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Protist, Vol.

169, 268–279, xx 2018
http://www.elsevier.de/protis
Published online date 20 March 2018

ORIGINAL PAPER

Morphology and Molecular Phylogeny of
Peritrich Ciliate Epibionts on Pelagic
Diatoms: Vorticella oceanica and
Pseudovorticella coscinodisci sp. nov.
(Ciliophora, Peritrichia)
Fernando Gómeza,1,2 , Lu Wangb,c,d,2 , and Senjie Linb,c

aCarmen Campos Panisse 3, E-11500 Puerto de Santa María, Spain
bState Key Laboratory of Marine Environmental Science and Marine Biodiversity and
Global Change Research Center, Xiamen University, Xiamen, China
cDepartment of Marine Sciences, University of Connecticut, Groton, CT, USA
dInstitute of Oceanography, Minjiang University, Fuzhou, China

Submitted December 13, 2017; received in revised form February 22, 2018; Accepted March 13, 2018
Monitoring Editor: Genoveva F. Esteban

Consortia of the peritrich ciliate Vorticella oceanica and the planktonic diatom Chaetoceros coarctactus
are reported from the South Atlantic Ocean. The morphologies of the constituent species were similar
to their counterparts from other geographical regions, revealing a widespread distribution in tropi-
cal waters. Besides, we describe Pseudovorticella coscinodisci sp. nov. that lives on Coscinodiscus
wailesii and other pelagic diatoms off Brazil. The zooids were 34–45 ␮m long, and 19–30 ␮m wide with
a stalk too short to coil. The cell surface showed a reticulate pellicle with 18–19 and 11–12 rows above
and below the aboral ciliary wreath, respectively. In the SSU rDNA phylogeny V. oceanica clustered
between the clades of Vorticella and Pseudovorticella/Epicarchesium, and Pseudovorticella coscin-
odisci sp. nov. clustered as a sister group of Pseudovorticella paracratera and P. sinensis. The V.
oceanica-C. coarctactus consortium likely represents an obligate species-specific mutualistic symbio-
sis as the constituents are not known as free-living forms. In contrast, the diatom hosts are known as
free-living forms in the consortia of Pseudovorticella coscinodisci. The new molecular data and species
description will be valuable dataset for future research on the diversity and ecological significance of
ciliate-diatom epibiotic consortia.
© 2018 Elsevier GmbH. All rights reserved.

Key words: Commensalism; epibiosis; molecular phylogenetics; new species; symbiosis; vorticellid.

Introduction
The surface waters of tropical oceans, usually
1
Corresponding author;
depleted in nutrients, are unfavourable environ-
2
F. Gómez and L. Wang contributed equally to this paper. ments for many organisms, especially the diatoms
e-mail fernando.gomez@fitoplancton.com (F. Gómez). that are well adapted to high-turbulent, nutrient-

https://doi.org/10.1016/j.protis.2018.03.003
1434-4610/© 2018 Elsevier GmbH. All rights reserved.
Ciliate Epibioses on Diatoms 269

rich waters (Margalef 1978). However, several cell, sometimes named V. oceanica, attached to
diatoms can proliferate in these unfavourable con- large drum-shaped diatoms of the genus Coscin-
ditions when living in mutualistic symbioses. Some odiscus Ehrenberg (De Faria and Da Cunha 1917;
diatoms live in symbiosis with diazotrophic filamen- Hallegraeff and Jeffrey 1984; Gárate-Lizárraga and
tous cyanobacteria to benefit from nitrogen fixation Muñetón-Gómez 2009; Navarro 1981). Molecular
of the symbiotic cyanobacteria (Carpenter et al. data are available for numerous species of vorti-
1999; Gómez et al. 2005), and other diatoms have cellids (Sun et al. 2012, 2016), while missing for
modified their morphologies to live in a phoresy the species that inhabit the open ocean as epibiont
commensalism such as Chaetoceros tetrastichon on diatoms. In this study, we generated the first
Cleve and C. dadayi Pavillard with the tintin- molecular phylogeny for V. oceanica and identified
nid Eutintinnus inquilinus (O.F. Müller) Schrank a new species of vorticellid ciliate that is epibiotic
(Gómez 2007) or Chaetoceros coarctactus Lauder with diatoms.
with the peritrich ciliate Vorticella oceanica O.
Zacharias (Nagasawa and Warren 1996).
Vorticella Linnaeus is one of the most diverse
genera of peritrichs with over 200 species (Noland
Results
and Finley 1931; Warren 1986). The genus includes Observations of Vorticella
non-colonial, solitary species with a cell body
(zooid) and a long, slender helically contractile stalk oceanica-Chaetoceros coarctactus
that is tethered to inanimate substrates or aquatic The consortia of V. oceanica-C. coarctactus were
organisms (Foissner et al. 1992; Lynn 2008; Warren found almost throughout the year in the sam-
1986). Foissner and Schiffmann (1974) erected pling site off Ubatuba, Brazil. In our recently
the genus Pseudovorticella Foissner & Schiffmann, collected material, V. oceanica was only observed
which is characterized by a reticulate silverline sys- attached to C. coarctactus. The diatom was never
tem, thus differentiating it from Vorticella, which observed devoid of Vorticella, and the ciliate was
has a transverse silverline system (Warren 1987). not observed as a free-living form or attached to
Vorticella and Pseudovorticella stay in clusters or other living or inanimate substrates. A diatom chain
groups considered as colonies, but they are not true of C. coarctactus usually reached 1–1.2 mm long
colonies because each cell has its own individual and composed of 25–30 cells. Each diatom cell
stalk. usually contained 3–4 individuals of V. oceanica.
The species of Vorticella and Pseudovorticella Exceptionally, the diatom chain reached up to 2 mm
tend to proliferate in bacteria-rich eutrophic environ- long and 35–40 cells (Fig. 1A). At this level, the
ments. In contrast, Vorticella oceanica Zacharias diatom chain tended to split into two separated con-
was described from the Sargasso Sea, one of sortia. In the upright position, the expanded stalk
the most oligotrophic regions of the world oceans. of V. oceanica was about 100–190 ␮m and the
Zacharias (1906), without illustration, described V. zooids protruded beyond the setae of the diatom
oceanica with zooids of 28 ␮m long and 22 ␮m in host (Fig. 1B–C). The stalk of the zooids con-
diameter, with a stalk in spiral of 80 ␮m attached tracted forming five active coils (including the ends)
to an indeterminate species of Chaetoceros. The (Fig. 1D).
lack of illustrations and an adequate description Vorticella oceanica sometimes shared the host
has led to a misunderstanding of V. oceanica with other organisms. We can observe sporadically
that was considered conspecific with the freshwa- clumps of sporocysts of other protists (Fig. 1E–F),
ter V. striata Dujardin (Noland and Finley 1931; eggs of crustaceans (Fig. 1G) and groups of a
Warren 1986). Nagasawa and Warren (1996) rein- photosynthetic dinoflagellate, tentatively identified
stated V. oceanica as an independent species that as Cochlodinium pirum (F. Schütt) Lemmermann
lives attached on the diatom Chaetoceros coarc- (Fig. 1H). The cluster constituted a suitable prey
tactus Lauder. The consortium of V. oceanica-C. for numerous pelagic predators (Fig. 1I–N). The
coarctactus is widespread in the warm oceans zooid of V. oceanica responded to the approach of
(Balech 1964; Crosby and Wood 1958; Cupp 1943; a predator with contraction of the stalk, remaining
Delsman 1939; Gárate-Lizárraga and Muñetón- over chain axis under the protection of the diatom
Gómez 2009; Hallegraeff and Jeffrey 1984; Karsten setae. Larva or adult stages of different species of
1907; Kimor et al. 1992; Koray 1988; Lee and copepods showed a common mechanism of pre-
Lee 2011; Margalef 1957; Navarro 1982; Paulmier dation by attaching to the rear of the consortium.
1993; Tester and Steidinger 1979). Some stud- In the case of small predators (copepod larvae),
ies have also reported a short-stalked vorticellid they remained attached to the diatom and travelled
270 F. Gómez et al.

Figure 1. Light micrographs of the consortium Vorticella oceanica and Chaetoceros coarctactus from the South
Atlantic Ocean off Brazil. Scale bars = 50 ␮m. A) Diatom chain in girdle view. B) Valve view. C–D) Oblique view.
E–F) Unidentified sporocites as epiphyte. G) Crustacean eggs as epiphyte. H) The arrows point dinoflagellates,
tentatively Cochlodiniuim pirum, as epiphyte. I–K) Copepod larva feeding on the zooids. L–N) Adult copepod
feeding on the zooids. O) Ethanol-preserved consortium. Note the coiled stalks. P) Zooids with stalk detached
by mechanical damage. Q) Host devoid of zooids. The arrow point the coiled residual stalks. R) Detached
zooids without stalk.

by the propulsion of the zooids (Fig. 1I–K). In the aged by mechanical injuries, detached individuals
case of large copepods, the predator transported may keep the stalk as a loose cable that did not
the consortium during its jumps (Fig. 1L–N). The contract (Fig. 1P). Our attempts to culture the V.
zooid responded to fixation with the contraction of oceanica-C. coarctactus consortium were not suc-
the stalk (Fig. 1O). When the consortium was dam-
Ciliate Epibioses on Diatoms 271

cessful. The zooids detached, leaving coiled stalks ris (Greville) Ralfs in Pritchard and Chaetoceros
attached to the diatom (Fig. 1Q–R). castracanei Karsten in order to test the attachment
The surface morphology of the cells of V. ocean- of zooids or telotrochs of V. oceanica into other
ica was examined using SEM (Fig. 2). The zooids diatoms. We did not observe V. oceanica on the
showed a total of 29–30 transverse striations, lack- frustules of these other diatoms.
ing the longitudinal striations. The striations in the
oral region of the cell were more widely spaced
than those in the aboral region (Fig. 2D–G). It Description of Pseudovorticella
was difficult to discern the position of the aboral coscinodisci sp. nov.
ciliary wreath (trochal band). In the few excep- The morphology and the molecular data indicated
tions (Fig. 2H), the striations in the pellicle of the that the short-stalked vorticellid cells that colonized
zooids were 19–20 from the peristome to the aboral Coscinodiscus spp. and other pelagic diatoms con-
ciliary wreath, and 10 to scopula (see Supplemen- stitute a distinct species unrelated to V. oceanica.
tary Material Fig. S1 in the online version at DOI: This vorticellid species differed from the other
10.1016/j.protis.2018.03.003). The stalk showed a known species of the genus Pseudovorticella that
conspicuous adhesive disc with a rugose surface live in benthic habitats attached to inanimate sub-
of about 4 ␮m in diameter (Fig. 2F). strates. This vorticellid species is here described as
Pseudovorticella coscinodisci sp. nov.
Molecular Phylogeny
We obtained the SSU rRNA gene sequences from Order: Peritrichida Stein, 1859
2 and 3 clones of consortia isolated on Octo- Suborder: Sessilina Kahl, 1933
ber 2, and November 27, 2015, respectively. The
sequences of V. oceanica branched with a strong Family: Vorticellidae Ehrenberg, 1838
support (Bootstrap Probability, BP 100%; Pos- Genus: Pseudovorticella Foissner & Schiffmann,
terior Probability, PP 1) with an environmental 1974
sequence (KT201577) isolated from a bacteria-rich
biofilm in a coastal area. The sequences of Pseudovorticella coscinodisci F. Gómez, Lu
V. oceanica constituted a new clade that clus- Wang & Senjie Lin, sp. nov. (Figs. 4, 5, 6D–E)
tered with moderate support (BP 72%; PP 0.98) DIAGNOSIS: Solitary vorticellid cells, usually
between the clade of Vorticella (containing the type, forming clusters, living as epibiont on the valve
the freshwater species V. convallaria Ehrenberg) of large marine planktonic diatoms of the genus
and the clade of Pseudovorticella/Epicarchesium Coscinodiscus and more sporadically in other
Jankowski (Fig. 3). In reference to branch distances diatom genera. The shape of the zooid was conical
between genera of other lineages (i.e., Carchesium and truncated at the scopula, and with a peristomal
Ehrenberg and Ophrydium Bory de St. Vincent) lip in the oral end. The living zooids were 34–45 m
and distances between different Vorticella species long, and 19–30 m wide. The widest part of the
in this tree, this topology could be interpreted as V. zooid was the peristomial collar of 25–35 m in
oceanica representing a distinct genus separated diameter. There was a single contractile vacuole
from other Vorticella species that formed another situated just beneath the peristome, and several
distinct subclade. food vacuoles. The length of the stalk varied from
about 5 m in some individuals, and more typically
Culturing Experiments one time the length of the zooids. The stalk is too
short to coil. Pellicular striations were arranged
The consortia of Vorticella oceanica-Chaetoceros
horizontally and vertically in a wall brick-like retic-
coarctactus were found almost throughout all the
ulation. The number of rows of the striations from
year, and co-occurred with the proliferation of Cos-
the peristomial area to the aboral ciliary wreath
cinodiscus wailesii Gran & Angst and other diatoms
was 18–19, and from the aboral ciliary wreath to
with a short-stalked vorticellid species as epibiont.
the scopula was 11–12. Telotrochs were observed.
Thus, it can be hypothesised that the zooids or
telotrochs of V. oceanica may be able to colo- HOLOTYPE: A SEM stub (registration number
nize different diatom hosts, and the length of the TIO201802) containing the holotype specimen
stalk may vary according to the nature of the host. (Fig. 5A) is deposited at the Third Institute of
We added consortia of V. oceanica-C. coarctac- Oceanography at Xiamen, China.
tus in diluted cultures of the diatoms C. wailesii, ICONOTYPE: Fig. 6E
Palmerina hardmaniana Hasle, Stephanopyxis tur-
272 F. Gómez et al.

Figure 2. Scanning electron microscopy of Vorticella oceanica. (AD) Adhesive Disc. (ACW) Aboral Ciliary
Wreath. (s) striation. (sc) scopula. (st) stalk. Scale bars = 10 ␮m (1 ␮m in the insets). A–B) Zooid with non-coiled
stalk. C, F) The inset shows the adhesive disk of the stalk. D, G) The inset shows the transverse striations.
E–F) Zooids with globular shape. H–I) Zooids showing prominent transverse striations.

TYPE HOST: Usually found as epibiont on the and Palmerina hardmaniana. In some periods, up
large marine diatom Coscinodiscus wailesii, occa- to 30% of the cells of C. wailesii show clusters
sionally on C. gigas and Palmerina hardmaniana, of Pseudovorticella coscinodisci sp. nov. attached
and more rarely in another pelagic diatoms. to both valve faces (Fig. 4A–C). There is some
TYPE LOCALITY: Off Ubatuba, Brazil, South variability in the length of the stalk, and some-
Atlantic Ocean (23◦ 32 20.15 S; 45◦ 5 58.94 W). times individuals show longer stalks in one diatom
valve when compared to those in the other valve
ETYMOLOGY: The epithet ‘coscinodisci’ (Fig. 4C–D). In a recently divided diatom, the new
refers to the type host, species of the frustule valve was not yet colonized by epibionts
diatom genus Coscinodiscus. Zoobank code: (Fig. 4E). The telotrochs actively moved on the
6DECD037-479B-4E98-A718-D4D82753D89E. surface of the diatom host (Fig. 4F–G). Exception-
ally, a single individual was observed attached to
the mantle or girdle bands of the diatom host. It
Morphological and Ecological Description should be noted that the girdle bands of the diatom
The consortia Vorticella oceanica-Chaetoceros are almost continuously growing and constituted
coarctactus co-occur with the proliferations of an instable substrate for the adhesive disk. The
free-living cells Coscinodiscus wailesii, accompa- stalk may bend, but it was too short to form a
nied by other large diatoms such as C. gigas spring (Fig. 4C–D). The zooids in the centre of
Ciliate Epibioses on Diatoms 273

Figure 3. SSU rDNA phylogenetic tree of ciliates with special focus on vorticellid peritrichs. Newly sequenced
species are shown in bold type. Each sequence name consists of taxon name, GenBank accession number,
sample number and SSU rDNA gene clone code. The bootstrap values ≥50 from ML analysis (right) and
posterior probabilities ≥0.9 (left) from Bayesian analysis are noted to the left of internodes. Scale bar = 0.02
substitutions per site (the phylogenetic tree is presented in nwk.format in the Supplementary Material).

the valve showed a perpendicular stalk and the the valve surface (Fig. 4M). The stalk was absent
mouth parallel to the valve face. In the periphery in unattached individuals (Fig. 4N–O). Clusters of
of the valve face, the stalk and/or zooid leaned these short-stalked vorticellid cells were occasion-
toward the periphery (Fig. 4H). In some individu- ally found on Coscinodiscus gigas (Fig. 4P) and
als, the stalk was very short (Fig. 4I–J). The cells Palmerina hardmaniana (Fig. 4Q–S). Exception-
showed a contractive vacuole (Fig. 4I, L), and sev- ally, one or two individuals were found attached
eral food vacuoles (Fig. 4K–M). There was not a to less common diatoms such as Stephanopy-
clear pattern in the distribution of the zooids on xis turris (Fig. 4T) and Chaetoceros castracanei
274 F. Gómez et al.

Figure 4. Light micrographs of Pseudovorticella coscinodisci sp. nov. attached to several diatom hosts. (CV)
Contractile vacuole. (FV) Food vacuole. (t) telotroch. Scale bar = 50 ␮m. A–O) Coscinodiscus wailesii. P) Cos-
cinodiscus gigas. R–S) Palmerina hardmaniana. T) Stephanopyxis palmeriana. U) Chaetoceros castracanei.
B–C) Note the different length of the stalks. D) Note the longer stalks in the zooid of one of the host valve. E)
Note the lower number of epiphytes in a recently formed diatom valve. F–G) The arrow points the telotroch.
I–M) Short-stalked cells. N–O) Detached zooids. Q) Co-occurrence of the diatoms Coscinodiscus wailesii and
Palmerina hardmaniana.
Ciliate Epibioses on Diatoms 275

Figure 5. Scanning electron microscopy of Pseudovorticella coscinodisci sp. nov. (AD) Adhesive Disc. (ACW)
Aboral Ciliary Wreath. (pl) peristomial lip. (s) striation. (sc) scopula. (st) stalk. A–D, H–O) Zooids. A) The inset
shows the surface reticulation. B) The inset shows the adhesive disc. E–G) Detail of the pellicle. Note the
reticulation. P–Q) Detail of the stalk. (A–D, H–O) scale bars = 10 ␮m (2 ␮m in the insets). (E–G, P–Q) = 2 ␮m.

Figure 6. Line drawings of Vorticella oceanica (A–C) and Pseudovorticella coscinodisci sp. nov. (D–E). A) Live
zooid redrawn from Nagasawa and Warren (1996, Fig. 5a). B) Redrawn from the SEM picture by Nagasawa
and Warren (1996, Fig. 6b). C) Based on Figure 2D. D) Based on Figure 4L. E) Holotype based on Figure 5A.
Scale bars = 10 ␮m.

(Fig. 4U). This suggests that these attachments of sii, Palmerina hardmaniana, Stephanopyxis turris
the zooids are accidental and these hosts are not and Chaetoceros castracanei growing in free-living
suitable for the development of the clusters. Our forms.
attempts to culture Pseudovorticella coscinodisci The surface morphology of the cells of Pseu-
as a free-living form or living on these diatoms dovorticella coscinodisci was examined using SEM
were unsuccessful. Instead our attempts resulted in (Fig. 5). Pellicular striations were arranged horizon-
cultures of the host diatoms Coscinodiscus waile- tally and vertically in a wall brick-like reticulation
276 F. Gómez et al.

Table 1. Comparison of Pseudovorticella coscinodisci sp. nov. with the closer Pseudovorticella relatives in the
molecular phylogenies. 1 x = 37.9, SD = 3.53, n = 10; 2 x = 25.5, SD = 3.38, n = 10.

Character P. coscinodisci sp. nov. P. paracratera P. sinensis P. punctacta
Body shape conical wide conical bell-shaped Conical or bell-shaped
Body length (␮m) 34–451 40–88 50–60 65–80
Body width (␮m) 19–302 64–144 35–45 55
Transverse rows 18–19 15–18 26–32 –
above trochal band
Transverse rows 11–12 9–11 12–15 –
below trochal band
Stalk as × times ×0.2–1 ×5–12 ×4–5 ×4–5
zooid length
Habitat On pelagic diatoms Marine benthic Marine benthic Marine benthic
Source This study Ji et al. (2004) Ji et al. (2003) Warren (1987)

(Fig. 5E–G). The aboral ciliary wreath was evident Discussion
in some individuals as a marked transversal con-
striction (Fig. 5A–D, M–O). The number of rows of All the known species of Vorticella and Pseudovor-
the striations from the peristomial area to the abo- ticella have been described in benthic habitats,
ral ciliary wreath was 18–19, and from the aboral with the exception of Vorticella oceanica that was
ciliary wreath to the scopula was 11–12 (see Fig. able to colonize the open ocean in a stable asso-
S1 as Supplementary Data in the online version at ciation with the diatom Chaetoceros coarctactus.
DOI: 10.1016/j.protis.2018.03.003). The adhesive Vorticella oceanica was described from the trop-
disc of stalk was 3–4 m in diameter (Fig. 5B, P–Q). ical Atlantic (Zacharias 1906) and Nagasawa and
We obtained two SSU rRNA gene sequences Warren (1996) re-described in detail its morphology
of Pseudovorticella coscinodisci sp. nov. collected from Japanese waters (Fig. 6A–B). The cell dimen-
on 18 December 2014 and 28 July 2015. The sions and surface pellicle striation were similar to
sequences branched with strong support in one of our observations from the tropical Atlantic Ocean
the subclades of Pseudovorticella/Epicarchesium (Fig. 6C). This suggests a widespread distribu-
(Fig. 3). Within the subclade Pseudovorticella cos- tion of the consortium V. oceanica-C. coartctactus.
cinodisci clustered most closely with Pseudovorti- We attempted to culture Chaetoceros coarctactus
cella paracratera Ji, Song & Warren (DQ662847) (Ji with a protocol successfully used in culturing the
et al. 2004) and P. sinensis Ji, Song & Al-Rasheid diatoms that were host of Pseudovorticella cos-
(DQ845295.1) (Ji et al. 2003), then to P. punctata cinodisci. Even adding turbulence, the attempts to
(Dons) Warren (DQ190466) (Warren 1987), and culture C. coarctactus yielded no success.
more distantly to other species identified as Pseu- We describe Pseudovorticella coscinodisci, a
dovorticella sp. and Epicarchesium abrae (Precht) second vorticellid species living as epibiont on
Ji, Song & Warren (Ji et al. 2003). The tree con- diatoms in the warm ocean water column (Figs
tained another distinct subclade, which comprised 4, 5, 6D–E). The closer relatives in the molec-
several sequences of Pseudovorticella that are ular phylogeny were P. paracratera, P. sinensis
not identified at the species level and Epicarche- and more distantly with P. punctacta. The zooid of
sium pectinatum Foissner, Berger & Schaumburg Pseudovorticella coscinodisci is smaller than these
(Fig. 3). Compared to distances between Vorticella species, and the stalk is too short to form a coil
species and between species in other lineages in (Table 1). This new species has been known at the
the tree, this two-subclade topology calls for the coasts of Brazil since one century ago (De Faria
split of the species of Pseudovorticella into two and Da Cunha 1917). Pseudovorticella coscin-
groups, tentatively two genera, and confirms the odisci has been illustrated from the Caribbean Sea
polyphyletic character of Epicarchesium. All the (Navarro 1981), Australia (Hallegraeff and Jeffrey
known species of Vorticella and Pseudovorticella 1984) and the Mexican Pacific (Gárate-Lizárraga
are solitary, non-colony forming, although they have and Muñetón-Gómez 2009). The distribution is
a gregarious behaviour forming clusters, while Epi- underestimated because the observations of live
carchesium is a colonial genus which zooids shared plankton are scarce in tropical waters. Most of the
the stalk. plankton studies are based on preserved mate-
Ciliate Epibioses on Diatoms 277

rial, and these delicate vorticellid cells were lost or 0.5% SDS; 200 ␮g mL−1 proteinase K) was used to re-suspend
too distorted to be recognized. Further studies are the cell pellets, the resuspension was transferred into a 1.5 mL
needed to evaluate the ecology of the consortia of tube and incubated at 56 ◦ C for two days. The supernatant was
removed into a new tube after centrifuging at 12000 rpm for
peritrich ciliates and pelagic diatoms in the tropical 5 mins. Ceramic beads (0.5 mm diameter) were added to the
ocean. lysate and the remaining intact cells were disrupted through
a FastPrep-24 bead mill (MP Biomedicals, Irvine, CA, USA)
at 6 m s−1 for 1 min. Subsequent DNA extraction followed a
CTAB method coupled with DNA Clean-up & Concentration
Methods column (Zhang et al. 2005). At the end of the extraction pro-
cess, DNA was eluted in 20 ␮L 10 mM Tris-HCl buffer (pH 8.0).
The SSU rRNA gene was amplified using the universal primers
Sampling and microscopy observations: Sampling was car-
18ScomF1 and 18ScomR1 (Wang et al. 2016). To avoid con-
ried out from December 2013 to December 2015 off Ubatuba
taminations with diatoms, peritrich-specific primers Peri947F
(23◦ 32 20.15 S; 45◦ 5 58.94 W). Consortia were collected
and Peri1403R (Liu and Gong 2012) were also used for target-
using a phytoplankton net (20 m mesh size) in surface waters.
ing peritrich SSU rRNA gene sequences. The PCR conditions
The living, concentrated samples were examined in Utermöhl
were: 94 ◦ C for 1 min, 35 cycles of 15 s at 95 ◦ C, 30 s at 56 ◦ C
chambers at a magnification of ×200 with inverted microscopes
and 90 s at 72 ◦ C, followed by an additional step of exten-
Eclipse TS-100 (Nikon Inc.) and Olympus IX73 (Olympus
sion at 72 ◦ C for 7 min. The resultant amplicons were purified
Inc., Tokyo, Japan), and photographed with a digital camera
using Clean & Concentrator column (Zymo Research, Orange,
(Cyber-shot DSC-W300; Sony, Tokyo, Japan) mounted on the
CA, USA) and directly sequenced with corresponding primers
microscope’s eyepiece.
(DNA Analysis Facility, Yale University, New Haven, USA). The
For scanning electron microscopy, the ciliate-diatom consor-
PCR products of SSU rDNA were cloned into a T-vector and
tia were isolated with a pipette and placed in filtered seawater
12–24 clones were picked up and sequenced for each sample.
with glutaraldehyde (final concentration of 5%). The sample was
Sequence reads were aligned and assembled using MEGA 6.0
filtered onto a 3 m pore size Nuclepore membrane filter, fixed
(Tamura et al. 2013). The newly generated sequences were
with osmium, dehydrated with a graded series of ethanol (30%,
deposited in DDBJ/EMBL/GenBank under accession numbers
50%, 70%, 90%, 95%, 99%, 100%) and critical-point-dried with
MH055797-MH055803.
CO2 . Filters were mounted on stubs, sputter-coated with gold
Phylogenetic analyses: SSU rDNA sequences
and viewed under a field-emission scanning electron micro-
related to our new sequences were identified using
scope Zeiss Supra 55 (Carl Zeiss Inc., Oberkochen, Germany).
BLAST and downloaded from GenBank database
Images were presented on a black background using Adobe
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). All available sequences
Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA,
of Vorticella, Pseudovorticella and representing sequences of
USA).
other clades of peritrich ciliates reported in Sun et al. (2016)
Cultures: Ciliate-diatom consortia were isolated to estab-
were included. Alignment was performed using CLUSTAL X
lish cultures. Each consortium was individually pipetted and
(Thompson et al. 1997) with default alignment parameters
successively placed in glass dishes with 0.2 ␮m filtered sea
to construct phylogenetic trees using Maximum Likelihood
water collected that day from the same locality to remove con-
(ML) and Maximum Parsimony methods. Maximum Likelihood
taminants. Finally, the consortia were placed in 6-well tissue
phylogenetic analysis of 18S rDNA was performed with a
culture plates in 0.2 ␮m-filtered seawater, and supplemented
PhyML web server (http://www.atgc-montpellier.fr/phyml/)
with supernatant water of aged cultures of microalgae (dinoflag-
using the general time reverse (GTR) model with estimated
ellates and diatoms) as a source of bacteria, and in f/2 medium
values of the proportion of invariants (I) and the gamma
with silicates. Several variations of this protocol were tested. In
shape parameter (G). The branch supports of the ML tree
all the cases, the culture plates were placed in an incubator used
were inferred using Shimodaira-Hasegawa-like (SH) aLRT and
for microalgae culturing, at 23 ◦ C, 100 ␮mol photons m−2 s−1
were estimated by performing 100 non-parametric bootstrap
from cool-white tubes, in a 12:12 L:D cycle. Taken into account
replicates with the same parameters mentioned above. In
that Chaetoceros coarctactus in its symbiotic form was fully
addition, a Bayesian Inference (BI) of phylogeny was performed
covered of plastids (including the setae), we attempted to cul-
with Mr. Bayes 3.1.2 (Huelsenbeck and Ronquist 2001), along
ture it following the protocol that successfully worked with other
with 500000 generations of MCMC with trees sampled every
diatoms. The diatom cells were placed in filtered seawater
1000 cycles, and the first 25% of the states discarded as
enriched with f/2 + Si medium. The diatom Coscinodiscus waile-
burn-in. The phylogenetic tree is presented in nwk. format in
sii proliferated as a free-living form, while the attempts with
the Supplementary Material.
Chaetoceros coarctactus failed. Alternatively, turbulence was
added with micro-bubble aeration without success.
DNA extraction, PCR and sequencing: Consortia of
Vorticella oceanica-Chaetoceros coarctactus or Pseudovor-
ticella coscinodisci-Coscinodiscus wailesii were individually Acknowledgements
micropipetted using a fine capillary into a clean chamber, and
washed several times through a series of drops of 0.2 ␮m-
filtered seawater to remove other organisms. Finally, the F.G. was supported by Brazilian Conselho Nacional
consortia were deposited into a 0.2 ml Eppendorf tube filled de Desenvolvimento Científico e Tecnológico [grant
with several drops of 100% ethanol. The sample was kept at number BJT 370646/2013-14] and a MEL Senior
room temperature and in darkness until the molecular analy- Fellowship at Xiamen University, China [grant num-
sis could be performed. Prior to PCR, the sample tube was
centrifuged and ethanol was evaporated by placing the tube ber MELRS1711]. L.W. and S.L. were supported
overnight in a desiccator at room temperature. Four hundred by the Chinese Visiting Scholarship Council and
␮L of DNA lysis buffer (10 mM Tris pH 8.0; 0.1 M EDTA pH 8.0; the Natural Science Foundation of China (grant #
278 F. Gómez et al.

41330959). This is contribution #2018259 of MEL, Ji D, Song W, Al-Rasheid KA (2003) Description of a marine
Xiamen University. peritrichous ciliate, Pseudovorticella sinensis n. sp. (Ciliophora,
Peritrichia) from China. J Eukaryot Microbiol 50:360–365
Ji D, Song W, Warren A (2004) Pseudovorticella paracratera
n. sp., a new marine peritrich ciliate (Ciliophora: Peritrichida)
from north China. Hydrobiologia 515:49–57
Appendix A. Supplementary data
Karsten G (1907) Das indische Phytoplankton nach dem
Material der deutschen Tiefsee-Expedition 1898-1899. Wis-
Supplementary data associated with this arti- senschaftliche Ergebnisse der Deutschen Tiefsee-Expedition
cle can be found, in the online version, at auf dem Dampfer “VALDIVA” 1898-1899 2:221–548
https://doi.org/10.1016/j.protis.2018.03.003.
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