Plankton Benthos Res 7(3): 111–125, 2012

Plankton & Benthos
Research© The Plankton Society of Japan

Discrepancy between conventional morphological systematics andnuclear phylogeny of tintinnids (Ciliophora: Choreotrichia)
TAKEHIRO K AZAMA1,*, SEIJI ISHIDA1, 2, SATOSHI SHIMANO3 & JOTARO URABE1,4
1

Graduate School of Life Sciences, Tohoku University, 6–3 Aoba, Aramaki, Aoba-ku, Sendai 980–8578, Japan
International Advanced Research and Education Organization, Tohoku University, 6–3 Aoba, Aramaki, Aoba-ku, Sendai
980–8578, Japan
3
Environment Education Center, Miyagi University of Education, 149 Aoba, Aramaki, Aoba-ku, Sendai 980–0845, Japan
4
Research Center for Marine Biology, Tohoku University, Asamushi, Aomori 039–3501, Japan
2

Received 9 February 2012; Accepted 23 April 2012
Abstract: Tintinnid ciliates have been traditionally classiﬁed according to morphological and morphometric features of the lorica. To examine if the morphological characteristics of loricae reﬂect the phylogenetic relationships,
we collected 23 tintinnid morphospecies (11 genera) and reconstructed phylogenetic trees based on 84 partial sequences of nuclear small subunit rRNA (SSrRNA) gene fragments of 44 morphospecies from a total of eight families(55 sequences from this study, and 29 sequences from the literature). We found that tintinnid ciliates could be classiﬁed into six clades consisting of ﬁve major clades. While one clade consisted of one family, each of the other ﬁve
clades consisted of more than two families. Although two families were found only in one clade, the remaining six
families were found in more than two clades. In addition, each of six morphospecies was found in more than two
major clades, indicating that they are polyphyletic. Principal coordinate analysis showed that morphology of the lorica overlapped substantially between the clades. These results imply that most of the morphological and morphometric traits of marine tintinnid species do not reﬂect SSrRNA genetic distances even at family levels. It is necessary to
identify new characteristics that reﬂect phylogenetic relationships robustly.
Key words: marine ciliates, morphological variation, nuclear SSrRNA, phylogeny, Tintinnida

Introduction
Planktonic ciliates with vase-shaped shells called loricae
are classiﬁed as tintinnids (order Tintinnida) and contain
about 1,200 species belonging to >100 genera and 15
families（Pierce & Turner 1993, Taniguchi 1997, Lynn2008）. These ciliates are <0.2 mm in cell size and inhabit
fresh water, coastal waters and the deep oceans (Beers &Stewart 1967, Heinbokel & Beers 1979, Uye et al. 1996).
Because their prey includes suspended bacteria (Gast 1985,
Karayanni et al. 2008), tintinnids are recognized as one of
the crucial members making up aquatic microbial loops(Legendre & Rassoulzadegan 1995). In addition, tintinnid
ciliates graze on phytoplankton (Rassoulzadegan & Etienne 1981) and often ingest more than 20% of the primary
production (Capriulo & Carpenter 1983, Verity 1985).
* Corresponding author: Takehiro Kazama; E-mail, kazama303@gmail.
com

There are even species that feed preferentially on dinoﬂagellate (Stoecker et al. 1981) and terminate algal blooming
such as red tides through their grazing pressure (Watras etal. 1985). Thus, tintinnid ciliates are an important microzooplankton component playing multiple roles in channeling and transferring energy and materials in aquatic foodwebs.
Tintinnid ciliate loricae are made of hyaline pseudochitin excreted from the cells and often ornamented by grains
and particles available from the surrounding environment.
Because the shape, size and structure of the lorica, as wellas the pattern and materials used for agglutination, are impressive and likely species-speciﬁc (Kofoid & Campbell
1939), classical taxonomy and systematics of tintinnid ciliates have been based on morphological characteristics ofthe loricae (Kofoid & Campbell 1939, Pierce & Turner
1993, Taniguchi 1997), although these characteristics are
not necessarily related to infraciliary structures of the cells
that are generally used in the taxonomy and systematics ofother Choreotrichia (Laval-Peuto & Brownlee 1986).

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Apart from these taxonomic studies, recent studies haveshown that some morphological characteristics of loricae
have important ecological implications. For example, a series of studies done by Dolan and colleagues showed thatlorica width at the oral part of tintinnid ciliates restricted
the size range of edible food and was highly related to
avoidance ability against copepod predation (Dolan et al.
2002, Dolan et al. 2006, Dolan et al. 2009, Dolan 2010).
These ﬁndings suggest that tintinnid species live in restricted ecological niches imposed by the morphology ofthe lorica as a phylogenetic constraint. Alternatively, tintinnid species may have evolved distinctive morphologicalcharacteristics of the lorica through natural selection toadapt to their local environment, regardless of phylogeny.
This possibility implies that some morphological characteristics may diverge between closely related species andconverge among distantly related species, and thus thatthey may not necessarily reﬂect the phylogenetic relationship among tintinnid species.
Recent developments in molecular-biology-based techniques have enabled us to analyze genetic distances between organisms and reconstruct phylogenetic trees without information on the morphological features (Edwards2009). DNA sequences of nuclear small subunit rRNA
(SSrRNA) gene fragments have been used to construct
phylogenetic trees of ciliates, including tintinnids (Snoeyenbos-West et al. 2002, Strüder-Kypke & Lynn 2003, Gaoet al. 2009). Strüder-Kypke & Lynn (2008) and Li et al.
(2009) reconstructed phylogenies based on the SSrRNA
gene sequences of several tintinnid species. However,since the number of species (DNA sequences) that they examined was limited, it is not yet clear to what extent themorphological characteristics of loricae reﬂect the phylogenetic relationships among tintinnid ciliates. In this study,
we analyzed the DNA sequence of the nuclear SSrRNA
gene fragments of 23 morphospecies (55 sequences) collected along the northeast coast of Japan. Incorporating sequence data from previous studies, we reconstructed SSrRNA phylogeny using a total of 44 tintinnid morphospecies (84 sequences). We then examined how well the morphological characteristics of the loricae reﬂect the phylogenetic relationships among these tintinnid ciliates.
Materials and Methods
Collection and identiﬁcation
Tintinnid ciliates used for genetic analyses were collected at several sites in Sendai Bay and its vicinities during the period from June to October 2009 (Fig. 1). Live tintinnids were collected by buckets or water samplers andconcentrated into plastic bottles using 20 μm mesh-sizenet. These live samples were observed under a light microscope at magniﬁcations of ×100 and ×400 to identify
morphospecies based on morphological characteristics ofthe loricae according to Taniguchi (1997). We also referred

to descriptions by several authors: Bakker and Phaff (1976)
for Tintinnopsis minuta Wailes, Hada (1937) for T. elongata Daday, Hada (1938) for T. karajacensis Brandt, Roxas(1941) for T. mortenseni Schmidt, and Cordeiro & Sassi(1997) for T. tubulosa Levander. In this study, specimensthat we could not identify to species level due to uncertainties in morphological characteristics were not used. Afterthe identiﬁcation, typical specimens of each morphospecies were photographed and individually ﬁxed with 99%
ethanol and stored for genetic analyses. Of the morphospecies examined, microscopic images of Steenstrupiellasteenstrupii (Claparède & Lachmann) Kofoid & Campbell
and T. lobiancoi Daday are not shown in Fig. 2 because wefailed to take well-focused photographs capturing, themorphological characteristics of these species.
DNA extraction, PCR ampliﬁcation, cloning and sequencing
Total genomic DNA was extracted from a single cellusing QuickExtract DNA Extraction solution (Epicenter,
Madison, WI, USA). Samples were incubated at 65°C for
2 h and at 95°C for 20 min in 20 μL of the QuickExtract
solution, and were stored at −20°C. We ampliﬁed a fragment of the nuclear small subunit ribosomal RNA (SSrRNA) gene from specimens using the nested PCR protocol
described by Shimano et al. (2008) except for Tintinnopsis
mortenseni and T. tenuis Stein. Each 30 μL of the ﬁrst PCR
reaction consisted of 3.0 μL of 10× Ex Taq buffer, 2.5 mMof each dNTPs, 0.3 μM of each primer of SR1 and SR12(Nakayama et al. 1996), 0.75 U of Ex Taq DNA polymerase(Takara, Tokyo, Japan), and 22.85 μL of extracted DNA.
Each 30 μL of the ﬁnal PCR reaction consisted of 3.0 μL of
10× Ex Taq buffer, 2.5 mM of each dNTPs, 0.3 μM of ciliate-speciﬁc primer CS322F (Puitika et al. 2007) and0.3 μM of SR12 (Nakayama et al. 1996), 0.75 U of Ex Taq
DNA polymerase (Takara, Tokyo, Japan), and 0.1 μL of theﬁrst PCR product. We puriﬁed the ﬁnal PCR products
using ExoSAP-IT (USB Cleveland, OH, USA), performed
cycle-sequencing reactions with a BigDye Terminator
Cycle sequencing kit (Applied Biosystems Inc., Foster
City, CA, USA), and obtained sequences in both directions
with an ABI Prism 3130 sequencer (Applied Biosystems
Inc). Following this protocol, the DNA sequences of T.
mortenseni and T. tenuis were not readable because ofoverlapping peaks in the sequence data. In these specimens, therefore, the ﬁrst PCR products were cloned using
the TOPO TA Cloning Kit (Invitrogen, Burlington, ON,
Canada). Subsequently, three colonies were chosen fromeach plate in the kit to re-amplify the SSrRNA gene fragment with the primers CS322F and SR12, and the puriﬁed
PCR products as above. Then, these products were sequenced as above. Within each of these specimens, difference in the sequence among colonies was less than 1% anddid not affect phylogenetic analyses.

Gene and morphology of tintinnids

113

Fig. 1. The location of Sendai Bay and Hiroura tidal ﬂat in Japan and of sites where samples were collected. SP1, Sendai
Port; SG1, Shiogama Port; TTR, Tetaru; ISM, Ishinomaki Port; MNG, Mangokuura: OSK, Oshika; ONG, Onagawa; GZN,
Gozen; A00, Hiroura tidal ﬂat; C00, river mouth adjacent to Hiroura tidal ﬂat.

Phylogenetic analyses
We obtained 55 sequences of the nuclear SSrRNA genefragments from 23 morphospecies in Sendai Bay andits vicinities. With sequence data from previous studies(Agatha & Strüder-Kypke 2007, Duff et al. 2008, Gao et al.
2009, Li et al. 2009, Snoeyenbos-West et al. 2002, StrüderKypke & Lynn 2003, 2008), we reconstructed SSrRNA
phylogeny using a total of 44 tintinnid morphospecies (84sequences). We aligned the sequences with the CLUSTALW algorithm (Thompson et al. 1994) and manually adjusted the alignment using BioEdit (Hall 1999) and MEGA
4 (Tamura et al. 2007). For phylogenetic analysis, we used
a 1,072 bp fragment of SSrRNA that was common among
the sequences examined. We selected a best-ﬁt model of
nucleotide substitution with KAKUSAN 4 (Tanabe 2007)

and reconstructed the phylogenetic tree applying Maximum-Likelihood (ML), Bayesian Inference (BI) and
Neighbor-Joining (NJ) methods. The ML tree was computed with 1,000 bootstrap replicates using TREEFINDER(Jobb 2008). BI analysis was performed using MrBayesver. 3.1.2 (Ronquist & Huelsenbeck 2003) with 2,000,000
iterations of Markov Chain Monte Carlo (MCMC) sampling. The convergence of MCMC parameters and effective sample size (>100, Kass et al. 1998) were conﬁrmed byTracer vl.5.0 (Rambaut & Drummond 2009). The NJ tree
was reconstructed with 1,000 bootstrap replicates and theKimura 2-parameter model of nucleotide substitution using
MEGA 4. Eight species of Strombidinopsidae, Hypotrichea, Oligotrichia and Stichotrichia (Strombidinopsis acuminate, S. jeokjo, Laboea strobili, Novistrombidium testaceum, Gastrostyla steinii, Cyrtohymena citrine, Eu-

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Fig. 2. Light microscope images of tintinnid species photographed under differential interference contrast. a. Amphorellopsis acuta, b. Amphorides amphora, c. Codonella amphorella, d. Codonellopsis morchella, e. Codonellopsis ostenfeldi, f. Eutintinnus lususundae, g. Favella azorica, h. Favella ehrenbergii, i. Favella taraikaensis, j. Helicostomella fusiformis, k. Leprotintinnus pullucidus, l. Stenosemella nivalis, m. Tintinnopsis corniger, n. Tintinnopsis dadayi, o. Tintinnopsis elongata, p. Tintinnopsis karajacensis, q. Tintinnopsis minuta, r. Tintinnopsis mortenseni, s. Tintinnopsis tenuis, t. Tintinnopsis tocantinensis, u.
Tintinnopsis tubulosa. Scale bars: 20 μm.

Gene and morphology of tintinnids

plotidium arenarium, Aspidisca steini) were used as outgroups.
Morphological and morphometric analyses
We selected 35 morphological traits of the lorica (Table
1) that have been incorporated into taxonomic keys forclassifying and identifying species, genera and familes oftintinnids in Taniguchi (1997), and used presence/absenceof these traits for the analysis (yes-1/no-0): if a specimen
had a particular morphological trait, we scored “1" for thatspecimen regarding that characteristic, otherwise wescored “0”. In addition to the scores of specimens collectedin this study, we also estimated the scores of morphospecies that were not collected in this study but were used forthe genetic analyses. There were determined based on descriptions, illustrations and photographs of the morphospecies from the literature. We obtained a matrix of binary
data on the lorica reﬂecting the morphology of a total of 44
morphospecies, and calculated pairwise similarity values
between the morphospecies by simple matching (Sokal &Michener 1958). Then, we performed principal coordinate(PCo) analysis based on the similarity matrix and calculated scores of the ﬁrst four PCo axes for each morphospecies. These statistical analyses were performed with the aidof the statistical software package R, version 2.10.0. (R Development Core Team 2010).
Using the photographs or illustrations, we measured
width and length of the loricae using the software package
Image J (Abramoff et al. 2004). Then we calculated the aspect ratio (AR), which is the ratio of the width to length ofthe lorica, because it has often been reported as one ofcharacters used who discribing tintinnids (Hada 1938,
Roxas 1941).
Results
Collected species
We collected and identiﬁed a total of 23 morphospecies(11 genera, 6 families) based on lorica morphology (Fig. 2,
Appendix Table 1). While we analyzed a nuclear SSrRNA
gene fragment from only a single specimen of each morphospecies of Amphorides amphora Claparède & Lachmann, Codonellopsis morchella (Cleve) Jörgensen, C. ostenfeldi (Schmidt) Kofoid & Campbell, Favella azorica(Cleve) Jörgensen, Steenstrupiella steenstrupii, Stenosemella nivalis Jörgensen, Tintinnopsis elongata and T. tubulosa due to their rare occurrence, we analyzed fragmentsfrom more than two specimens each for the other morphospecies. We obtained a total of 55 partial sequences fromthe 23 morphospecies (Appendix Table 1).
Phylogene tic analyses
The phylogenic tree of nuclear SSrRNA gene fragments(1,072 bp) was reconstructed using 84 unique sequencesfrom 44 morphospecies belonging to a total of eight fami-

115

lies with the maximum-likelihood (ML), Bayesian inference (BI), and neighbor-joining (NJ) methods (Fig. 3, Appendix Fig. 1, Appendix Fig. 2). The sequences used inthese analyses were 55 sequences from the 23 morphospecies collected for this study, and 29 sequences from 28
morphospecies reported in the literature (eight morphospecies were common to this study). A general time-reversible
plus Gamma distributed model (GTR+G) was selected as
the best model of nucleotide substitution for both ML andBI analyses according to Bayesian Information Criteria(Tanabe 2007). The BI and NJ trees were mostly congruent
with the ML tree. Mean and maximum genetic distances in
the ML tree within the order Tintinnida were 4.2% and39%, respectively. Five major clades (A–E) were supported
by higher bootstrap values of ML (>65%) and NJ (>53%)
and clade credibility values of BI (>0.98, Fig. 3, Appendix
Fig. 1, Appendix Fig. 2). In clade A, a paraphyletic groupthat was genetically distant from other morphospecies(>8%) was treated as subclade A-1 and the residuals weregrouped as subclade A-2. The subclades were also supported by higher bootstrap values of ML (82%) and NJ(80%) and clade credibility values of BI (0.65). However
there was a slight difference between these trees. For example, T. beroidea EF123709 belonged to subclade A-1 in
the ML tree and the NJ tree but was placed under subclade
A-2 in the BI tree.
Each clade consisted of several morphospecies belonging to more than two families. The exception was clade B,
which contained only morphospecies from the family Ptychocylididae, which is characterized by a hyaline lorica
with prongs and visible reticulation. However, morphospecies of Ptychocylididae were also found in subclade A-2.
Similarly, although each of the families Rhabdonellidae
and Dictyocystidae were found only in one clade, the otherfamilies were found spread over at least two clades.
Among clades (A–E), subclade A-2 was the most diverseand consisted of morphospecies belonging to eight families. Clade E was the most ancestral and consisted of thefamilies Tintinnidiidae and Codonellidae. The morphospecies of the family Codonellidae were found in all clades
except for clade B.
Some morphospecies were found in more than two
clades (Fig. 3): Amphorellopsis acuta Kofoid & Campbell
was found in subclade A-2 and clade D; both Favella ehrenbergii Claparéde & Laachmann and F. taraikaensis
Hada were found in subclade A-2 and clade B; Helicostomella fusiformis (Meunier) Jorgensen was found in subclades A-1 and A-2; Tintinnopsis dadayi Bonnetto & Ezcurra de Drago was found in subclades A-1 and A-2, andclade C; T. karajacensis appeared in subclade A-1, clade E
and clade F, and had morphological variation in terms oflorica length. An elongated type of this species occurred inclade D and clade E (AB640659: Fig. 4a) while a short lorica type occurred in subclade A-1 (AB640660: Fig. 4b).

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Fig. 3. A phylogenetic tree by the maximum-likelihood (ML) method for tintinnid nuclear SSrRNA sequences. The numbers
at nodes represent ML bootstrap support values of more than 50%. Red circles denote morphospecies, which were newly sequenced and analyzed in this study. All of pairwise genetic distances among the ﬁve major clades (Clade A to E) and two subclades (A-1, A-2) were more than 8%. Polyphyletic morphospecies have been highlighted with the same color.

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Gene and morphology of tintinnids

Table 1. Morphological characteristics of the lorica that have been used for classifying and identifying species (S), genera (G) andfamilies (F) of tintinnids, and eigen vector values of the characteristics to the ﬁrst four axes (PCo I to IV) of the principal coordinate
analysis. The levels of the top four loadings to each axis are denoted by bold letters.
Trait
No.

Loricaportion1

Surface

2
3
4
5
6
7

Morphology
Lorica agglutinates substances or grains.
Lorica has patterns (ex: band or strial) on thesurface.
Lorica has fenestrae.
Lorica is annulated with 5–10 spiral turns inanterior part.
Lorica is almost hyaline with hardly any visible
reticulation.
Lorica is ornamented with vertical striae.
Lorica is shaped with thick annular bulge.

Classicication level
F

G

*

26
27
28
29
30
31
32
33
34
35

Prong

Aboral part is closed-end.
Aboral part is abruptly narrowing orrounded-end.
Aboral part is moderately tapering-end.
Aboral part is closed square shape.
Aboral part is ﬂaring (only opened-end shape).
Aboral part is sharp or has a prong.
Lorica has a prong.
Prong is distinctly elongated.
Prong is ornamented by segments, needle or skirt.
Prong is ornamented by ridges.
Ridge part of prong is longer than one third lengthof body.

Morphological and morphometric analyses
Relationships among the traits of lorica morphology
were summarized well according to several axes of theprincipal coordinate (PCo) analysis (Table 1). The ﬁrst four

−0.1660.057−0.108

0.001

*

*

−0.263−0.003−0.015−0.023−0.184

0.211

0.064

0.120

−0.275−0.251

0.010
0.101

−0.054−0.041

0.005
0.023

*
*
*

0.948
−0.137
0.449
0.234

0.139
−0.093−0.228
0.073

0.044
0.178
−0.470−0.455−0.118−0.3100.138−0.117*0.293−0.491

0.329

0.089

*

0.129

−0.380

0.177

0.285

*
*

−0.228−0.269−0.0670.006−0.097−0.037

0.003
0.011

*

0.116

−0.403−0.002

0.332

*
*
*
*

−0.143−0.054−0.235−0.265−0.004−0.191−0.033−0.017

0.210
0.260
0.037
−0.019−0.172−0.226−0.177−0.101*−0.200−0.0490.072−0.216**0.026−0.257−0.249−0.2930.057
0.009−0.107−0.108−0.0190.114
0.001−0.084

0.924

0.077

0.032

−0.016

*

0.862

0.116

−0.071−0.119****−0.206−0.279−0.2640.422−0.047−0.018−0.0010.299

0.057−0.064−0.0770.353

0.111−0.006−0.043
0.085

*
*
*
*

−0.014−0.014−0.265−0.287

0.442
0.442
0.045
−0.0080.078
0.078−0.058−0.0740.226
0.226−0.004−0.023*−0.287−0.008−0.074−0.023****

Aboral−0.086−0.257

Lorica has oral collar or ﬂare.
Oral part of collar is narrower than bowl
diameter.
Oral part of collar is wider than bowl diameter.
Oral part is cone-shaped.
Oral part is distinctly funnel-shaped (ﬂaring
steeply).
Lorica has inner-collar (double oral rims).
Collar is distinctly partitioned against bowl.
Collar has no fenestrae.
Collar has many fenestrae (porous).
Inner-collar is longer than one third the length ofbody.
Collar with substances or grains on the surface.
Collar has minutely dentate oral rim.
Coller is annulated with spiral turns.

25−0.024

0.176

Oral

22
23
24

0.619

0.068

12

21

PCo IV

0.274

*

17
18
19
20

PCo III−0.141

Lorica is rounded shape in cross-section.
Bowl is thickened at central or anterior part.
Bowl is thickened at posterior part.
Bowl width is longest at posterior part.

16

PCo II

*

Bowl

14
15

PCo I

*

*

8
9
10
11

13

S

Eigen vectors

*
*
*
*

*

*

*

axes (I–IV) explained >75% of variance in the lorica morphology. The ﬁrst principal coordinate axis (PCo I) was
mainly correlated to morphological traits Nos. 1, 8, 25 and26 in Table 1. Thus, large PCo I values reﬂected such loricae that are agglutinated and have a rounded shape incross

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T. K azama et al.

icant difference was detected in the AR among the clades(p=0.016, Kruskal-Wallis test).
Discussion

Fig. 4. Morphospecies found in two different clades. a. Tintinnopsis karajacensis (AB640660) in subclade A-1, b. T. karajacensis (AB640661) in clade E in Fig. 3. Scale bars: 20 μm.

section with a closed aboral part that is round or narrows
in shape. PCo II was correlated mainly with morphologicaltraits Nos. 12, 14, 31 and 32 and described loricae withslightly ﬂaring oral portions or no collar, and with an elongated prong. The PCo III was correlated mainly with morphological traits Nos. 10, 11, 12 and 30 and described loricae with an oral collar, a sharpened aboral part and a bowlthat is not thickened or wide at the posterior part. The PCo
IV was correlated mainly with morphological traits Nos. 9,13, 16 and 18 and described hyaline loricae with a funnelshaped oral part that was not separated from the bowl, andthe bowl is not thickened in any part. We calculated valuesof the PCos axes of the 44 morphospecies, and plotted
these in two-dimensional planes of paired axes according
to clades (Fig. 5a, c, d) together with associations of themain morphological traits correlated to these axes (Fig. 5b,d, f). Morphospecies in clades A–D overlapped each otherin all the graphs. Among these, morphospecies from subclade A-2 were scattered most widely over the two-dimensional trait space, indicating that this subclade included
members with a variety of loricae that covered largely all
the morphological variations found in the other clades.
Clade B had higher values for PCo II compared withclades C and D (Fig. 5b), because morphospecies of thisclade had loricae with a sharpened aboral part or with aprong, and these characteristics were not found in the latter
two clades. However, there were no large differences in
PCo I, III and IV among these clades (Fig. 5d, f).
Morphometric analyses are shown in Fig. 6. Again, subclade A-2 showed large variations in the morphometrics ofthe loricae and covered the morphometric variations foundin subclade A-1 and the other clades. In general, morphospecies in clade B had longer loricae compared with thosein subclade A-1, clade C or clade D (Fig. 6a). In addition,
morphospecies in clades C and D tended to have loricae
with narrow width relative to the length, as shown by alarger AR (width: length ratio, Fig. 6b), although no signif-

Previous studies on the phylogeny of the Choreotrichia(including the order Tintinnida) were unable to clearly elucidate the phylogeny within the Tintinnida based on nuclear SSrRNA gene fragments (Snoeyenbos-West et al.
2002, Strüder-Kypke & Lynn 2003, 2008, Gao et al. 2009,
Li et al. 2009), because they analyzed a limited number oftintinnid species. Here we newly analyzed 55 sequences of
the SSrRNA gene fragments of tintinnid species (23 morphospecies, 11 genera and 6 families) collected in Sendai
Bay in Japan and its vicinity. Incorporating sequence datafrom the previous studies, we reconstructed a phylogenetic
tree composed of 84 sequences from 44 morphospecies intotal of eight families, and enhanced our understanding oftintinnid phylogeny. The results showed that at least six
families (Codonellidae, Codonellopsidae, Ptychocylididae,
Tintinnidiidae, Tintinnidae and Metacylididae) are polyphyletic.
We analyzed whether or not the morphology and morphometry of the loricae are traits reﬂecting genealogicalrelationships among tintinnid ciliates. The results showedthat among the six clades that we identiﬁed genetically,
subclade A-2 was the most diverse in terms of lorica morphology and morphometry. Because subclade A-2 is a large
paraphyletic group within clade A, variation in lorica morphologies within subclade A-1 was contained within that ofsubclade A-2. Although lorica morphology did not overlap
between clade B (consisting of Ptychocylididae alone) andclade C (the sole clade containing Eutintinnus), or between
clade B and clade D (consisting mainly of Tintinnidae),their lorica morphologies overlapped those of clade A to alarge degree. Furthermore, even though lorica length andwidth variation also differed between clade B and clade C,
there was signiﬁcant overlapping with clade A-2. These results suggest that there is no systematic relationship between lorica morphology and nuclear SSrRNA, althoughsome morphological features are useful to taxonomicallyseparate species in clade B from those in clade C and clade
D: species in the former clade can be distinguished fromthose in the latter two clades in that the loricae haveprongs, although the same characteristic is also found inspecies belonging to clade A.
Because agglutinated or hyaline lorica is a very noticeable features, it has been used as a key character separating Codonellidae, Codonellopsidae and Tintinnidiidae
from other families of Tintinnida (Kofoid & Campbell
1939). However, morphospecies of Codonellidae, Codonellopsidae and Tintinnidiidae belonged to polyphyleticclades and some clustered with morphospecies belonging
to other families lacking substrates on te lorica surface.
Moreover, there was no genetic difference between twomorphospecies Codonellopsis ostenfeldi (Codonellopsidae,

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119

Fig. 5. Results of principal coordination analysis for morphologies of loricae in tintinnids. Loadings of major morphologicaltraits in the ﬁrst four PCo axies are shown by lines with arrows in two-dimensional ordination spaces determined by the paired
axes (a, c and e). Loadings of eigen vectors are multiplied by 3 for comparison and morphological traits are denoted by the
numbers in Table 1. Ranges and values of morphospecies in different clades shown in Fig. 3 to these axes are shown by plots
and lines with different colors (b, d and f). The ordination spaces are shown as PCo I against PCoII (panel a and b), PCo III(panel c and d) and PCo IV (panel e and f). Values in parentheses are the percentage contribution of PCo axes.

a family with substrates on the lorica surface) and Helicostomella fusiformis (Metacylididae, a family without
substrates on the lorica surface), or between the two morphospecies Tintinnopsis dadayi (Codonellidae, a familywith substrates on the lorica surface) and Amphorides amphora (Tintinnidae, a family without substrates on the lorica surface). These results indicate that the presence or absence of substrates on the lorica surface may be a trait thathas evolved independently in different lineages without
being constrained phylogenetically, and thus should not be
used as a key trait for tintinnid taxonomy at generic, fam-

ily or even species levels. It is known that Favella campanula Schmidt can change the morphology of the lorica
through metamorphosis (Gold 1969). Tintinnopsis produce
agglutinated loricae with particle or mineral grains in natural settings, but have hyaline loricae when cultured in thelaboratory without a supply of substrates for the grains(Gold 1968, 1973). It therefore seems that the type of lorica(hyaline or agglutinated) is not necessarily geneticallyﬁxed and could be a plastic trait.
Strüder-Kypke & Lynn (2008) suggested that T. beroidea and T. dadayi belonged to the “true” Tintinnopsis

120

T. K azama et al.

Fig. 6. Relationship between length and width of lorica (a) and box plots (median, quantiles and range) of the aspect ratios(=lorica width/length) in different clades shown in Fig. 3 (b).

genus as a monophyletic group and were phylogeneticallydistinguishable from other species of Tintinnopsis. In thisstudy, however, T. dadayi AY143562 was genetically moresimilar to Codonellopsis ostenfeldi and Helicostomella fusiformis than to T. beroidea EF123709. Thus, the species of“true” Tintinnopsis are genealogically unrecognizable. Due
to morphological and genetic diversity, the genus Tintinnopsis has been thought to be paraphyletic (Laval-Peuto &Brownlee 1986, Agatha & Riedel-Lorje 2006, StrüderKypke & Lynn 2008). In this study, morphospecies of Tintinnopsis were found not only in clade A-1 but also inclades A-2, C, D and E, which contained species from various families. This result implies that this genus is polyphyletic rather than paraphyletic, and is composed of genetically diverse species. According to classical classiﬁcation,
Tintinnopsis spp. have three typical characteristics of thelorica: agglutination of particles or grains, absence of a hyaline collar, and a non-funnelled shape at the oral part.
This study suggests that these morphological features are
not phylogenically ﬁxed, but are traits that have converged
among genealogically different tintinnid groups.
Recently, Kim et al. (2010) showed that specimens ofFavella ehrenbergii from the coastal waters of Incheon,
Korea differed genetically from those collected by Snoeyenbos-West et al. (2002) along the east coast of North
America. According to the nuclear SSrRNA phylogeny,
specimens of F. ehrenbergii from Korea were close to F.
panamensis Kofoid & Campbell and F. campanula, while
specimens of F. ehrenbergii from eastern North America
were close to F. taraikaensis (from northern China) andMetacylis sp. Since lorica morphology is very similar between F. ehrenbergii and F. taraikaensis, Kim et al. (2010)
suggested that Snoeyenbos-West et al. (2002) might havemisidentiﬁed their species. However, both specimens of F.
ehrenbergii and F. taraikaensis from Sendai Bay were
found not only in clade A but also in clade B, and formed
subclades within each clade with other morphospecies.
These results imply that Favella is not a monophyletic
genus and it is hard to identify species of this genus accurately according to the morphology of the lorica.
We also found that morphospecies of Amphorellopsis
acuta, Helicostomella fusiformis, Tintinnopsis dadayi andT. karajacensis were polyphyletic and contained diverse

lineages. The differences in partial sequence of SSrRNA
among specimens within each of these morphospeices cannot be explained due to PCR or sequence reading errors
because these differences (>8%) were much larger thanthose cause by such errors (<1%). This result implies that
there are many cryptic species within the Tintinnida, justas with other planktonic taxa such as diatoms (Amato et al.
2007), copepods (Chen & Hare 2008) and aloricate ciliates(Katz et al. 2005). On the one hand, studies with laboratory cultures of tintinnid species have shown that size andmorphology of the lorica can vary greatly even within asingle species (Gold 1968, 1973, Kim et al. 2010). On theother hand, the present study showed that even if there areno notable differences in the size and shape of the loricae
between tintinnids, they do not necessarily share the samedescendant and thus the same genealogical position. Therefore, characteristics of the lorica are not necessarily ﬁxed
traits and both divergence and convergence in the morphology of loricae has occurred in tintinnids due to evolutionary processes. For these reasons, most morphologicalfeatures of the lorica are not useful for classifying tintinnid
species, even at the family level. To classify species of Tintinnida on a morphological basis without discrepancy withgenetic data (Mcmanus & Katz 2009), it may be better touse cytological traits such as inner cell form, cell size andcilia, although only a few studies have as yet conducted
cytological analyse, and only for a limited number of tintinnid species (Laval-Peuto & Brownlee 1986, Agatha &Riedel-Lorj 2006, Agatha & Strüder-Kypke 2007).
Acknowledgements
We are grateful to Satoshi Chiba, Wataru Makino, andYoshihisa Suyama for their discussion and technicalsuggestions. We also thank Masakado Kawata and Noe
Matsushima for their help in the genetic analyses, andTakuya Sato and Makoto Abe for their help with laboratory work. This study was funded in part by a Grant-inAid for Scientiﬁc Research B (No. 22370007) from MEXT
Japan to JU. TK, SI and JU were supported by the Global
COE program “Centre for ecosystem management adapting to global change” (J03) of MEXT Japan.

Gene and morphology of tintinnids

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Appendix Table 1. DDBJ accession numbers, sampling sites (see Fig.1), sampling dates, sequence lengths, and GC
contents of nuclear SSrRNA sequences from specimens obtained in this study.
Species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55

Amphorellopsis acuta
Amphorellopsis acuta
Amphorides amphora
Codonella amphorella
Codonella amphorella
Codonella amphorella
Codonellopsis morchella
Codonellopsis ostenfeldi
Eutintinnus lususundae
Eutintinnus lususundae
Favella azorica
Favella ehrenbergii
Favella ehrenbergii
Favella taraikaensis
Favella taraikaensis
Helicostomella fusiformis
Helicostomella fusiformis
Leprotintinnus pellucidus
Leprotintinnus pellucidus
Leprotintinnus pellucidus
Steenstrupiella steenstrupii
Stenosemella nivalis
Tintinnopsis corniger
Tintinnopsis corniger
Tintinnopsis dadayi
Tintinnopsis dadayi
Tintinnopsis dadayi
Tintinnopsis dadayi
Tintinnopsis dadayi
Tintinnopsis elongata
Tintinnopsis karajacensis
Tintinnopsis karajacensis
Tintinnopsis karajacensis
Tintinnopsis karajacensis
Tintinnopsis karajacensis
Tintinnopsis karajacensis
Tintinnopsis karajacensis
Tintinnopsis lobiancoi
Tintinnopsis lobiancoi
Tintinnopsis lobiancoi
Tintinnopsis lobiancoi
Tintinnopsis lobiancoi
Tintinnopsis lobiancoi
Tintinnopsis minuta
Tintinnopsis minuta
Tintinnopsis mortenseni clone-1
Tintinnopsis mortenseni clone-2
Tintinnopsis mortenseni clone-3
Tintinnopsis tenuis clone-1
Tintinnopsis tenuis clone-2
Tintinnopsis tenuis
Tintinnopsis tenuis
Tintinnopsis tocantinensis
Tintinnopsis tocantinensis
Tintinnopsis tubulosa

Accession
Nos.

Site

Date

Length(bp)

GC content(%)

AB640624
AB640625
AB640626
AB640627
AB640628
AB640629
AB640630
AB640631
AB640632
AB640633
AB640634
AB640635
AB640636
AB640637
AB640638
AB640639
AB640640
AB640641
AB640642
AB640643
AB640647
AB640648
AB640676
AB640678
AB640651
AB640652
AB640653
AB640654
AB640655
AB640656
AB640657
AB640658
AB640659
AB640660
AB640661
AB640662
AB640663
AB640664
AB640665
AB640666
AB640667
AB640668
AB640669
AB640670
AB640671
AB640672
AB640673
AB640674
AB640679
AB640680
AB640681
AB640682
AB640649
AB640650
AB640683

SP1
OSK
C00
TTR
TTR
TTR
SP1
SP1
SP1
SP1
C00
SP1
SP1
SG1
SG1
SP1
SP1
SG1
SG1
SG1
C00
C00
MNG
C00
TTR
TTR
SG1
SG1
SG1
SG1
C00
ISM
TTR
TTR
SG1
SG1
SP1
C00
SP1
ONG
GZN
GZN
GZN
ISM
TTR
C00
C00
C00
C00
C00
ONG
ONG
C00
A00
SP1

13-Aug26-Aug26-Aug22-Oct22-Oct22-Oct13-Aug
3-Sep
12-Jul12-Jul
28-Jun12-Jul12-Jul
22-Oct22-Oct
3-Sep
3-Sep
3-Sep
3-Sep
3-Sep
13-Aug26-Aug26-Aug26-Aug
3-Sep
3-Sep
3-Sep
3-Sep
3-Sep
3-Sep
13-Aug
3-Sep
3-Sep
3-Sep
3-Sep
3-Sep
3-Sep
12-Jul12-Jul26-Aug22-Oct22-Oct22-Oct3-Sep
3-Sep
28-Jun28-Jun28-Jun28-Jun28-Jun26-Aug26-Aug13-Aug26-Aug
3-Sep

1371
1288
1330
1366
1333
1353
1092
1312
1319
1345
1641
1337
1353
1354
1334
1299
1349
1349
1320
1334
1329
1303
1289
1295
1346
1344
1320
1379
1375
1303
1286
1295
1323
1312
1382
1363
1375
1333
1276
1277
1373
1343
1370
1330
1375
1254
1237
1237
1296
1250
1320
1322
1343
1364
1305

46.90
48.91
45.94
47.80
48.16
48.04
45.42
47.26
49.13
49.52
47.17
47.94
48.04
48.08
48.50
48.65
46.70
49.59
49.62
49.48
49.36
46.97
48.41
46.80
46.66
46.73
47.35
49.17
47.27
47.51
46.66
47.03
47.92
47.10
48.12
46.96
46.76
47.56
46.71
47.22
47.41
47.13
47.30
48.42
48.36
46.81
47.13
47.13
47.30
46.80
46.67
47.28
47.51
47.95
48.05

124

T. K azama et al.

Appendix Fig. 1. Phylogenetic tree by the Bayesian Inference method for tintinnid nuclear SSrRNA sequences. The
numbers at nodes represent Bayesian clade credibility values.

Gene and morphology of tintinnids

125

Appendix Fig. 2. Phylogenetic tree by Neighbor-Joining (NJ) method for tintinnid nuclear SSrRNA sequences.
The numbers at nodes represent NJ bootstrap support values.