The morphology and phylogeny of two euplotid ciliates, Diophrys

Transcription

International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2757–2773
DOI 10.1099/ijs.0.039503-0
The morphology and phylogeny of two euplotid
ciliates, Diophrys blakeneyensis spec. nov. and
Diophrys oligothrix Borror, 1965 (Protozoa,
Ciliophora, Euplotida)
Xiaozhong Hu,1,2 Jie Huang1 and Alan Warren2
Correspondence
Xiaozhong Hu
1
xiaozhonghu@ouc.edu.cn
2
Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of
China, Qingdao 266003, PR China
Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK
The morphology, infraciliature and molecular phylogeny of two marine ciliated protozoans,
Diophrys blakeneyensis spec. nov. and Diophrys oligothrix Borror, 1965, isolated from British salt
marshes, were investigated using microscopic observations of live and protargol-impregnated
specimens, and by small subunit (SSU) rRNA gene sequence analysis. Diophrys blakeneyensis
spec. nov. is characterized as follows: cell oval to rectangular in outline; size variable,
approximately 60–180 ¾ 30–80 mm in vivo; adoral zone comprising about 45 membranelles;
usually five frontal, two ventral, five transverse, two left marginal and three caudal cirri; five dorsal
kineties with more than 10 dikinetids each; 7–23 spherical to ellipsoid macronuclear nodules in a
ring-like pattern; marine biotope. The population of Diophrys oligothrix described here
corresponds well with previous populations in terms of its general morphology and ciliary pattern,
in particular the continuous ciliary rows on the dorsal side with loosely arranged cilia. The main
differences between the present and previously reported populations are the broader buccal field
and greater number of dorsal kineties in the present population, both of which are regarded as
population-dependent features. Phylogenetic analyses based on SSU rRNA gene sequence data
demonstrate that Diophrys blakeneyensis is most closely related to Diophrys oligothrix, and both
organisms cluster with two congeners with high bootstrap support within a larger group that
contain the core species of the Diophrys-complex. Cladistic analysis based on morphological and
morphogenetic data broadly agree with the SSU rRNA gene sequence phylogeny. Both analyses
suggest that the genus Diophrys may be polyphyletic.
INTRODUCTION
Diophrys-like spirotrich ciliates are commonly found worldwide in marine and estuarine biotopes, especially sandy
beaches and salt marshes (Agamaliev, 1971; Alekperov, 1984;
Alekperov & Asadullayeva, 1999; Aliyev, 1990; Borror, 1963,
1965a, 1965b, 1972; Budenbrock, 1920; Carey, 1992; Curds
& Wu, 1983; Czapik, 1981; Dragesco, 1963; Dragesco &
Dragesco-Kernéis, 1986; Fauré-Fremiet, 1964; FernandezLeborans & Novillo, 1994; Ganapati & Rao, 1958; Hartwig,
1973, 1974; Hill, 1981; Hu, 2008; Kahl 1932; Kattar 1970;
Mansfeld 1923; Petz et al., 1995; Raikov & Kovaleva, 1968;
Ruinen, 1938; Shen et al., 2011; Song & Packroff, 1993, 1997;
Song & Wilbert, 1994, 2002; Song et al., 2007, 2009a, 2009b).
Abbreviations: BI, Bayesian inference; SSU rRNA, small subunit rRNA.
The GenBank/EMBL/DDBJ accession numbers for the SSU rRNA
gene sequences of Diophrys blakeneyensis and Diophrys oligothrix are
JN172996 and JN172995, respectively.
039503 G 2012 IUMS
Members of the genus Diophrys are characterized by having
a prominent right posterior-lateral concavity where the
sickle-shaped caudal cirri are located. In the past 30 years
there have been two taxonomic revisions of the genus
Diophrys (Curds & Wu, 1983; Song et al., 2007). Until 2010,
eleven nominal species of Diophrys were recognized, six of
which show highly divergent patterns of infraciliature,
especially with respect to the numbers and distribution of
frontoventral and marginal cirri. Jankowski (1979) erected a
separate genus, Paradiophrys, for these divergent species.
Subsequently Hill & Borror (1992) erected a third genus,
Diophryopsis, and divided the Diophrys-like species among
these three genera. This classification was accepted by
Jankowski (2007) who erected the subfamily Diophryinae to
include these three genera, a decision that was supported by
Shao et al. (2010). Recently, three more Diophrys-like genera
have been established, namely Apodiophrys, Heterodiophrys
and Pseudodiophrys (Jiang & Song, 2010; Jiang et al., 2011).
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X. Hu, J. Huang and A. Warren
These studies, however, did not support the monophyly of
the subfamily Diophryinae. Furthermore, the validity of the
genera Apodiophrys and Paradiophrys, which are mainly
based on morphological characters, was challenged by the
molecular data (Jiang et al., 2011). These conflicts between
the morphological and molecular data cannot be resolved
for a variety of reasons including the well-known limitations
of single gene genealogies and a lack of data for a sufficient
number of species from a sufficient range of geographical
locations.
During a faunistic survey of ciliates in salt marshes along the
eastern and southern coasts of the UK, several species of
Diophrys were isolated and studied using light microscopy.
In addition the small-subunit rRNA (SSU rRNA) gene of
some isolates was successfully sequenced. In this paper we
document the morphology and SSU rRNA gene sequences
of two Diophrys species, Diophrys blakeneyensis spec. nov.,
and Diophrys oligothrix. The molecular phylogeny of each
was analysed based on SSU rRNA gene sequence data.
Evolutionary relationships among the Diophrys-like ciliates
are discussed in the light of morphological, morphogenetic
and molecular data.
METHODS
Morphological studies. Populations of D. oligothrix Borror, 1965
and D. blakeneyensis spec. nov. were collected on 4 August 2010 from
the intertidal region of a salt marsh near Blakeney village, Norfolk,
UK (1u 19 E; 52u 579 N). This region of the salt marsh was dominated
by the glasswort, Salicornia europaea. A second population of D.
blakeneyensis spec. nov. was collected on 30 September 2010 from the
intertidal region of a salt marsh at West Wittering, near Chichester
Harbour, Hampshire, UK (0u 549 W; 50u 469 N) where sea purslane,
Atriplex portulacoides, was dominant. Samples were collected at low
tide and comprised small volumes of wet sediment covered with algal
mat. Seawater from adjacent marsh ditches was also collected from
which environmental data, i.e. water temperature, pH and salinity
were recorded. The seawater samples were also used for the
maintenance of ciliates in the laboratory.
In the laboratory, subsamples were diluted using seawater collected in
situ. Specimens were isolated with a micropipette and examined in
vivo using bright-field and Nomarski differential interference contrast
microscopy (Xu et al., 2011). The protargol impregnation method of
Wilbert (1975) was used in order to reveal the infraciliature. Counts
and measurements of stained specimens were performed at a
magnification of 61250. Drawings were made with the help of a
camera lucida. Terminology and systematics mainly follow Jankowski
(2007) and Lynn (2008).
DNA extraction and PCR amplification. Specimens were individu-
ally isolated and washed three times in autoclaved filtered seawater.
Two to four cells of each of the two species collected at Blakeney were
applied to Indicating FTA Elute Cards (WB120411; Whatman) and
allowed to dry for at least 3 h at room temperature prior to
processing or storage. Elution of DNA from Indicating FTA Elute
Cards was carried out according to the manufacturer’s instructions.
Primer 82F (59-GAAACTGCGAATGGCTC-39) and the universal
eukaryotic primer Euk B (Medlin et al., 1988) were used to amplify
the SSU rRNA gene using the cycling parameters: 5 min at 94 uC; 35
cycles of 30 s at 94 uC, 1 min at 60 uC and 2 min at 72 uC; and one
cycle of 7 min at 72 uC. PCR products corresponding to the expected
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size were separated by agarose gel electrophoresis. Cleaned DNA was
cloned into the pMD18-T vector (Takara Biotechnology) and transformed into a competent Escherichia coli strain. Sequencing in both
directions was carried out on an ABI 3730 automatic sequencer
(Applied Biosystems).
Phylogenetic analyses. In order to analyse the molecular phylogeny
of D. blakeneyensis spec. nov. and D. oligothrix, their SSU rRNA gene
sequences were aligned with SSU rRNA gene sequences of 40 spirotrich
ciliates obtained from GenBank (for accession numbers see Fig. 7).
Alignments were constructed, using MUSCLE v3.7 with default parameters and were manually refined in BioEdit 7.0.5.2 (Hall, 1999).
Preliminary maximum-likelihood (ML) analyses performed online on
the CIPRES Portal v.2.0 (URL: http://www.phylo.org/sub_sections/
portal) with RAxML-HPC BlackBox 7.2.7 (Stamatakis, 2006;
Stamatakis et al., 2008) showed that different selections of representative taxa (e.g. 42, 49, 58, 66 and 67 species) resulted in trees with
similar topologies. Based on these preliminary analyses, we selected a
set of 42 SSU rRNA gene sequences for further phylogenetic analyses
with Protocruzia adherens and Protocruzia contrax as the outgroup
species. The resulting alignment included 1737 characters and is
available from the authors upon request. Maximum-likelihood bootstrapping analyses were carried out with 100 replicates using RAxML
with the setting as described in Stamatakis (2006) and Stamatakis et al.
(2008). Bayesian inference (BI) was performed with MrBayes v3.1.2
(Ronquist & Huelsenbeck, 2003) using the GTR+I+G model as
selected by AIC in MrModeltest v2.0 (Nylander, 2004). Four
simultaneous chains were run for 1 000 000 generations sampling
every 100 generations. The first 25 % of sampled trees were discarded as
burn-in prior to constructing a 50 % majority rule consensus tree.
Cladistic analysis. A cladistic analysis of 17 species, representing
seven genera of uronychiids, was carried out based on morphological
and morphogenetic data, both from the present investigation and from
previously published studies. These include species of the six Diophryslike genera plus members of the genus Uronychia. The phylogenetic
relationships within the family Uronychiidae were elucidated by
applying Hennig’s argumentation method (Hennig 1982; for details see
Agatha, 2004). For discussions on the justification for determining the
apomorph and plesiomorph, see Agatha (2004); Berger (2008); Borror
& Hill (1995); Miao et al. (2007), and Shao et al. (2008).
RESULTS
Diophrys blakeneyensis spec. nov. (Figs 1, 2 and
3; Table 1)
Diagnosis. Medium- to large-sized Diophrys, approximately
60–180650–150 mm in vivo; body elliptical in outline and
slightly greyish to yellowish; pellicle flexible, with underlying
granules closely spaced and arranged in lines or around
membranelles, cirri and dorsal cilia; approximately 45 adoral
membranelles; six to nine frontoventral, two or three marginal, five transverse and three caudal cirri; five dorsal kineties;
macronuclear apparatus comprising 7–23 spherical to ellipsoidal nodules generally arranged in a ring; marine habitat.
Type locality. Intertidal region of a salt marsh near Blakeney
village, Norfolk, UK (1u 19 E; 52u 579 N). Seawater collected
from marsh ditches nearby was 18 uC, pH ca. 8.0 and salinity
29 %.
Type specimens. A protargol slide with the holotype specimen
is deposited in the Natural History Museum, London, UK,
International Journal of Systematic and Evolutionary Microbiology 62
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Two euplotid ciliates from salt marshes
Fig. 1. Morphology of Diophrys blakeneyensis spec. nov. from life (a–c) and after protargol impregnation (d–i). (a) Ventral view
of a typical individual. (b, c) Note the arrangement of cortical granules around the transverse cirri (b) and around the dorsal
bristles and among the dorsal kineties (c). (d) Ventral view of a specimen just after division, showing infraciliature and nuclear
apparatus (outlined); arrowhead indicates caudal cirri, double-arrowhead marks membranelle at distal end of adoral zone. (e)
Ventral view of a cell with 6 frontal cirri; arrows show micronuclei. (f) Ventral view of an individual with 4 frontal cirri and 3
marginal cirri (arrowheads); arrows show micronuclei, double-arrowhead marks membranelles at distal end of adoral zone. (g)
Ventral view of a cell with reduced ventral cirrus (arrow); arrowheads show micronuclei, double-arrowheads mark membranelles
on dorsal side. (h, i) Ventral and dorsal views of the same specimen, showing infraciliature and nuclear apparatus; arrow
indicates micronucleus. AZM, adoral zone of membranelles; CC, caudal cirri; DK1–5, dorsal kinety 1–5; EM, endoral
membrane; FC, frontal cirri; Ma, macronucleus; PM, paroral membrane; TC, transverse cirri, VC, ventral cirri. Bars, 40 mm.
with registration number NHMUK 2011.10.28.1. Two paratype slides are deposited in the Laboratory of Protozoology,
Ocean University of China, Qingdao, China with registration
numbers Bla20108452 and Chi20109301.
Etymology. Named after the village where the species was
first discovered.
Description. Cell size is highly variable, approximately 60–
180650–150 mm in vivo; body shape oval to rectangular
http://ijs.sgmjournals.org
(Figs 1a, 2a–e, h, i). Anterior end with a conspicuous, thin
collar (Figs 1a and 2e) accompanied by ridges; cilia of
apical membranelles emerge near the ridges. Posterior end
bluntly pointed with three strong caudal cirri emerging
from concave area on right dorsal side (Figs 1a, 2a, e, 2h
arrow, 2i double-arrowhead). Dorsoventrally flattened with
ventral side flat and dorsal side bulging evenly when viewed
from lateral aspect. In ventral view, two longitudinally
oriented ribs are visible, right rib starting near distal end of
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Fig. 2. Photomicrographs of Diophrys blakeneyensis spec. nov. from life. (a) Ventral view, showing two ribs, right rib starting
from distal end of adoral zone, left rib starting near posterior portion of buccal field (arrows). (b) Dorsal view, showing
membranelles at distal end (arrows) and caudal cirri (arrowhead). (c, d, f) Ventral view of different specimens to show different
body shapes and the arrangement of cirri; arrows in (c, d) and arrowheads in (f) mark marginal cirri; arrowheads in (c) show
membranelles at distal end of adoral zone, arrows in (f) indicate frontal cirri. (e) Ventral view, showing dorsal cilia (arrows) and
undulating membranes. (g) Ventral view of portion of a contracted cell, showing cortical granules around cirri and
membranelles (arrows). (h) Dorsal view, showing membranelles at the apical end of adoral zone (arrowheads) and sickleshaped caudal cirri derived from right posterior-lateral concavity (arrow). (i) Dorsal view, showing membranelles at distal end
(arrow) and right posterior-lateral concavity (double-arrowhead). (j) Ventral view of portion of cell, showing macronuclear
nodules (arrows). (k) Ventral view of a plumper body; arrows show dorsal cilia, arrowheads indicate marginal cirri, doublearrowhead marks ingested diatom. (l) Dorsal view of portion of cell to show densely aligned cortical granules. (m) Cortical
granules (arrowheads) around dorsal bristles (arrows) making rosettes. UM, undulating membranes. Other abbreviations as in
Fig. 1. Bars, 50 mm.
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Fig. 3. Photomicrographs of Diophrys blakeneyensis spec. nov. after protargol impregnation. (a) Ventral view, showing the
general ciliature and nuclear apparatus; arrow marks membranelles at distal end of adoral zone. (b) Ventral view of an individual
with only one ventral cirrus (arrow). (c, f) Ventral view of specimens with seven and six frontal cirri, respectively. (d) Ventral view,
showing fibres from the two rightmost transverse cirri (arrowhead) and two marginal cirri (arrows). (e) Left lateral view, noting
two marginal cirri (arrows) and the leftmost dorsal kinety shortened posteriorly. (g, j, k) Dorsal (g) and ventral (j, k) views of the
same specimen, showing infraciliature and dorsal kineties (arrows). (h, i) Ventral view of different cells, showing shape variance
of micronuclei (arrows). (l) Ventral view of portion of cell, showing paroral and endoral membranes. (m) Details of macronuclear
nodules and micronuclei (arrows). (n) Ventral view of proximal portion of cell, showing ventral, transverse and marginal cirri.
(o) Ventral view of proximal portion of cell with three marginal cirri. (p–r) Ventral (p, q) and dorsal (r) views of the same specimen,
showing infraciliature and nuclear apparatus; arrows in (p) and (r) indicate dorsal kineties while in (q) marks marginal cirri;
arrowhead in (q) shows reduced ventral cirrus. (s) Ventral view, arrows show spherical micronuclei. MC, marginal cirri. Other
abbreviations as in Fig. 1. Bars, 50 mm.
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Table 1. Morphometric characterization of Diophrys blakeneyensis spec. nov. (upper row: Blakeney population; middle row: West
Wittering population) and Diophrys oligothrix (lower row)
Data based on protargol-impregnated specimens.
specimens examined.
Characteristic
Body length (mm)
Body width (mm)
Length of adoral zone of membranelles (mm)
Number of adoral membranelles
Number of frontoventral cirri
Number of transverse cirri
Number of left marginal cirri
Number of caudal cirri
Number of dorsal kineties
Number of macronuclear nodules
Number of micronuclei
Number of basal body pairs in DK1
CV,
Coefficient of variation; DK, dorsal kinety; Max, maximum; Min, minimum; n, number of
Min
Max
Mean
SD
SE
CV
n
62
78
60
36
45
41
40
46
38
40
38
10
6
7
7
5
5
5
2
2
2
3
3
3
5
5
5
7
14
2
1
2
2
9
10
6
13
14
6
11
11
6
10
9
7
8
6
6
172
154
106
144
112
68
96
92
66
49
51
39
9
7
7
5
5
5
3
3
2
3
3
3
5
5
5
19
23
3
4
6
4
15
18
10
18
22
10
19
19
11
14
16
10
11
12
10
113.5
117.6
85.6
76.8
75.5
54.2
67.7
72.3
53.6
44.9
44.3
34.7
7.1
7
7
5
5
5
2.0
2.0
2
3
3
3
5
5
5
12.1
16.9
2.1
2.5
4.3
3.0
12.3
14.0
7.9
16.3
17.2
7.9
13.8
14.8
8.4
11.7
12.7
8.4
9.6
9.9
7.3
26.97
20.47
13.79
21.94
15.75
8.50
13.03
11.44
9.00
2.47
3.22
2.39
0.49
0
0
0
0
0
0.20
0.20
0
0
0
0
0
0
0
3.32
2.60
0.29
0.89
0.96
0.63
1.49
1.84
1.02
1.31
2.39
1.23
1.58
2.41
1.46
1.21
1.96
1.04
0.91
1.64
1.16
5.39
4.17
2.94
4.48
3.21
1.90
2.61
2.33
1.92
0.50
0.66
0.51
0.10
0
0
0
0
0
0.04
0.04
0
0
0
0
0
0
0
0.66
0.53
0.06
0.20
0.20
0.19
0.34
0.39
0.24
0.32
0.52
0.29
0.37
0.50
0.35
0.29
0.41
0.24
0.24
0.36
0.28
23.8
17.4
16.1
28.6
20.9
15.7
19.2
15.8
16.8
5.5
7.3
6.9
6.9
0
0
0
0
0
10.0
10.0
0
0
0
0
0
0
0
27.4
15.4
13.8
35.6
22.3
21.0
12.1
13.1
12.9
8.0
13.9
15.6
11.4
16.3
17.4
10.3
15.4
12.4
9.5
16.6
15.9
25
24
22
24
24
20
25
24
22
24
24
22
25
24
22
25
24
22
25
24
22
25
24
22
25
24
21
25
24
22
20
23
11
19
22
18
17
21
18
18
23
18
17
23
18
15
21
17
adoral zone, left rib starting near posterior portion of
buccal field (Figs 1a, 2a, arrows). Wide depression in
central region of cell between ribs. Five strong transverse
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cirri located in posterior half of cell within wide depression;
several short ridges between transverse cirri (Figs 1a and
2c).
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Numerous tiny yellow–greenish cortical granules, about
0.5 mm across, beneath pellicle, often grouped around bases
of membranelles, cirri and dorsal bristles (Figs 1b, c, 2g
arrows, 2m arrowheads) or arranged in lines both on ventral
and dorsal sides (Figs 1c and 2l) rendering the cell yellowish
at low magnification. Beneath this layer of cortical granules
are larger granules (possibly mitochondria), about 1.5 mm in
size, densely arranged all over the body both on ventral and
dorsal sides. Cytoplasm colourless or yellowish and transparent, although endoplasm contains numerous granules
(1–8 mm in diameter) and several food vacuoles that
typically contain diatoms. Contractile vacuole not recognized. 7–23 macronuclear nodules (Ma), globular to
ellipsoid, 10–20 mm long, generally arranged in a ring
(Figs 1d–g, i, 2j arrows; 3a–c, h, i, p–s); one to six spherical
micronuclei closely associated with macronuclear nodules
(Figs 1e, f, i, arrows, 1g arrowheads, 3h, i, m, s arrows).
Locomotion by rapid crawling on substrate or by swimming.
Cilia of anterior and distal membranelles approximately 20–
25 mm long in vivo. Frontal, ventral and left marginal cirri
also ca 20–30 mm in length in vivo. Transverse and caudal
cirri very strong with cilia 30–40 mm and 25–35 mm long in
vivo, respectively. Dorsal cilia easily seen in live specimens,
about 5–7 mm long in vivo (Figs 2e, k arrows).
Buccal field extending over 60 % of body length and about
65 % of body width. Adoral zone composed of 38–51
membranelles, only two or three of which at distal end reach
right side of body (Figs 1d, f double-arrowhead, 2c arrowheads, 2i, 3a arrow); 10–14 apical membranelles are located
on dorsal side (Figs 1g double-arrowheads, 2h arrowheads).
Apical membranelles comprise three equal-length rows of
kinetosomes, ventral membranelles 3- or 4-rowed with two or
three long rows and one short row. Paroral membrane (PM)
distinctly curved, anterior half composed of many oblique,
short rows of 3 or 4 kinetosomes; endoral membrane (EM)
about 60 % length of PM and comprises two rows of densely
arranged kinetosomes; PM and EM optically intersect near
their posterior ends (Figs 1e, h, 3l). Four to seven (usually
five) frontal cirri (FC) arranged in a group in anterior region
of frontal area, one to three (usually two) ventral cirri (VC)
located together as ‘pre-transverse cirri’ (Figs 1d–h, 2f arrows,
2c, k, 3b arrow, 3c arrows, 3j, o, q, s). Invariably five transverse
cirri (TC; Figs 1d–g, 2c, d, f, k; 3a, c, j, o, q); two bundles of
conspicuous fibres emerge from two rightmost TC and
extend to cirrus III/2 (Fig. 3a, n, 3d arrowhead). Usually two
(rarely three) widely spaced left marginal cirri (MC), one near
proximal portion of adoral zone, the other about level with
transverse cirri (Figs 1d, e, g, h, 1f arrowheads, 2c, d arrows,
2f, k arrowheads, 3c–e, q arrows, 3o). Three caudal cirri
(CC) at right cell margin (Figs 1d arrowhead, e–h). Five
dorso-lateral kineties (DK) each with about 10–17 dikinetids
and conspicuously shortened anteriorly; leftmost and rightmost kineties also shortened posteriorly; three central kineties
terminate near posterior pole (Figs 1d, h, i, 3g, k, p, r arrows).
Environmental data. For details of the Blakeney site, see
‘Type Locality’ above. Seawater was collected from marsh
ditches near the sampling site at West Wittering. The water
temperature was 22 uC, pH ca. 8.0, and salinity 35 %.
Diophrys oligothrix Borror, 1965 (Figs 4 and 5;
Table 1)
Description. Cell size variable, about 70–120630–60 mm in
vivo; body shape oval, ratio of length to width ca. 2 : 1 (Fig.
4a, b, d, e, g, h). Anterior end with a conspicuous, thin collar
(Fig. 4f arrow) accompanied by ridges (Fig. 4c arrowheads);
cilia of apical membranelles emerge near these ridges (Fig. 4h
arrowheads). Posterior end bluntly pointed with three strong
caudal cirri emerging from a concave area on right dorsal
side (Figs 4c, e, h, n arrow). Dorsoventrally flattened with
ventral side flat and dorsal side bulging evenly (Fig. 4i). In
ventral view two ribs are visible, right rib starting from distal
end of adoral zone, left rib starting near posterior portion of
buccal field (Figs 4a, b, e, g arrow); wide depression in central
region of cell between the two ribs. Row of five strong
transverse cirri emerge from within wide depression in
posterior half of cell (Fig. 4r); several short ridges between
transverse cirri that are sometimes visible after protargol
impregnation (Figs 4p arrows, 5b, f arrowheads).
Numerous tiny yellow–greenish cortical granules (approximately 0.5 mm across) beneath pellicle, often grouped around
bases of membranelles (Fig. 4k arrowheads), cirri (Fig. 4j
arrowheads) and dorsal bristles (Fig. 4o arrowheads) or arranged in lines on dorsal side (Fig. 4s) rendering cell yellowish
at low magnification. Beneath this layer of cortical granules are
larger granules (possibly mitochondria), approximately 1.5 mm
in size, disc-like with a central depression, densely arranged all
over body on both ventral and dorsal sides (Fig. 4r arrows).
Cytoplasm colourless or yellowish and transparent, containing
several food vacuoles and numerous granules 1–8 mm in
diameter (Fig. 4l). Contractile vacuole not recognized. Usually
two sausage-shaped macronuclear nodules, one located in
anterior half of cell near right margin, the other positioned in
posterior half near left margin (Figs 4l, 5a, f); anterior nodule
occasionally with a constriction in middle portion (Fig. 5k
arrow) or divided into two parts (Fig. 5g). Two to four
spherical or ellipsoid micronuclei adjacent to macronuclear
nodules (Fig. 5k arrowheads). Locomotion by rapid crawling
on substrate or by swimming. Feeds on bacteria, flagellates and
small ciliates, e.g. scuticociliates (Fig. 5d).
Cilia of anterior and distal membranelles approximately 20–
25 mm long in vivo. Frontal, ventral and left marginal cirri
also ca 20–30 mm in length. Transverse and caudal cirri very
strong with cilia 30–40 mm and 25–35 mm long, respectively.
Dorsal cilia readily visible in live specimens, approximately
5–7 mm long (Fig. 4q, s arrowheads).
Buccal field ca. 60 % of body length and ca. 50 % of body
width (Fig. 4a, b, d–f). Adoral zone comprising 31–39
membranelles, of which only two or three distalmost ones
reach right side of body (Fig. 5g arrowhead, 5j doublearrowhead); eight or nine apical membranelles located on
dorsal side; apical membranelles comprise three equal-length
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Fig. 4. Photomicrographs of Diophrys oligothrix from life. (a, b, d, e, g, m, n) Ventral view of different cells with variable body
shape, showing cirral pattern and two ribs, one near right cell margin and the other near left cell margin (arrow in a, b, e and g),
two marginal cirri (arrowheads in m and n), caudal cirri (arrow in n). (c) Ventral view, showing apical membranelles located near
ridges (arrowheads). (f) Ventral view, showing thin collar (arrow) where apical membranelles (double-arrowhead) are located.
Arrowheads show marginal cirri. (h) Ventral view, showing cirri-like apical membranelles (arrowheads). (i) Right ventro-lateral
view. (j) Ventral view of portion of cell, showing small cortical granules around cirri (arrowheads). (k, p) Ventral view of anterior
(k) and posterior (p) portion of cell, showing membranelles at distal end (arrow in k), short ridges between transverse cirri
(arrows in p) and small cortical granules around cirri (arrowheads). (l) View of portion of cell, showing one sausage-like
macronuclear nodule. (o) Dorsal view of portion of cell, showing small cortical granules arranged in lines and clustered around
dorsal bristles. (q, s) Dorsal view of portion of cell, showing dorsal bristles and small cortical granules. (r) Ventral view of portion
of cell, showing transverse cirri and large granules (arrows). MN, macronuclear nodule. Other abbreviations as in Fig. 1. Bars,
50 mm.
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Fig. 5. Photomicrographs of Diophrys oligothrix after protargol impregnation. (a, e) Ventral view of two different specimens,
showing the ciliature; arrows show marginal cirri. (b) Ventral view of portion of cell; arrows show fibres from two rightmost
transverse cirri, which extend anteriorly to the frontal cirrus IV/2; arrowheads mark short ridges between transverse cirri. (c)
Ventral view of an individual, arrow marks fragmented marginal cirrus. (d) View of portion of cell, showing ingested
scuticociliates. (f) Ventral view of posterior portion of cell; dorsal kinety 1 terminates at level of transverse cirri; arrowheads point
to short ridges between transverse cirri. (g) View of individual with three macronuclear nodules; arrowhead marks membranelles
at distal end. (h, i) Ventral view of anterior portion of the same cell, showing membranelles at distal end (arrows) and frontal cirri
(arrowheads). (j, m) Ventral (j) and dorsal (m) view of the same specimen, showing infraciliature; arrow shows the conspicuously
shortened dorsal kinety 1; arrowhead marks right rib; double-arrowhead indicates membranelles at distal end of adoral zone;
Ventral cirri are enclosed by broken line. (k) View of a specimen with two sausage-shaped macronuclear nodules and two
micronuclei (arrowheads); arrow shows a constriction in the anterior macronuclear nodule; double-arrowhead marks dorsal
kinety 5, which starts at anterior one-third of cell. (l) Ventral view of anterior portion of cell; arrow marks the reduced frontal
cirrus; arrowhead indicates fibres projecting anteriad. MC, marginal cirri; MN, macronuclear nodule. Other abbreviations as in
Fig. 1. Bars, 50 mm.
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Fig. 6. Morphology of five closely related species from life (a, d, g, j, l) and after protargol impregnation (b, c, e, f, h, i, m, n). (a–c)
Diophrys appendiculata (from Song & Packroff, 1997). (d–f) Diophrys scutum (from Song & Packroff, 1997). (g–i) Diophrys
apoligothrix (from Song et al., 2009a). (j, k) Paradiophrys multinucleata (from Hartwig, 1973, originally named Diophrys
multinucleata). (l–n) Diophrys oligothrix (from Song & Packroff, 1997). Bar, 25 mm.
rows of kinetosomes (Fig. 5i arrows), ventral membranelles 3or 4-rowed (i.e. two or three long rows and one short
row). Paroral membrane distinctly curved, its anterior half
composed of many oblique, short rows of three or four
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kinetosomes; endoral membrane approximately 65 % length
of paroral, with two rows of densely arranged kinetosomes;
paroral and endoral optically intersect near their posterior
ends (Fig. 5a, c, e, j, l). Five frontal cirri grouped in anterior
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Fig. 7. The maximum-likelihood (ML) tree inferred from the small subunit rRNA gene sequences of 42 spirotrich ciliates,
showing the positions of Diophrys blakeneyensis spec. nov. and Diophrys oligothrix (bold) and their relatives (boxed). Numbers
near nodes are non-parametric bootstrap values for ML and posterior probability values for Bayesian inference (BI). Filled circles
indicate full support in all analyses; filled rectangles represent 99 % for ML and 1.00 for BI. All branches are drawn to scale. Bar,
two substitutions per 100 nt positions. Protocruzia contrax and Protocruzia adherens were the outgroup taxa.
region of frontal area (Fig. 5a, e, j, h, i arrowheads). Two
ventral cirri located together as ‘pre-transverse cirri’ (Fig. 5e, f,
j). Five transverse cirri (Fig. 5a–c, e, f, j) with two bundles of
conspicuous fibres that emerge from rightmost two and
extend to cirrus III/2 (Fig. 5b arrows). Two widely spaced left
marginal cirri, one near proximal portion of adoral zone, the
other approximately level with transverse cirri (Fig. 5a, e
arrows, 5j); posterior marginal cirrus occasionally fragmented
(Fig. 5c arrow). Five dorso-lateral kineties, each comprising
seven or eight dikinetids on average and conspicuously
shortened anteriorly; leftmost and rightmost kineties shortened posteriorly, three central kineties terminate at posterior
pole of cell (Fig. 5f, m, j, arrow, k double-arrowhead). Three
caudal cirri (Fig. 5e, j).
Environmental data. Seawater collected from marsh ditches
near the sampling site was 18 uC, pH ca. 8.0, and salinity 29 %.
Molecular data and phylogenetic analyses based
on SSU rRNA gene sequences (Fig. 7)
The SSU rRNA gene sequences of D. blakeneyensis spec. nov.
and D. oligothrix were deposited in GenBank with accession
numbers JN172996 and JN172995, respectively. The length
and DNA G+C content of the SSU rRNA genes were
1647 bp and 44.51 % for D. blakeneyensis spec. nov. and
1574 bp and 44.66 % for D. oligothrix.
The BI and ML trees inferred from SSU rRNA gene sequences had similar topologies for the Diophrys-complex
group regardless of the number of taxa included in the
preliminary analyses (i.e. 70, 60 or 24 taxa). As shown in Fig.
7, D. blakeneyensis spec. nov. grouped with D. oligothrix
(Qingdao population) with low support (0.61 BI, 54 % ML),
and then clustered with D. oligothrix (Blakeney population)
with high support (1.00 BI, 99 % ML). The next closest
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Table 2. Morphological and morphogenetic characters on which the phylogenetic tree (Fig. 8) of representatives of uronychiids is
based
For discussions on justification for determining apomorphic vs plesiomorphic character states, see Agatha (2004), Berger (2008), Borror & Hill
(1995), Miao et al. (2007), and Shao et al. (2008).
Character
1. Left marginal cirri located:
2. Left marginal row
3. Caudal cirri from:
4. Body
5. Marginal anlagen
6. Dorsal cilia
7. Cell surface
8. Dorsal ciliary rows
9. Frontal cirri distributed:
10. Undulating membrane
11. Macronucleus
12. Adoral zone of membranelles
Apomorph
Plesiomorph
Subcaudally
Absent
Single anlage
Rigid
Absent
Long and rigid
Sculptured
Fragmented
In a pattern
One highly reduced
In more than two parts
Bipartite
Post-buccally
Present
Two or more anlagen
Flexible
Present
Short and flexible
Not sculptured
Continuous
Randomly
Two
In two parts
In one part
Fig. 8. Cladistic relationships of 17 species of the family Uronychiidae representing the six Diophrys-like genera plus Uronychia,
based on morphological and morphogenetic information. See Table 2 for an explanation of the characters used. For discussions
on characters used in this analysis see Agatha (2004), Berger (2008), Borror & Hill (1995), Miao et al. (2007), Shao et al.
(2008). Abbreviations for taxa: Aova, Apodiophrys ovalis; Dapo, Diophrys apoligothrix; Dapp, Diophrys appendiculata; Dbla,
Diophrys blakeneyensis; Dhys, Diophryopsis hystrix; Doli, Diophrys oligothrix; Dpar, Diophrys parappendiculata; Dscu,
Diophrys scutum; Hzhu, Heterodiophrys zhui; Para, Paradiophrys; Pnig, Pseudodiophrys nigricans; Uro, Uronychia.
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Table 3. Morphometrical and morphological comparison of eight related and well-described diophryid species
Taxa: 1, Diophrys blakeneyensis nov. spec.; 2, D. oligothrix; 3, D. oligothrix; 4, D. oligothrix; 5, D. appendiculata; 6, D. parappendiculata; 7, Pseudodiophrys nigricans; 8, Diophrys apoligothrix; 9, D.
scutum; 10, Diophryopsis hystrix. B, Bipartite; NB, non-bipartite; NS, non-sculptured; S, sculptured.
Characteristic
1
2
3
4
5
6
7
8
9
10
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Size in vivo
60–180650–150 70–120630–60 50–100630–60 79–106650–60 30–70630–55 120–180660–120 60–90640–50 60–110640–70 140–200680–150 30–40620–
(mm)
30
NB
NB
NB
NB
NB
B
B
B
NB
B
Adoral zone of
membranelles
Adoral
38–51
31–39
26–35
40–45
35–44
54–68
22–27
25–31
61–73
15–18
membranelles
Dorsal kinety
5
5
4–5
4
5
5–6
4–5
5
6–9
5
Left marginal
2, postorally
2, postorally
2, postorally
2, postorally
2, postorally
2, postorally
2, postorally
1, postorally
2, postorally
1, postorally
cirri
Frontoventral
7, grouped
7, grouped
7, grouped
7, grouped
7, grouped
7, grouped
7, grouped
7, grouped
7, grouped
9, mostly in 2
cirri
rows
Transverse cirri
5
5
5
5
5
5
5
5
5
5
Macronucleus
7–23
2
2
2
2
2
2
2
2
2
NS
NS
NS
NS
NS
NS
S
S
NS
NS
Body surface
Dorsal ciliature
DK rows
DK rows
DK rows
DK rows
DK rows
DK rows
DK rows
DK rows
DK rows
DK rows
continuous; cilia
continuous;
continuous;
continuous;
fragmented;
fragmented; cilia
continuous;
continuous;
continuous; cilia continuous;
short, flexible
cilia short,
cilia short,
cilia short,
cilia short,
short, flexible
cilia short,
cilia short,
short, flexible
cilia long,
flexible
flexible
flexible
flexible
flexible
flexible
rigid
Data source
This study
This study
Song & Packroff Borror (1965a)
Shen et al. (2011) Jiang et al.
Song &
Song et al.
Song & Packroff Song et al.
(1997)
Packroff
(2011)
(2009a)
(1997)
(2007)
(1997)
relatives were Diophrys parappendiculata and Diophrys
appendiculata, the type species of Diophrys. This subclade
was sister to a group composed of Diophrys scutum, Diophrys
apoligothrix, Diophryopsis hystrix and Pseudodiophrys nigricans. Heterodiophrya zhui, the type species of the genus
Heterodiophrya, was the next closest relative to this large
clade which, along with another major clade consisting of
other Diophrys-like species and four species of the genus
Uronychia, represented the family Uronychiidae.
Cladistic analysis based on morphological and
morphogenetic characters (Table 2, Fig. 8)
The morphological and morphogenetic characters used for
the cladistic analysis are shown in Table 2. The tree based
on this analysis showed that D. blakeneyensis spec. nov. is
most closely related to D. oligothrix which together form a
sister group to D. appendiculata and D. parappendiculata
(Fig. 8). The genus Diophrys is polyphyletic when D.
apoligothrix and D. scutum are included.
DISCUSSION
Diophrys blakeneyensis spec. nov. and its
position in the SSU rRNA tree (Figs 6, 7 and 8;
Table 3)
The two populations of Diophrys blakeneyensis spec. nov.
correspond well in terms of their living morphology, buccal
apparatus and somatic ciliature so they are very likely
conspecific. Morphometric investigations showed that only
three of 16 characters, i.e. the numbers of transverse cirri,
caudal cirri and dorsal kineties, are invariable within populations of D. blakeneyensis spec. nov. which suggests that
these characters can be used to distinguish among species of
the genus Diophrys (Table 1). Three other traits, i.e. the
numbers of adoral membranelles, left marginal cirri and
frontal cirri, varied little between the two populations of D.
blakeneyensis spec. nov. thus supporting their conspecificity.
Of the remaining characters, the numbers of macronuclear
nodules and dikinetids in each dorsal kinety are relatively
highly variable within both populations but they overlap
considerably, again supporting their conspecificity (Table 1).
The genus Diophrys sensu Hill & Borror (1992) is characterized by having two groups of frontoventral cirri, usually one
or two left marginal cirri near the proximal portion of the
adoral zone, two separate undulating membranes and three
posterior-laterally located caudal cirri. Based on this definition, eight species have previously been assigned to Diophrys.
Of these, only Diophrys salina lacks any infraciliature
information; however, it clearly differs from D. blakeneyensis
spec. nov. in its small body size (30–40 mm), well-developed
anterior adoral membranelles, and short and fine frontoventral and transverse cirri (Ruinen, 1938).
Partial information is available on the infraciliature of Diophrys
peloetes which may or may not be a separate species (Song et al.,
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2007). It resembles D. blakeneyensis spec. nov. in terms of body
shape and size and the pattern of its ventral somatic infraciliature. However, the former has more distal adoral membranelles extending far onto the right ventral side (10 from
illustration vs 2 or 3) and 8 dorsal kineties all of which are
parallel to the main body axis (vs invariably 5 DK that curve
posteriorly toward the right margin) (Borror, 1965b).
The remaining six species of the genus Diophrys are all
well-described. Morphological data for five of these, plus
Pseudodiophrys nigricans and Diophryopsis hystrix for which
molecular data are also available, are summarized in Table
3 along with data for D. blakeneyensis spec. nov. Among
the six Diophrys species, D. oligothrix and D. blakeneyensis
spec. nov. are more closely related to one another than to
any other congener in terms of morphological characters
(Table 3; Figs 6 and 8). They have a similar body shape and
cirral pattern (e.g. continuous dorsal kineties, numbers and
arrangement of frontoventral and left marginal cirri) but
differ in the number of macronuclear nodules (usually two
vs usually more than ten). Their close relationship was also
supported by the SSU rRNA gene sequence data with D.
blakeneyensis spec. nov. having 99.1 % and 99.2 % similarities to each of two populations of D. oligothrix, i.e.
differences of 14 nt from our new isolate of D. oligothrix
JN172995, and 13 nt from D. oligothrix DQ353850.
Each of the five other species of Diophrys constantly has
two macronuclear nodules and thus cannot be confused
with D. blakeneyensis spec. nov. Additionally, the latter can
be distinguished from its various congeners by a combination of the following features: (1) the number of adoral
membranelles (ca. 45 vs 61–73 in D. scutum; Song &
Packroff, 1997; Fig. 6d–f); (2) the number of marginal cirri
(usually two vs invariably one in D. apoligothrix; Song et al.,
2009a; Fig. 6h, i); (3) dorsal cilia distributed sparsely and
evenly (vs dorsal cilia in groups in D. appendiculata and D.
parappendiculata; dorsal cilia densely spaced in D. scutum)
(Shen et al., 2011; Song & Packroff, 1997; Fig. 6a–c); (4) cell
surface not sculpted (vs cell surface conspicuously sculpted in
D. apoligothrix; Song et al., 2009a; Fig. 6g). The separation of
D. blakeneyensis spec. nov. from D. apoligothrix, D. scutum,
D. parappendiculta and D. appendiculata was also justified by
their positions both in the SSU rRNA gene tree and the
morphology/morphogenetic-based tree (Figs 7 and 8).
Although SSU rRNA gene sequence data are not available for
Diophrys japonica, this species can be easily separated from
D. blakeneyensis spec. nov. by the numbers of macronuclear
nodules (2 vs 7–19), left marginal cirri (invariably 1 vs 2),
and dorsal kineties (invariably 4 vs invariably 5) and in the
presence (vs absence) of the fragment kinety (Hu, 2008).
Paradiophrys multinucleata resembles D. blakeneyensis spec.
nov. in having multiple macronuclear nodules (Hartwig,
1973; Fig. 6j, k). The former, however, can easily be
separated from the latter by the arrangement of the
macronuclear nodules (packed together in the central region
of the body vs in a ring-like pattern), body shape (broadly
oval with truncated apical end vs oval to rectangular), the
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number of transverse cirri (4 vs 5), and the number and
position of the marginal cirri (2 postbuccal vs 3 subcaudal)
(Hartwig, 1973).
The identity and phylogenetic position of
Diophrys oligothrix (Figs 6, 7 and 8; Table 3)
Diophrys oligothrix was first reported from a marsh pool in
New Hampshire, USA (Borror, 1965a). It was subsequently
isolated and redescribed from coastal waters in Qingdao,
China and seawater ponds on King George Island, Antarctica
(Song & Packroff, 1997; Song & Wilbert, 1994, 2002). Our
population corresponds well with previous populations in
terms of body size and shape and its ciliary pattern, especially
the continuous ciliary rows on the dorsal side with loosely
arranged cilia. There are, nevertheless, three minor differences with the Blakeney population having a relatively longer
buccal field (ca. 60 % of body length vs about 50 % of body
length in other populations), 31–39 adoral membranelles (vs
26–37 in the Qingdao and Antarctic populations, 40–45 in
the New Hampshire population), and invariably five dorsal
kineties (vs four or five, usually four in other populations).
Based on previous studies (Curds & Wu, 1983; Song et al.,
2007) these variations can be considered as populationdependent, so these populations are very likely conspecific
(Table 3; Fig. 8). Based on SSU rRNA gene sequence data,
however, the two populations of D. oligothrix do not
group together, the Qingdao isolate clustering first with D.
blakeneyensis spec. nov. Possible explanations for this
inconsistency include: (1) the phylogeny based on SSU
rRNA gene sequence data may not reflect the true relationships between these two species, especially given the low
support value for this node (54 % ML, 0.61BI); (2) the two
sequenced isolates of D. oligothrix might represent cryptic
species. Neither of these hypotheses can be tested presently
because the type population of D. oligothrix has yet to be
analysed genetically so its phylogenetic position on the SSU
rRNA gene tree is not known. The morphological data do not
justify the establishment of a novel species for the population
isolated in the present study so we identify it as a population
of D. oligothrix.
Note on the phylogenetic positions of the genera
Pseudodiophrys, Diophryopsis and Diophrys
Diophrys apoligothrix is similar to Pseudodiophrys nigricans
with the two species sharing a number of morphological
characters including: (i) bipartite adoral zone of membranelles; (ii) sculpted cell surface; (iii) frontoventral cirri in
two groups; (iv) continuous dorsal ciliary rows; (v) five
transverse cirri; and (vi) two macronuclear nodules (Jiang
et al., 2011; Song et al., 2009a; Table 3). However, the former
differs from the latter in having: (1) two undulating membranes (vs a single, highly reduced undulating membrane), the
latter character being an apomorphy according to Agatha
(2004) and Berger (2008), and; (2) one (vs two) left marginal
cirrus. Thus, the genus Pseudodiophrys is not congeneric with
Diophrys. Interestingly, Pseudodiophrys groups very closely
with Diophrys apoligothrix in both the morphological/morphogenetic tree and the SSU rRNA gene tree (Figs 7 and 8) so
it is possible that Pseudodiophrys is a subgenus of Diophrys.
However, such a decision should await further data, in particular SSU rRNA gene sequence data for other species of
Diophrys and in particular D. japonica, which closely resembles
D. apoligothrix in having one left marginal cirrus and five
dorsal kineties. Such data should help to resolve the true
position of D. apoligothrix and its relationship to Pseudodiophrys.
Diophryopsis hystrix resembles Diophrys in having two
undulating membranes, three posterior-laterally located
caudal cirri, and two groups of frontoventral cirri, so they
are related. The former, however, distinctly differs from the
latter by its asymmetrical (vs oval to rectangular) body
shape, highly developed spine-like (vs short and flexible)
dorsal cilia, anterior frontoventral cirri in two rows (vs
non-rowed), highly reduced (vs highly developed) paroral
membrane, and the origin of the left marginal cirri which
derive from the dorsal kinety (vs marginal) anlage (Table
3). Furthermore, Diophryopsis branches early within the
clade of Diophrys-like uronychiids in both the morphological and SSU rRNA gene trees (Figs 7 and 8). These data
support the separation of Diophryopsis from Diophrys at the
generic level (Song et al., 2007; Shao et al., 2010).
All available data indicate that the genus Diophrys is not
monophyletic, but rather a moderately supported polyphyly
(Figs 7 and 8). Furthermore the validity of the genera
Pseudodiophrys and Diophryopsis, which were established
mainly by morphological data, is still questionable since it has
previously been reported that single-gene analyses of this
group can suffer from phylogenetic artefacts (Yi et al., 2009).
Therefore, more comprehensive taxon sampling, coupled
with multi-gene analyses, will be required to resolve the phylogenetic relationships among the Diophrys-like hypotrichs.
ACKNOWLEDGEMENTS
This work was supported by a Marie Curie International Incoming
Fellowship within the 7th European Community Framework Programme (awarded to X. H.), and the Natural Science Foundation of
China (Project numbers: 40976075 and 41176119; both to X. H.). We are
grateful to University College London, UK and in particular Dr Terry
Preston (UCL), for help with sampling at Blakeney by allowing use of the
Blakeney Point Field Station.
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The morphology and phylogeny of two euplotid ciliates, Diophrys

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