A., Roberts, D. & Hu, X. 2007. Redescriptions oftwo marine planktonic ciliates from China, Parastrombidium faurei (Kahl,1932) Maeda, 1986 and Strombidium capitatum (Leegaard, 1915) Kahl,1932 (Ciliophora, Oligotrichea). Eur. J. Protistol., 43:27–35.

Received: 01/19/10, 07/16/10, 10/10/10; accepted: 10/12/10

49LIU ET AL.—LYNNELLA SEMIGLOBULOSA N. G., N. SP.