Protist, Vol. 163, 560–573, July 2012
http://www.elsevier.de/protis
Published online date 16 December 2011
ORIGINAL PAPER
Ultrastructure and Molecular Phylogeny of
Thaumatomonads (Cercozoa) with Emphasis on
Thaumatomastix salina from Oslofjorden, Norway
Shuhei Otaa,1 , Wenche Eikrema,b , and Bente Edvardsena
aMarine Biology, Department of Biology, University of Oslo, P.O. Box 1066 Blindern, NO-0316
bNIVA, Norwegian Institute for Water Research, Gaustadalléen 21, NO-0349 Oslo, Norway
Oslo, Norway
Submitted May 24, 2011; Accepted October 24, 2011
Monitoring Editor: Michael Melkonian
A culture of Thaumatomastix was isolated from a sediment sample collected in Oslofjorden and
established as a monospecific strain (UIO286). Based on this culture, light and transmission electron
microscopy and phylogenetic analyses were carried out. Thaumatomastix species are confined within
the order Thaumatomonadida of the class Imbricatea and phylum Cercozoa. They are heterotrophic
and their cell bodies are covered with silica scales. Observations of thin sections as well as whole
mounts indicate that the morphology and ultrastructure of UIO286 is identical to T. salina, which was
initially described from salt pools in Denmark. Detailed examination revealed some new features such
as the presence of pseudopodia and silica deposition vesicles producing spine scales. The phylogeny
presented here includes ribosomal DNA sequences from both imbricatean cultures and environmental samples. The 18S rDNA phylogenetic tree suggests that (i) Thaumatomastix is paraphyletic within
the Thaumatomonadida clade, (ii) there is no close affinity between T. salina and other cultured and
sequenced strains, but it is closely related to a sequence obtained from environmental DNA; we propose the present strain to serve as a reference culture of Thaumatomastix species and T. salina.
Further, we discuss the distribution, habitats, and evolution of scale formation among euglyphids and
thaumatomonads.
© 2011 Elsevier GmbH. All rights reserved.
Key words: Cercozoan diversity; heterotrophic flagellate; Imbricatea; Oslofjorden; Thaumatomastix; ultrastructure.
Introduction
The class Imbricatea (Cercozoa) is characterized
by solitary cells carrying silica scales and currently
comprises four orders: Euglyphida, Spongomonadida, Thaumatomonadida, and Marimonadida
1
Corresponding author. Present address: Department of Integrated Biosciences, Graduate School of Frontier Sciences,
University of Tokyo, FSB-601, Kashiwanoha, Kashiwa, Chiba
277-8562, Japan
e-mail [email protected], ota [email protected]
(S. Ota).
© 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.protis.2011.10.007
(Cavalier-Smith and Chao 2003; Howe et al. 2011).
Although Euglyphida and Thaumatomonadida are
scaly cercozoans, they differ morphologically; the
euglyphids are testate filose amoebae (Meisterfeld
2000), whereas the thaumatomonads are scaly
flagellates or amoeboflagellates (Patterson and
Zölffel 1991). Well-known thaumatomonad genera are Allas Sandon (1927), Thaumatomonas
de Saedeleer (1931) and Thaumatomastix
Lauterborn (1899), which are biflagellate heterotrophic flagellates with siliceous body scales.
The genus Thaumatomonas is distinguished from
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 561
Thaumatomastix by the absence of flagellar
scales and by possession of a very short anterior
flagellum (e.g., Wylezich et al. 2007). However,
some Thaumatomastix species lack flagellar
scales and have a short anterior flagellum (e.g.,
T. tripus = Chrysosphaerella tripus; Takahashi and
Hara 1984), indicating that genus delimitation may
be imprecise and further examination is needed
using both morphological and molecular data.
More recently, Howe et al. (2011) transferred
nine Thaumatomastix species (basionyms:
T.
dybsoeana,
T.
formosa,
T.
fragilis,
T. fusiformis, T. gloenlandica, T. igloolika,
T. nigeriensis, T. spinosa, and T. splendida)
to the genus Reckertia (Conrad 1920). Moreover,
Howe et al. (2011) pointed out that the transfer of
Chrysosphaerella triangulata (Balonov 1980) to
Thaumatomastix (Beech and Moestrup 1986) was
incorrect. Consequently, six species are currently
recognized in the genus Thaumatomastix. Marine
Thaumatomastix species have been recorded
in pelagic temperate to polar environments at
salinities ranging from 2 to 35 PSU (Beech and
Moestrup 1986; Ikävalko 1998; Moestrup 1979;
Takahashi and Hara 1984; Thomsen et al. 1995;
Thomsen and Ikävalko 1997; Vørs 1992a,b, 1993).
There have been some reports on Thaumatomastix morphology using whole-mount transmission
electron microscopy of collected field material (e.g.,
Beech and Moestrup 1986; Thomsen et al. 1995),
whereas only a few previous studies have provided
phylogenetic information of Thaumatomastix:
For instance, Wylezich et al. (2007) proposed
a phylogenetic position of Thaumatomonadida
when describing Thaumatomonas coloniensis,
and Chantangsi et al. (2010) and Cavalier-Smith
and Chao (2003) proposed a global cercozoan
phylogeny. However, there has been no previous
study integrating morphology, ultrastructure and
molecular phylogeny in Thaumatomastix species.
During a sampling cruise in outer Oslofjorden
in June 2010, we collected water and sediment
samples, from which we established over 60
cultured strains. One of the strains (UIO286) isolated from the sediment samples was identified
as Thaumatomastix salina based on morphology.
Thaumatomastix salina was originally described
from marine salt pools in Denmark as the chrysophyte Chrysosphaerella salina (Birch-Andersen
1973). Later Beech and Moestrup (1986) reexamined C. salina and its allies in detail by
whole-mount electron microscopy as well as light
microscopy, and found that C. salina was heterotrophic and showed considerable resemblance
in spine morphology to Thaumatomastix species.
Consequently, C. salina was transferred to the
genus Thaumatomastix and new combinations
were provided (Beech and Moestrup 1986). Here
we examined the morphology, ultrastructure, and
the 18S rDNA sequence of cultured T. salina
(UIO286) and linked morphology-based identification to molecular phylogeny. In addition, we
observed silicon deposition vesicles (SDVs) closely
associated with mitochondria, which has been
described previously in thaumatomonads (Karpov
1990, 1993, 2000; Karpov and Zhukov 1987;
Moestrup 1982; Swale and Belcher 1974, 1975).
Results
Nomenclatural Note
Thaumatomastix salina (Birch-Andersen 1973:
142) Beech and Moestrup 1986: 866 emend. S.
Ota, Eikrem et Edvardsen
Basionym: Chrysosphaerella
Andersen 1973: 140
salina
Birch-
Emended diagnosis: Cells solitary, heterotrophic,
spherical to elliptical, 8-13 ␮m (mean = 10.7 ␮m,
n = 10) long, 5.3-9.1 ␮m (mean = 7.5 ␮m, n = 10)
wide. Two flagella of unequal length, posterior flagellum naked, longer than body (c. 15 ␮m); anterior
flagellum short (c. 2.5 ␮m) with flagellar scales. Cell
movement gliding with rapid flicking motion. Flagellate cells may transform to amoeboid cells with
branching pseudopodia. Cell cytoplasm with one
to three (or sometimes more) orange bodies. Cell
surface covered with one layer of inter-connected
spine scales and elliptical scales. Spines 3.59.3 ␮m (mean = 5.5 ␮m, n = 15) long and 0.1 ␮m
wide. Basal part with two discs, proximal disc
0.5 ␮m; distal disc 0.3 ␮m in diameter. Tip of spine
scale with three ridges. Spineless scales elliptical
and two-tiered, 1.0-1.6 ␮m (mean = 1.3 ␮m, n = 15)
long, 0.6-1.1 ␮m (mean = 0.9 ␮m, n = 15) wide with
rims. Elliptical plates with perforations (sometimes
non-perforated). Scales produced within silica
deposition vesicles closely associated with mitochondria. Ejectosome-like organelles elongated
with fibrous and amorphous content. Microbody
located in proximity of nucleus.
Lectotype (ICZN): Birch-Andersen 1973, Bot.
Tidsskr. 68: p. 142, fig. 3; Bramsnæsvig, Sjælland,
Denmark (for nomenclatural stability under the
ICZN, we designated a lectotype here).
Habitat: marine or brackish
Distribution (salinity): Denmark (Birch-Andersen
562 S. Ota et al.
colored bodies were often observed in the peripheral region of the cell (Supplementary Figure S1).
Occasionally flagellated cells switched to an amoeboid cell type and vice versa. The amoeboid cells
changed rapidly to the flagellated stage (approximately 40 seconds, see Supplementary Movie S2).
The amoeboid cell type possessed branching pseudopodia that attached to the substratum (Fig. 1D).
Cells often moved by gliding or creeping on the
substratum; the long flagellum was trailing on
the substrate when swimming (see Supplementary
Movie S3).
TEM Whole Mounts
Figure 1. Light micrographs of Thaumatomastix
salina. A, B. Elliptical cells. Spine scales are visible
in A. C. Ovoid cell with flagellum. D. Cell possessing
pseudopodia (arrows).
1973, 3.4-24.0 PSU; Vørs 1992a, 22-25 PSU), Finland (Thomsen 1979, 1.2-6.6 PSU; Vørs 1992b,
5-6 PSU), Greenland (Thomsen and Ikävalko
1997, 25 PSU), Japan (Takahashi and Hara 1984,
30-33 PSU), New Zealand (Moestrup 1979, 35
PSU), Norway (this study, 34 PSU)
DNA sequence information: The accession number FR846196 is a nuclear 18S rDNA sequence of
Thaumatomastix salina.
Reference strain: UIO286 at the University of
Oslo Algal Culture Collection. A duplicate strain is
available from the Culture Collection of Algae and
Protozoa, UK under the code CCAP1973/1.
Morphology
Cells were solitary, colorless, spherical to slightly
elliptical, 8-13 ␮m (mean = 10.7 ␮m, n = 10) long,
5.3-9.1 ␮m (mean = 7.5 ␮m, n = 10) wide, and with
two unequal flagella (Fig. 1A-C). The long flagellum (posterior flagellum; PF) was longer than the
body length (c. 15 ␮m), and the short flagellum (c.
2.5 ␮m; anterior flagellum, AF) was difficult to see
under the light microscope (Fig. 1C). The cells were
covered by two scale types. Spine scales were visible (Fig. 1A), but the other type, i.e. elliptical plate
scales, were rarely observed in the light microscope. One to three (or sometimes more) orange
In the TEM whole mount preparations, the anterior
and posterior flagella and two types of body scales
were revealed (Fig. 2A, B). The PF was naked (i.e.,
smooth), whereas the AF was covered with small
ovoid or elliptical scales with a ridge in the center
of the scale (Fig. 2C). In some cases, however, the
short anterior flagellum was lacking scales.
Plate and spine scales covered the entire cell
surface (Fig. 2A, B). The elliptical two-tiered
plate scales were of almost uniform size, 1.0-1.6
(mean = 1.3, n = 15) ␮m long, 0.6-1.1 (mean = 0.9,
n = 15) ␮m wide (Fig. 3A-D). The semi-elliptical
regions were perforated and the number of perforations varied from approximately 7-33 (Fig. 3A-D)
and non-perforated zones were present around its
minor axis (Fig. 3A-D). Sometimes the perforations were aligned along the semi-elliptical regions
circumferentially (Fig. 3D). Scales without perforations as shown by Takahashi and Hara (1984, fig.
14) were rarely observed (not shown). In lateral
view, the scale appeared as two fused plates, each
of them had a dish-like structure with a marginal
rim (Fig. 3E). The spine scales varied in length
(3.5-9.3 ␮m; mean = 5.5 ␮m, n = 15) and the distal part had three ridges (Fig. 3F, G). The central
ridge was electron opaque, located longitudinally
in the center of the spine (Fig. 3F, G). The proximal
end (facing the cell membrane) of the spine scale
was enlarged and enclosed with a proximal plate
(Fig. 3H). A collar-like structure (pterygoid plate)
was observed, which was located near the proximal
plate, and the diameter of the pterygoid plate was
approximately two-thirds that of the proximal plate
(Fig. 3H).
TEM Thin Sections
The general ultrastructure of a typical cell is shown
in Figure 4A. Cells were covered with body scales
of two types, arranged in a single layer and tightly
inter-connected (Fig. 4B, C). A nucleus was located
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 563
Figure 2. Transmission electron micrographs of Thaumatomastix salina. Whole mounts. A. Whole cell, showing
the long (arrows) and short flagellum (arrowhead). B. Detail of the cell surface, showing spine scales (arrows)
and elliptical scales (arrowheads). C. Higher magnification view if the short flagellum, showing flagellar scales.
The arrow indicates a ridge in the center of the scale.
in the anterior part of the cell (Fig. 4A). Frequently
one, or sometimes more vacuoles were observed,
notably in the peripheral region of the cell (Fig. 4A).
A microbody with amorphous content was located
near the nucleus (Figs 4A, 5F). Occasionally, some
roughly cylindrical ejectosome-like structures composed of fibrous and amorphous material was
observed in vesicles located immediately under the
plasma membrane (Fig. 4D). Some of the vesicles
containing ejectosome-like structures were open to
the exterior of the cell (Fig. 4D); while in others
the membrane was intact (Fig. 4E). Flat (occasionally elliptical or irregularly shaped) vesicles
with no apparent content were often observed just
underneath the plasma membrane (Fig. 4F). The
two flagella emerge from the bottom of a furrowlike invagination at the anterior end of the cell
(Fig. 4G).
Table 1. Result of the AU test comparing alternative positions.
Hypothesis
Tree topology
p-values
A
B
C
D
E
F
G
((((Tha + Spo) + Mar) + N/C) + Eug) + Cer
(Tha + Spo) + (Eug + (N/C + Mar)) + Cer
((((Tha + Spo) + N/C) + Mar) + Eug) + Cer
((((Tha + Spo) + Eug) + N/C) + Mar) + Cer
((((Tha + Eug) + Spo) + N/C) + Mar) + Cer
((((Tha + Spo) + Mar) + Eug) + N/C) + Cer
((((Tha + Spo) + N/C) + Eug) + Mar) + Cer
0.737
0.514
0.314
0.315
0.250
0.069
0.029*
*Hypothesis is rejected at the significance level of 0.05. Tha = Thaumatomastix salina; Spo = Spongomonadida;
Mar = Marimonadida; N/C = Nudifila + Clautriavia; Eug = Euglyphida; Cer = Cercomonadida. Scaly taxon is
shown in bold. Hypothesis D represents the topology as inferred the present ML analyses. Supplementary
Table 1 shows schematic trees corresponding to the present topology.
564 S. Ota et al.
Figure 3. Transmission electron micrographs of Thaumatomastix salina. Whole mounts. A-D. Front view of
elliptical scales. E. Side view of the elliptical scale, showing a dish-like structure with a marginal rim (arrows).
F. Whole spine scale. G. Detail of the distal part of the scale. The central ridge is electron opaque (arrows). H.
Detail of proximal part of the scale, showing a proximal plate (arrowhead) and collar-like structure (arrow).
Many mitochondrial profiles with tubular cristae
were dispersed irregularly throughout the cytoplasm. SDVs were closely associated with mitochondria (Fig. 5A-C) and both types of scales (spine
and elliptical scales) were developed in mitochondria associated SDVs. One of the two plates of the
two-tiered elliptical scales was observed developing in the SDV. The dorsal side of the scale was
directed towards the mitochondrial matrix and the
dorsal ridge projections often invaginated into the
mitochondrial matrix (Fig. 5B-C). When the scale
was mature, the SDV moved away from the mitochondrion and the scale was transported to the
surface (Fig. 5C-E). One vesicle contained one
plate (i.e., one of the two parts of the scale), and
the SDVs containing mature plates were located
immediately under the plasma membrane during
their transfer to the surface (Fig. 5E). SDVs containing spine scales were deeply invaginated into the
mitochondria during the development of the scale
(Fig. 5F). When the scale was mature, the SDVs
containing spine scales were also transferred to the
surface (Fig. 5G).
Phylogeny
Figure 6 shows the inferred imbricatean phylogeny
and phylogenetic position of the present strain of
Thaumatomastix salina (UIO286). The Bayesian
inference and maximum likelihood (ML) analyses showed that the monophyly of Euglyphida
was robustly recovered (99% ML bootstrap values (BS); 1.00 Bayesian posterior probabilities
(BPP)), but the monophyly of the Thaumatomonadida was weekly to moderately supported (64%
BS; 0.99 BPP). Within the Thaumatomonadida,
clades A, B, C and D were identified with relatively high support values (≥85% BS; 1.00 BPP);
clade A was sister to clade B represented only
by environmental sequences. Clade C comprised
the species Reckertia filosa and some environmental sequences. Clade D comprised the present
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 565
Figure 4. Transmission electron micrographs of Thaumatomastix salina. Thin sections. A. General ultrastructure, showing nucleus (n), mitochondria (m), microbody (mb) and vacuole (v). B. Detail of the cell surface.
Arrows indicate a longitudinal section of a spine scale. C. Detail of the cell surface, showing that elliptical scales
are tightly inter-connected (arrows). D, E. Ejectosome-like organelle. F. Detail of the cell surface, showing vesicles just under the plasmalemma (arrows). G. Transverse section of the furrow-like invagination including the
two flagella (f).
species (T. salina) and an environmental sequence
(AY180026). Thaumatomastix sp. (GQ144681) and
Peregrinia clavideferens (DQ211593) branched
basally within the Thaumatomonadida clade. Spongomonas solitaria (Spongomonadida) was sister to
the Thaumatomonadida with moderate Bayesian,
but weak ML support.
To examine alternative tree topologies, we carried out approximately unbiased (AU) test (Table 1;
see also Supplementary Table S4). Hypothesis A
566 S. Ota et al.
Figure 5. Transmission electron micrographs of Thaumatomastix salina. Thin sections. A. Mitochondrion with
early stage of silicon deposition vesicles (SDVs) (arrows). B. Mature elliptical scale in an SDV. C. Mature elliptical
scale in an SDV, about to leave the mitochondrion (arrow). D. Higher magnification view of the SDV. Arrows
indicate membrane of the SDV. E. Mature elliptical scale being transported to the surface (arrows). Arrowheads
indicate an elliptical scale outside the cell. F. Mitochondrion with spine scale in an SDV (arrowheads). Arrows
indicate an SDV including a spine scale, and the double-arrow indicates an SDV including an elliptical scale,
mb = microbody. G. Mature spine scale (proximal part) in the cytoplasm.
(Euglyphida as basal position within Imbricatea)
had the highest p-value amongst the alternative
hypotheses. Hypothesis G (Marimonadida as basal
within the Imbricatea) was rejected at the 5% level.
Hypothesis F (Nudifila + Clautriavia as basal within
the Imbricatea) was not rejected, but the p-value
was relatively close to the 5% level (Hypothesis
F = 6.9% of p-value).
Discussion
Identification
Within the thaumatomonads, scale morphology
and the presence or absence of spine scales are
essential in species identification (Table 2). The
present species has two types of body scales:
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 567
Thaumatomonas sp. SCCAP T1 AY496046
23/-
Thaumatomonas seravini AY496044
41/0.85
Thaumatomonas sp. U42446
53/0.75
Thaumatomonas coloniensis DQ211591
76/0.98
Thaumatomonas sp. ATCC50250 AF411261
Thaumatomonas sp. AF411260
A
25/-
Thaumatomonas sp. TMT002 DQ980486
71/-
Thaumatomonas seravini AF411259
100/1.00 79/0.99
Allas diplophysa AF411262
69/1.00
Uncultured Amb_18S_1434 EF023966
Uncultured Elev_18S_7011 EF025032
55/0.83
Thaumatomonadida
B
Uncultured Amb_18S_1319 EF023867
Uncultured 3b-D9 FN690393
44/-
Uncultured 5-B11 FN690394
50/0.72
C
Uncultured 8-32 FN690392
92/1.00
Reckertia filosa AY268040
53/0.88
Thaumatomastix salina UIO286 FR846196
85/1.00
Uncultured CCW52 AY180026
64/0.99
D
Thaumatomastix sp. GQ144681
39/0.70
Peregrinia clavideferens DQ211593
x2
Spongomonadida
Spongomonas solitaria CCAP 1971/1 HQ121435
93/1.00
Euglypha rotunda CCAP1520/1 X77692
Euglypha rotunda AJ418782
9/-
Euglypha rotunda AJ418784
43/0.56
Trinema enchelys AJ418792
44/0.78
Corythion dubium EF456751
Euglypha acanthophora AJ41878
25/0.50
Euglyphida
Assulina muscorum AJ418791
99/1.00
Ovulinata parva strain CCAP1554/1 HQ121432
80/0.94
Paulinella chromatophora X81811
x2
Cyphoderia ampulla GU228896
Nudifila producta CCAP1911/1 HQ121434
38/0.74
Clautriavia biflagellata FJ919772
N/C clade
Pseudopirsonia mucosa AJ5611
85/1.00
Auranticordis quadriverberis EU484394
98/1.00
54/97/1.00
Marimonadida
Cercomonas plasmodialis AF411268
Cercomonas LargeSA AF411266
Cavernomonas mira FJ790718
Sarcomonadea (outgroup)
Eocercomonas sp. AF411269
Uncultured eukaryote clone I_4_50 AB534514
0.1
Figure 6. RAxML phylogenetic tree of imbricateans and sarcomonads (out group) based on 18S
rDNA sequences (40 OTUs and 1,419 nucleotide positions). Accession numbers are given after the
species/environmental clone names. Bootstrap values/Bayesian posterior probabilities are shown near the internal nodes. Black dots on nodes indicate support values of 100%/1.00. The present species (Thaumatomastix
salina) is shown in bold.
spine scales and elliptical scales, and so do
Thaumatomastix salina, T. bipartita and T. setifera
as well. Thaumatomastix bipartita is distinguished
from T. salina by the possession of a bipartite
structure in the spine scales (Beech and Moestrup
1986). The AF of T. salina is very short and difficult
to see in light microscopy, whereas in T. setifera the
AF is longer and both flagella are clearly visible.
Consequently, the present isolate (UIO286) is
assigned to T. salina. Our isolate had the ability to
transform into an amoeboid stage and it formed
pseudopodia. In addition, an ejectosome-like
568 S. Ota et al.
simultaneously, suggesting that one mitochondrion-associated SDV can form one type of scales
only. However, Moestrup (1982) noted that in Reckertia sagittifera both body scales and flagellar
scales were formed in the same mitochondrionassociated SDV simultaneously, and thus further
observations are required to confirm this accordingly. Recently, mitochondria-associated SDVs
were also reported by Zolotarev et al. (2011) in
Thaumatomastix sp. from the White Sea. Although
we do not know the physiological advantages of
mitochondrion-associated SDVs, we infer that the
SDVs need some functions of the mitochondria to
develop scales. This feature is only observed in
the thaumatomonad lineage and appears unique
among the unicellular eukaryotes. Evolutionary
aspects are discussed below.
organelle was observed. All together these novel
phenotypes called for an emended diagnosis of T.
salina, which was provided above. The morphology
of the body scales may vary and Takahashi and
Hara (1984) showed that the elliptical body scales
may even lack perforations. Our observations
revealed that the number of perforations was
variable, suggesting that non-perforated body
scales are within the intra-species variation.
The ejectosome-like organelle observed in T.
salina was located just beneath the plasma membrane. It may also resemble a stage of endoor exocytosis. Since we are not convinced that
this organelle is an ejectosome, we use the
term “ejectosome-like organelle”. The longitudinal
outline was slightly cylindrical and contained a
structure that had both fibrous and amorphous
components. In contrast, the cylindrical ejectosomes previously known from cercozoan genera
(e.g., Gyromitus, Thaumatomonas and Bigelowiella
[chlorarachniophytes]) have a homogeneous and
somewhat electron-opaque structure (CavalierSmith et al. 2008; Howe et al. 2011; Moestrup
and Sengco 2001; Swale and Belcher 1975). This
organelle might be a unique ultrastructural feature
in Thaumatomastix.
Both elliptical and spine scales were formed
in SDVs associated with mitochondria. In the
present study we did not observe a mitochondrionassociated SDV forming both types of scales
Phylogenetic Relationship
The imbricatean (Euglyphida, Thaumatomonadida,
Spongomonadida, and Marimonadida) phylogeny
based on 18S rDNA sequences presented here
is roughly consistent with previously published ML
trees (Cavalier-Smith and Chao 2003; Ekelund
et al. 2004; Howe et al. 2011; Lara et al. 2007;
Wylezich et al. 2007) and provides new insight
into the phylogenetic position of T. salina. Thaumatomastix salina was found to be sister to an
uncultured environmental DNA sequence CCW52
Table 2. Comparison of morphology in Thaumatomastix species.
Thaumatomastix References
species
Number
of body
scale
types
Type of
body
scale
Spines Habitat
type
Other remarks
T. bipartita
Beech and Moestrup,
1986
Takahashi and Hara,
1984; Beech and
Moestrup, 1986
(Birch-Andersen,
1973) Beech and
Moestrup, 1986
Lauterborn, 1899
2
elliptical
Yes
Marine
Bifurcated spines
2
triangular
Yes
Marine
2
elliptical
Yes
Marine
Flagella present;
spine
length = 3.4-6.6 ␮m
-
Unknown
?
Yes
Marine
Takahashi and Hara,
1984; Beech and
Moestrup, 1986
Mikrjukov, 2002
2
triangular
Yes
Marine
2
triangular
Yes
Marine
T. patelliformis
T. salina
T. setifera
T. tripus
T. tauryanini
Both flagella
observed
Flagella present;
spine
length = 7.0-19 ␮m
no flagella observed
Note: Thaumatomastix species are listed alphabetically. Some Thaumatomastix species are unlisted here: T.
punctata is nomen nudum, T. thomseni was transferred to the Reckertia as R. thomseni (Karpov 2011), and T.
triangulata was dealt with Chrysophaerella species according to Howe et al. (2011).
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 569
(AY180026) originating from a marine anoxic environment off Cape Cod, NE USA (Stoeck and
Epstein 2003) with robust statistical support. The
present analyses also demonstrate that clade B
is composed only of environmental sequences
derived from soil samples (Lesaulnier et al. 2008);
and there are few isolated cultures of taxa closely
related to T. salina, probably due to the relative
difficulty of isolating and culturing Thaumatomastix species. In the present analyses Peregrinia
clavideferens is the deepest branch in the thaumatomonads; this is also consistent with the tree of
Wylezich et al. (2007) (Gyromitus sp. HFCC94 was
re-described and renamed as Peregrinia clavideferens by Howe et al. (2011)).
The well-supported clade A mainly comprises
Thaumatomonas species, but also contains strain
ATCC50250 (originally labeled as “Thaumatomastix sp.”) and Allas diplophysa. As noted previously
by Cavalier-Smith and Chao (2003) and Wylezich
et al. (2007), the present analyses also show that
there are very small differences in 18S rDNA within
clade A (e.g., more than 99% similarity of 18S
rDNA sequences between “Thaumatomastix” sp.
strain ATCC50250 and Thaumatomonas coloniensis). More recently, Howe et al. (2011) provided a
comprehensive taxonomy of filoseans (Cercozoa),
where they noted the misidentification of strain
ATCC50250 “Thaumatomastix sp.”, which is actually a Thaumatomonas species. Accordingly, we
also relabeled strain ATCC50250 of the present
OTU as Thaumatomonas in the phylogenetic analyses. Howe et al. (2011) also dealt with the taxonomical and nomenclatural confusion that surrounds
the morphologically similar genera Allas and Thaumatomonas. They suggested that there are no morphological grounds for retaining Thaumatomonas
as a genus distinct from Allas, which concurs with
a high degree of similarity in 18S rDNA sequences
between the genera. However, the authors retain
both genera because of nomenclatural stability; i.e.,
they did not regard the more recent name Thaumatomonas (widely used name) as junior synonym,
and considered ATCC50365 as a reference culture
of the genus Allas based on the novel scale type
discovered in strain ATCC50365 (Howe et al. 2011).
We basically agree with this idea; however further
culture-based studies are needed prior to a revision of the taxonomy of thaumatomonads, notably
the Allas-Thaumatomonas complex.
Because the basal branching was poorly
resolved in the present phylogenetic analyses,
we carried out an approximately unbiased (AU)
test to compare seven alternative tree topologies. Although hypothesis D, corresponding to
the present RAxML tree (the best tree) was not
rejected, hypothesis A had the highest p-values.
Hypothesis G (Marimonadida as basal position)
was rejected at the 5% level. Although hypothesis F
(Nudifila + Clautriavia as basal position within Imbricatea) was not rejected at the significance level of
5%, the p-value was relatively low (p-value = 6.9%).
Among hypotheses A, B, C, D, and E, hypothesis
E is the only topology where Thaumatomonadida and Euglyphida are monophyletic; the others
are topologies that represent a paraphyletic relationship in Thaumatomonadida and Euglyphida.
Moreover, the topology of hypothesis B is consistent with recently published trees (Chantangsi et al.
2010; Howe et al. 2011). The previous phylogenetic
analyses as well as the present AU test suggest
that scale-less imbricateans are likely to be positioned between the thaumatomonad and euglyphid
lineages (see below).
Evolution of Scale Formation
As suggested by Wylezich et al. (2007), the process of scale formation is completely different in
euglyphids and thaumatomonads; euglyphid body
scales are formed in vesicles associated with
the Golgi body (Meisterfeld 2000), whereas thaumatomonad body scales are formed in vesicles
associated with mitochondria (Karpov 1990, 1993,
2000; Karpov and Zhukov 1987; Moestrup 1982;
Swale and Belcher 1974, 1975; this study). In addition, the morphology of both groups is very distinct;
the euglyphids are testate amoebae (Lara et al.
2007), whereas the thaumatomonads are scalebearing flagellates (Swale and Belcher 1974, 1975;
Thomsen et al. 1995; Wylezich et al. 2007; this
study). In the present phylogenetic analyses, the
Thaumatomonadida and Euglyphida clades were
separately recovered with high Bayesian statistical
supports (but low to high ML support), suggesting
that they are distinct lineages. Moreover, the AU
test suggested that several scale-less lineages are
placed between these clades. Based on the AU
test, the scale-less genera Spongomonas, Pseudopirsonia (Kühn et al. 2004) and Auranticordis
(Chantangsi et al. 2008) are likely to be positioned
between the thaumatomonad and euglyphid lineages (Table 1; Hypotheses A, B, C, and F). Taking
these facts into account, it is deduced that scale
formation within the Imbricatea has distinct origins
rather than a single origin, and scale formation
might have evolved at least twice among the imbricateans. Our results comply with Patterson’s view
that the formation of scales in association with
570 S. Ota et al.
mitochondria may be a synapomorphy for thaumatomonads (Patterson 1999).
Distribution and Habitat of
Thaumatomonads
Our study from Oslofjorden suggested that thaumatomonads here are restricted to sediments as
benthos, as no thaumatomonad cells or sequences
were detected from water samples of any depths
or size fractions (Ota et al. unpublished data).
This may be consistent with previous reports
by Thomsen et al. (1995) and Thomsen and
Ikävalko (1997), in which Thaumatomastix species
were found from sea ice cores as benthos.
However, some previous studies reported that
Thaumatomastix species occurred in water samples as plankton (Beech and Moestrup 1986;
Birch-Andersen 1973; Takahashi and Hara 1984).
Wylezich et al. (2007) showed that the contribution
of thaumatomonads to the pelagic heterotrophic
protist community was on average only about 4%
in brackish water and 0.1% to 4% in fresh water
samples, whereas maximum abundances of thaumatomonads were recorded in the detritus layer or
sediments. Based on their observations, they indicated that re-suspension of detrital particles could
significantly contribute to the distribution of thaumatomonads.
As mentioned above, the uncultured clone
CCW52 (the closest relative of T. salina) originated
from a marine anoxic environment. In addition, the
uncultured marine eukaryote clones GoC4_B04
(FJ153695) and GoC4_A11 (FJ153693), having
91% and 90% similarity in partial 18S rDNA
sequences with Thaumatomastix sp. (GQ144681),
respectively (determined by BLAST search), originated from suboxic and anoxic waters of the
Gotland Deep (Baltic Sea) (Stock et al. 2009),
suggesting that Thaumatomastix and its relatives
have a wide distribution including suboxic and
anoxic environments. Further studies including
environmental DNA libraries (e.g., cloning and
454-pyrosequencing) would elucidate habitat preferences and community structures of cercozoans
and thaumatomonads (Thaumatomonas, Thaumatomastix, and Allas).
Concluding Remarks
The environmental sequencing approach is widely
used in marine ecology and environmental sciences to explore protist biodiversity, and genetic
data are rapidly accumulating in databases from
environmental DNA clone libraries (e.g., Lepère
et al. 2009; Massana et al. 2011; Sauvadet et al.
2010) and recently also from pyrosequencing (e.g.,
Bråte et al. 2010; Shalchian-Tabrizi et al. 2010).
These are powerful approaches that uncover
the biodiversity of marine environments, but still
many environmental DNA sequences are of uncertain affinities, and it is important that molecular
data is linked to morphological identification. The
present strain UIO286 isolated from Oslofjorden
was identified as Thaumatomastix salina based on
comprehensive morphological studies. The phylogenetic analyses demonstrated that T. salina strain
UIO286 is sister to one environmental sequence
that is as-yet uncultured. Thus, we propose UIO286
as a reference culture of T. salina for further environmental and phylogenetic studies. Isolating and
culturing efforts are still required to understand protist diversity in marine environments.
Methods
Sampling and culture: T. salina, strain UIO286 (isolator’s reference number: OF258) originated from a sediment sample
collected by B. Edvardsen 23 June 2010 during a BioMarKs
cruise with R/V Trygve Braarud in the outer Oslofjorden at a
station (59.254604N, 10.711379E, 103 m depth) north east of
Rauøy in Østfold County, Southern Norway. The sediment was
collected using a Gemini corer, from which the upper 2 cm was
transferred to a flask added bottom seawater (34 PSU, 6 ◦ C)
and kept cold (∼6 ◦ C) and dark until isolation.
T. salina was isolated by S. Ota using the micropipette
method (Andersen and Kawachi 2005), and the culture was
maintained at 17 ◦ C in sterile filtered seawater (30 PSU salinity)
added yeast extract (final conc. 25 mg/L; Becton, Dickinson and
Company) and pepton (final conc. 50 mg/L; Becton, Dickinson
and Company) or half-strength ES medium (Kasai et al. 2009)
added yeast extract (final conc. 25 mg/L) and pepton (final conc.
50 mg/L).
Light microscopy (LM): Living cells were observed under
a Zeiss Axio Scope A1 microscope (Carl Zeiss, Oberkochen,
Germany) equipped with Nomarski differential interference contrast and phase contract optics. Light micrographs were taken
with a Nikon D5000 digital camera (Nikon, Tokyo, Japan).
Transmission electron microscopy (TEM): For whole
mounts preparation, droplets of cells fixed with 5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA; final
conc.) were placed on grids covered by a formvar film. After
1-2 h, the droplets were removed and the grids were washed
twice in distilled water. Then the cells were stained in saturated
aqueous uranyl acetate (Merck, Darmstadt, Germany) for about
90 s, and washed with distilled water. For thin section, 50% glutaraldehyde was added directly into the culture and fixed 1 h at
room temperature (glutaraldehyde final conc. 2.5%). The cells
were washed four times (5 min each) with 0.1 M sodium cacodylate buffer (pH 7.8) (Agar Scientific Ltd., Essex, UK). After
centrifugation and removal of the supernatant, the pellet was
post-fixed in 1% (final conc.) osmium tetroxide overnight at 4 ◦ C.
The cells were rinsed three times with the same buffer, dehydrated through a graded ethanol series (30%, 50%, 70%, 90%,
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 571
and 100% × 4; 15 min each) on ice, and transferred to propylene oxide twice (15 min each). Samples were left overnight in
a 1:1 mixture of propyleneoxide and Epon’s resin (EM Bed-812
based on EPON-812, Sigma), followed by incubation in 100%
Epon’s resin overnight at room temperature, and polymerized at
60 ◦ C overnight (12 h or more). Ultrathin sections were cut on
a Reichert Ultracut S ultramicrotome (Leica, Vienna, Austria)
using a diamond knife. The sections were mounted on copper
grids coated with polyvinyl formvar films, and stained in saturated aqueous uranyl acetate (30 min) and lead citrate (3 min)
(Reynolds 1963). Sections and whole mount preparations were
observed using a FEI/PHILIPSCM-100 TEM (Hillsboro, Oregon, USA) at 80 kV at the Electron Microscopy Unit, Dept. of
Molecular Sciences, University of Oslo.
DNA extraction, polymerase chain reaction (PCR), and
sequencing: Cells were collected from a two-weeks-old raw
culture growing in a 50 ml culture flasks (Nunc, Roskilde, Denmark) containing 10-20 ml medium and genomic DNA was
extracted using the E.Z.N.A.TM SP Plant DNA kit (OmegaBio-Tek, Norcross, GA, USA) according to the manufacturer’s
protocol. The SSU (18S) nuclear ribosomal encoding region
was amplified using primers 1F and 1528R described in
Edvardsen et al. (2003), but with the 5 PRIME Taq DNA
polymerase (5 PRIME, Hamburg, Germany). The PCR condition was as follows: initial denaturation at 94 ◦ C for 3 min,
34 cycles (denaturation at 94 ◦ C for 30 s, annealing at 54 ◦ C
for 30 s, extension at 68 ◦ C for 1 min), and final extension at
68 ◦ C for 10 min. PCR products were run on a 0.8% agarose
gel and checked for purity and correct fragment length. The
PCR products were purified using ExoSAP-IT® (USB Corp.,
Cleveland, OH, USA) and bidirectionally sequenced using an
Applied Biosystems 3730 analyzer (Applied Biosystems, CA,
USA) sequencing device at the Department of Biology, University of Oslo.
Phylogenetic analyses: Sequences of the 18S rDNA were
aligned using ClustalX v. 2.0 (Larkin et al. 2007) and manually
edited using BioEdit v. 7.0.5.3 (Hall 1999). Maximum likelihood
(ML) analysis was carried out using RAxML v. 7.0.3 (Stamatakis
2006). The general time reversible model with parameters
accounting for invariable sites (I) and gamma-distributed (G)
rate variation across sites with four discrete rate categories
was used for the 18S rDNA dataset. The bootstrap analyses
were done in 100 replicates for the ML analysis. Bayesian
inference under the same evolutional model was performed
with MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). Two
Markov Chain Monte Carlo (MCMC) runs each with four chains
were performed for 1,000,000 generations, where the average standard deviation of split frequencies was 0.009024.
Trees were sampled every 100 generations. Bayesian posterior
probabilities were calculated from the majority rule consensus of the tree sampled after the initial burn-in phase. In
the present Bayesian and ML analyses, taxon sampling was
mainly based on the previous trees of Lara et al. (2007)
and Wylezich et al. (2007) in a Monadofilosan context, and
cercomonad sequences were used as outgroup. Some environmental (uncultured) sequences were also included in the
analyses. The Bayesian posterior probabilities were annotated
onto the ML tree. Bayesian phylogenetic analyses were carried
out on the University of Oslo Bioportal (www.bioportal.uio.no).
The SSU rDNA alignment is available from TreeBASE
(http://purl.org/phylo/treebase/phylows/study/TB2:S11905).
For examination of alternative trees, site-wise log-likelihoods
of seven alternative trees were calculated using TREE-PUZZLE
5.2 (Schmidt et al. 2002) with the same evolutionary model,
and approximately unbiased (AU) tests were performed using
CONSEL v.0.1i (Shimodaira and Hasegawa 2001).
Acknowledgements
We are most grateful to Tove Bakar at the Electron Microscopy Unit, University of Oslo, for her kind
help with the transmission electron microscope.
We would like to thank Simon Dittami and three
anonymous reviewers for critical reading of the
manuscript. We also thank Elianne Sirnæs Egge
and Sissel Brubak for their kind supports and technical assistance. This work was supported by the
EU BiodivERsA project BioMarKs (2008-2976562739-34).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
doi:10.1016/j.protis.2011.10.007.
References
Andersen RA, Kawachi M (2005) Traditional Microalgae
Isolation Techniques. In Andersen RA (ed) Algal Culturing Techniques. Elsevier, Academic Press, Amsterdam, pp 83–100
Balonov IM (1980) On a new species of the genus
Chrysosphaerella (Chrysophyta). Bot Zh 65:1190–1191
Beech PL, Moestrup Ø (1986) Light and electron microscopical observations on the heterotrophic protist Thaumatomastix
salina comb. nov. (syn. Chrysosphaerella salina) and its allies.
Nord J Bot 6:865–877
Birch-Andersen P (1973) Chrysosphaerella salina. A new
species of the Chrysophyceae from salt marsh pools. Bot
Tidsskr 68:140–144
Bråte J, Logares R, Berney C, Ree DK, Klaveness D, Jakobsen KS, Shalchian-Tabrizi K (2010) Freshwater Perkinsea and
marine-freshwater colonizations revealed by pyrosequencing
and phylogeny of environmental rDNA. ISME J 4:1144–1153
Cavalier-Smith T, Chao EE (2003) Phylogeny and classification of phylum Cercozoa (Protozoa). Protist 154:341–358
Cavalier-Smith T, Lewis R, Chao EE, Oates B, Bass D (2008)
Morphology and phylogeny of Sainouron acronematica sp. n.
and the ultrastructural unity of Cercozoa. Protist 159:591–620
Chantangsi C, Esson HJ, Leander BS (2008) Morphology and
molecular phylogeny of a marine interstitial tetraflagellate with
putative endosymbionts: Auranticordis quadriverberis n. gen. et
sp. (Cercozoa). BMC Microbiol 8:e123
Chantangsi C, Hoppenrath M, Leander BS (2010) Evolutionary relationships among marine cercozoans as inferred from
combined SSU and LSU rDNA sequences and polyubiquitin
insertions. Mol Phylogenet Evol 57:518–527
Conrad W (1920) Sur un flagella nouveau à trichocystes, Reckertia sagittifera, n. g., n. sp. Bull Acad Roy Sci Belg 6:541–555
572 S. Ota et al.
De Saedeleer H (1931) Notes de Protistologie IV. Thaumatomonas lauterbornii, n.g., n. sp., flagella nouveau. Recueil
L’Instit Zool Torley Rousseau 3:89–103
Lepère C, Vaulot D, Scanlan DJ (2009) Photosynthetic
picoeukaryote community structure in the South East Pacific
Ocean encompassing the most oligotrophic waters on Earth.
Environ Microbiol 11:3105–3117
Edvardsen B, Shalchian-Tabrizi K, Jakobsen KS, Medlin
LK, Dahl E, Brubak S, Paasche E (2003) Genetic variability
and molecular phylogeny of Dinophysis species (Dinophyceae)
from Norwegian waters inferred from single cell analyses of
rDNA. J Phycol 39:395–408
Lesaulnier C, Papamichail D, McCorkle S, Ollivier B, Skiena
S, Taghavi S, Zak D, van der Lelie D (2008) Elevated atmospheric CO2 affects soil microbial diversity associated with
trembling aspen. Environ Microbiol 10:926–941
Ekelund F, Daugbjerg N, Fredslunda L (2004) Phylogeny of
Heteromita, Cercomonas and Thaumatomonas based on SSU
rDNA sequences, including the description of Neocercomonas
jutlandica sp. nov., gen. nov. Eur J Protistol 40:119–135
Massana R, Pernice M, Bunge JA, del Campo J (2011)
Sequence diversity and novelty of natural assemblages
of picoeukaryotes from the Indian Ocean. ISME J 5:
184–195
Hall TA (1999) BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/98/NT.
Nucleic Acids Symp Ser 41:95–98
Meisterfeld R (2000) Testate Amoebae with Filopodia. In Lee
JJ, Leedale GF, Bradbury P (eds) An Illustrated Guide to the
Protozoa. Society of Protozoologists, Lawrence, KS, USA, pp
1054–1084
Howe AT, Bass D, Scoble JM, Lewis R, Vickerman K,
Arndt H, Cavalier-Smith T (2011) Novel cultured protists identify deep-branching environmental DNA clades of Cercozoa:
new genera Tremula, Micrometopion, Minimassisteria, Nudifila,
Peregrinia. Protist 162:332–372
Mikrjukov R (2002) Thaumatomastix tauryanini sp. n. (Protista, Sarcomonadea, Thaumatomonadida) is a new species of
phagotrophic amoebo-flagellate from the White Sea sediments.
Zool Zhur 81:261–265
Ikävalko J (1998) Further observations on flagellates within
sea ice in northern Bothnian Bay, the Baltic Sea. Polar Biol
19:323–329
Moestrup Ø (1979) Identification by electron microscopy of
marine nanoplankton from New Zealand, including the description of four new species. N Z J Bot 17:61–95
Karpov SA (1990) Analysis of the orders Phalansteriida, Spongomonadida и Thaumatomonadida. Zool Zhurn 69:5–12 (in
Russian)
Moestrup Ø (1982) Flagellar structure in algae: a review,
with new observations particularly on the Chrysophyceae,
Phaeophyceae (Fucophyceae), Euglenophyceae and Reckertia. Phycologia 21:427–528
Karpov SA (1993) The ultrathin structure of colourless flagellate Thaumatomonas seravini. Tsitologia 35:8–11 (in Russian)
Karpov SA (2000) Flagellate Phylogeny: Ultrastructural
Approach. In Leadbeater BSC, Green JC (eds) The Flagellates.
Systematics Association Spec. Publ. Taylor & Francis, London,
pp 336–360
Karpov SA (2011) Order Thaumatomonadida. In Protista III.
Handbook on Zoology. KMK Scientific Press. Saint Petersburg,
Moscow (in Russian) in press
Karpov SA, Zhukov BF (1987) Cytological peculiarities of
colourless flagellate Thaumatomonas lauterborni. Tsitologia
29:1168–1171 (in Russian)
Kasai F, Kawachi M, Erata M, Mori F, Yumoto K, Sato M,
Ishimoto M (2009) NIES-collection list of strains, 8th edition.
Jap J Phycol (Sôrui) 57(Suppl.):1–350
Kühn S, Medlin L, Eller G (2004) Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from
nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa
(Drebes) comb. nov. Protist 155:143–156
Lara E, Heger TJ, Mitchell EA, Meisterfeld R, Ekelund
F (2007) SSU rRNA reveals a sequential increase in shell
complexity among the euglyphid testate amoebae (Rhizaria:
Euglyphida). Protist 158:229–237
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez
R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W
and Clustal X version 2.0. Bioinformatics 23:2947–2948
Lauterborn R (1899) Protozoen-Studien IV. Flagellaten aus
dem Gebiete des Oberrheins. Zeitschr wiss Zool 65:369–391
Moestrup Ø, Sengco M (2001) Ultrastructural studies on
Bigelowiella natans, gen. et sp. nov., a chlorarachniophyte flagellate. J Phycol 37:624–646
Patterson DJ (1999) The Diversity of Eukaryotes. Am Nat
154:96–124
Patterson DJ, Zölffel M (1991) Heterotrophic Flagellates of
Uncertain Taxonomic Position. In Patterson DJ, Larsen J (eds)
The Biology of Free-Living Heterotrophic Flagellates. Clarendon Press, Oxford, pp 427–476
Reynolds ES (1963) The use of lead citrate at high pH as
an electron opaque stain in electron microscopy. J Cell Biol
17:208–212
Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics
19:1572–1574
Sandon H (1927) The Composition and Distribution of the Protozoan Fauna of the Soil. Oliver and Boyd, Edinburgh
Sauvadet A-L, Gobet A, Guillou L (2010) Comparative analysis between protist communities from the deep-sea pelagic
ecosystem and specific deep hydrothermal habitats. Environ
Microbiol 12:2946–2964
Schmidt HA, Strimmer K, Vingron M, von Haeseler A
(2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics
18:502–504
Shalchian-Tabrizi K, Reier-Røberg K, Ree DK, Klaveness
D, Bråte J (2010) Marine-freshwater colonizations of haptophytes inferred from phylogeny of environmental 18S rDNA
Sequences. J Eukaryot Microbiol 4:1144–1153
Ultrastructure and Molecular Phylogeny of Thaumatomastix salina 573
Shimodaira H, Hasegawa M (2001) CONSEL: for assessing
the confidence of phylogenetic tree selection. Bioinformatics
17:1246–1247
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed
models. Bioinformatics 22:2688–2690
Stoeck T, Epstein S (2003) Novel eukaryotic lineages inferred
from small-subunit rRNA analyses of oxygen-depleted marine
environments. Appl Environ Microbiol 69:2657–2663
Stock A, Jürgens K, Bunge J, Stoeck T (2009) Protistan diversity in suboxic and anoxic waters of the Gotland Deep (Baltic
Sea) as revealed by 18S rRNA clone libraries. Aquat Microb
Ecol 55:267–284
Swale EMF, Belcher JH (1974) Gyromitus disomatus Skuja
– a free living colourless flagellate. Arch Protistenkd 116:
211–220
Swale EMF, Belcher JH (1975) Gyromitus limax nov. sp.
– free living colourless amoebo-flagellate. Arch Protistenkd
117:20–26
Takahashi E, Hara S (1984) Two new marine species of
Chrysosphaerella (Chrysophyceae) with a reinvestigation of C.
salina. Phycologia 23:103–109
Thomsen HA (1979) Electron microscopical observations on
brackish-water nannoplankton from the Tvärminne area, SW
coast of Finland. Acta Bot Fenn 110:11–37
Thomsen HA, Ikävalko J (1997) Species of Thaumatomastix
(Thaumatomastigidae, Protista incertae sedis) from the Arctic
sea ice biota (North-East Water Polynya, NE Greenland). J Mar
Sys 10:263–277
Thomsen HA, Kosman C, Ikävalko J (1995) Three new
species of Thaumatomastix (Thaumatomastigidae, Protista
incertae sedis), a ubiquitous genus from the Antarctic ice biota.
Eur J Protistol 31:174–181
Vørs N (1992a) Heterotrofe protister (ekskl. dinoflagellater,
loricabærende choanoflagellater og ciliater). In Thomsen HA
(ed) Plankton i de indre danske farvande. Havforskning fra
Miljøstyrelsen Nr. 11, Copenhagen, pp 195–250
Vørs N (1992b) Heterotrophic amoebae, flagellates and heliozoa from the Tvärminne area Gulf of Finland, in 1988-1990.
Ophelia 36:1–109
Vørs N (1993) Heterotrophic amoebae, flagellates and heliozoa
from Arctic marine waters (North West Territories, Canada and
West Greenland). Polar Biol 13:113–126
Wylezich C, Mylnikov AP, Weitere M, Arndt H (2007)
Distribution and phylogenetic relationships of freshwater
thaumatomonads with a description of the new species Thaumatomonas coloniensis n. sp. J Eukaryot Microbiol 54:347–357
Zolotarev VA, Myl’nikova ZM, Myl’nikov AP (2011) The
ultrafine structure of amoeboid flagellate Thaumatomastix sp.
(Thaumatomonadida (Shirkina) Karpov, 1990). Inland Water
Biol 4:287–292
Available online at www.sciencedirect.com