Morphological and molecular study of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic)

Abstract

The presence of the benthic dinophysoid dinoflagellate Sinophysis canaliculata has been reported in the Canary Islands (eastern central Atlantic) in live field observations and on fixed macroalgae samples from intertidal ponds (26 sampling sites from El Hierro, Tenerife, Gran Canaria, Fuerteventura and Lanzarote islands). In vivo Sinophysis cells were typically pale pink colored. Light microscopy (LM) and scanning electron microscopy (SEM) micrographs showed a small characteristic narrow hypothecal cut that matched the original description of S. canaliculata. SSU rRNA gene (rDNA) nuclear phylogeny showed that S. canaliculata is closely related to S. microcephalus. Sinophysis specimens displayed cyanobacterial endosymbionts with orange autofluorescence from phycoerythrins. SSU rDNA analyses of the cyanobionts nearly matched a former sequence obtained from S. canaliculata in the Pacific Ocean (Japan). Sinophysis canaliculata survived up to five months in the original seawater samples. During that period cyanobionts were always present and maintained their orange autofluorescence, although the pink colour gradually vanished (<1 month) in most individuals. Molecular similarity of Sinophysis cyanobionts from the Canary Islands and Japanese waters suggest a deterministic relationship, likely a temporary maintenance inside their host via some specific grazing system. This article is protected by copyright. All rights reserved.

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NOTE
MORPHOLOGICAL AND MOLECULAR STUDY OF THE CYANOBIONT-BEARING
DINOFLAGELLATE SINOPHYSIS CANALICULATA FROM THE CANARY ISLANDS (EASTERN
CENTRAL ATLANTIC)
1
Marıa Garcıa-Portela
2
Instituto Espa~
nol de Oceanografia (IEO), Centro Oceanogr
afico de Vigo, Subida a Radio Faro 50, Cabo Estay, Canido, 36390
Vigo, Spain
Pilar Riobo
Instituto de Investigaciones Marinas, CSIC, Eduardo Cabello, 6, 36208 Vigo, Spain
and Francisco Rodrıguez
Instituto Espa~
nol de Oceanografia (IEO), Centro Oceanogr
afico de Vigo, Subida a Radio Faro 50, Cabo Estay, Canido, 36390
Vigo, Spain
The presence of the benthic dinophysoid
dinoflagellate Sinophysis canaliculata has been
reported in the Canary Islands (eastern central
Atlantic) in live field observations and on fixed
macroalgal samples from intertidal ponds (26
sampling sites from El Hierro, Tenerife, Gran
Canaria, Fuerteventura and Lanzarote islands). In
vivo Sinophysis cells were typically pale pink
colored. Light microscopy and scanning electron
microscopy micrographs showed a small
characteristic narrow hypothecal cut that matched
the original description of S. canaliculata. SSU
rRNA gene (rDNA) nuclear phylogeny showed that
S. canaliculata is closely related to S. microcephalus.
Sinophysis specimens displayed cyanobacterial
endosymbionts with orange autofluorescence from
phycoerythrins. SSU rDNA analyses of the
cyanobionts nearly matched a former sequence
obtained from S. canaliculata in the Pacific Ocean
(Japan). S. canaliculata survived up to 5 months in
the original seawater samples. During that period
cyanobionts were always present and maintained
their orange autofluorescence, although the pink
color gradually vanished (<1 month) in most
individuals. Molecular similarity of Sinophysis
cyanobionts from the Canary Islands and Japanese
waters suggest a deterministic relationship, likely a
temporary maintenance inside their host via some
specific grazing system.
Key index words: Canary Islands; cyanobiont; di-
noflagellates; phylogenetics; Sinophysis
Abbreviations: BI, Bayesian inference; LM, light
microscopy; ML, maximum likelihood; SEM, scan-
ning electron microscopy
The dinoflagellate genus Sinophysis has nine
accepted species (S. canaliculata,S. ebriola,S. grandis,
S. microcephalus,S. minima,S. stenosoma,S. verrucu-
losa,S. hoppenrathiana, and S. vespertilio; Hoppenrath
2000, Selina and Hoppenrath 2004, G
omez et al.
2012, Chom
erat et al. 2009, Chom
erat 2016). The
holotype, S. microcephalus (China; Nie and Wang
1944), is typically found in tropical waters linked to
detritus and its closely related species, S. canalicu-
lata, is a cosmopolitan temperate-warm organism
described in La Reunion Island (Indian Ocean)
stuck to macroalgae or associated with sediments
(Quod et al. 1999, Hoppenrath 2000). During a
benthic survey in the Canary Islands (2015), it was
reported the widespread occurrence of Sinophysis cf.
canaliculata. These exhibited internal globules
resembling the cyanobacterial-like endosymbionts
reported by Escalera et al. (2011) in Japan. Morpho-
logical and molecular analyses were performed on
Sinophysis to confirm its identity. The cyanobionts
were also studied using microscopy (light micro-
scopy (LM), epifluorescence microscopy and scan-
ning electron microscopy (SEM) techniques), and
sequencing of SSU rRNA gene (rDNA).
Samples were collected between September and
October 2015 (Fig. S1 in the Supporting Informa-
tion). Sinophysis cells were obtained from macroal-
gae in intertidal ponds (0–2 m depth) at Charco
Manso (El Hierro), Costa Blanca (Lanzarote) and
Tajao (Tenerife). Temperatures varied from 23°Cto
26°C in the sampling stations, with an increasing
trend from the eastern to the western islands. In El
1
Received 30 May 2016. Accepted 17 November 2016.
2
Author for correspondence: e-mail maria.garcia@vi.ieo.es.
Editorial Responsibility: S. Lin (Associate Editor)
J. Phycol. *, ***–*** (2017)
©2016 Phycological Society of America
DOI: 10.1111/jpy.12508
1

Hierro Island, Sinophysis cells were qualitatively
abundant in comparison with other locations. In
particular, in the northern station “Charco Manso”
the largest abundances were observed and quantita-
tive data were recorded, with densities of 125–471
cells g
1
(wet weight macroalgae). Live samples
were poured into 50 mL plastic flasks (Corning, NY,
USA) and kept at room temperature until their
examination in the laboratory (<1 week). A small
aliquot (15 mL) was fixed with Lugol’s Acid Solu-
tion.
Measurements of 17 vegetative cells and eight
endosymbionts were made with an Axiocam HRc
digital camera (Zeiss, Germany) in bright field with
a LM Leica DM LA (Leica Microsystems GmbH,
Wetzlar, Germany) at 63x. The autofluorescence of
Sinophysis cyanobionts was observed with blue excita-
tion (Ex: 488 nm, Em: 450–490 nm, barrier
515 nm; same equipment as above). Sample pro-
cessing for SEM followed Escalera et al. (2011) and
was carried out on samples from Tajao (Tenerife)
and Charco Manso (El Hierro). Filters were stuck
on the stubs and coated with gold with a K550 X
sputter coater (Emitech Ltd., Ashford, Kent, UK)
and observed with a FEI QUANTA 200 scanning
electron microscope (FEI Company, Hillsboro, OR,
USA). Areolae and pores (n=40) were measured
in three cells. The number of pores and areolae per
surface units (lm
2
) was calculated from six cells
(three in the right side view and three in the left),
counting the pores and areolae enclosed in 10 lm
2
.
Average of pores and areolae per lm
2
per cell were
calculated according to left and right sides. Narrow
thecal cut length was given as the average of mea-
surements in five cells. All Sinophysis cells used in
this study were similar and resembled S. cf. canalicu-
lata: a left sided u-shape cut located in the center of
the cell was visible in all of them. Only occasionally,
a few specimens with Sinophysis ebriola-like morphol-
ogy were observed.
The host nuclear SSU rDNA (18S) and cyanobac-
terial endosymbiont 16S rRNA genes were amplified
from single cells of Sinophysis using primers Euk-A/
Euk-B (Medlin et al. 1988) and CYA106F/CYA781
(N€ubel et al. 1997), respectively. The DNA amplifi-
cation conditions followed Medlin et al. (1988) and
N€ubel et al. (1997). Amplicons were purified with
ExoSAP–IT (USB Corp., Cleveland, OH, USA) and
sent to LIGHT run sequencing services (GATC Bio-
tech AG, Germany). The 18S rRNA sequence was
compared with 15 other sequences from Sinophysis
spp. The original and final alignment for the SSU
rDNA phylogeny consisted of 570 nt. A Kimura 2-
parameter model (Kimura 1980) with a gamma-
shaped parameter (G =0.21) was selected using
MEGA 6 software. The phylogenetic relationships
were determined using maximum likelihood (ML)
(MEGA 6) and Bayesian inference (BI) method with
a general time reversible model (Mr.Bayes v3.1;
Huelsenbeck and Ronquist 2001). Identity between
sequences was calculated using BioEdit v.7.2.5 with
a BLOSUM62 similarity matrix. Net average distance
between different groups of taxa was calculated on
MEGA 6. 16S rRNA gene of Sinophysis endosym-
bionts were compared with 43 records of 16S rRNA
cyanobacterial sequences. The original sequences of
Sinophysis endosymbionts (labeled as I and II,
respectively) consisted of 544 and 654 nt. The final
alignment for the 16S phylogeny consisted of 415
positions. A Kimura 2-parameter model (Kimura
1980) with gamma-shape parameter (G =0.90) and
proportion of invariable sites (I =0.55) was selected
on MEGA 6 and phylogenetic relationships were
carried out as above.
According to LM and SEM analysis Sinophysis cells
from the Canary Islands were almost circular to
slightly ovoid with an average length (antero-poster-
ior axis) of 43.44 2.77 lm and depth of
43.23 2.50 lm, being the length/depth ratio
1.52. A U-shaped slit was located within the center
on the left side of the cells. This character matched
FIG. 1. Micrographs of live Sinophysis canaliculata cells. (A and
D) LM micrographs. (B and E) UV excitation showing green aut-
ofluorescence from two (white arrows in micrograph B) or one
(white arrows in micrograph E) semicentral bodies and (C and
F) epifluorescence microscopy with blue light excitation showing
internal orange autofluorescence globules, SB: 10 lm.
2MAR
IA GARC
IA-PORTELA ET AL.

the original description of S. canaliculata (Quod
et al. 1999), and we will designate onwards as the
Sinophysis collected in this study. Freshly collected
Sinophysis cells exhibited a pale pink color (Fig. S2
and Video S1 in the Supporting Information) not
reported before in this species that turned brown/
greenish or colorless after a few weeks in the labora-
tory. S. canaliculata cells survived in the original sea-
water samples at 25°C for 5 months in 12:12 L:D
photoperiod but neither those or their endosym-
bionts released from the host could be maintained.
Pink color in S. canaliculata was likely due to phy-
coerythrins from their orange autofluorescent cya-
nobionts (Escalera et al. 2011), also revealed in our
study under blue light excitation. A similar brown/
greenish color has been also observed in S. micro-
cephalus specimens from the Arabian Sea (M. Sabur-
ova pers. comm.). The shift from reddish to
greenish color has also been described in other
Dinophysiales like in Dinophysis caudata (Park et al.
2008). The kleptoplastids could only regain reddish
color by engulfing new cryptophyte plastids via the
ciliate Mesodinium rubrum. Our observations also
suggest that cyanobionts in S. canaliculata are of
temporary nature and need to be periodically
acquired, directly or by a vector organism. Kidney-
shape internal globules fluoresced in orange under
blue light excitation (length 5.82 0.62 lm and
width 3.83 0.24 lm) and were more concentrated
near the cingulum (Fig. 1, C and F). Sinophysis also
had one or two semicentral dark brown bodies
(Fig. S3, A and D in the Supporting Information)
with green autofluorescence under UV excitation
(Fig. 1, B and E) but not under blue light, indicat-
ing that phycoerythrins were absent (Escalera et al.
2011).
Cells were bound by an ornamented theca with a
strong areolated surface (Fig. S4, A and B in the
Supporting Information). Pores were randomly
located inside and among the areolae (Fig. S4D).
The number of pores and areolae on the right
side were 5.53 0.94 and 0.95 0.18 per lm
2
,
respectively, and on the left side, were 7.14 2.25
and 1.25 0.52 per lm
2
, respectively. The areolae
and pores diameter were 1.02 0.22 lm and
0.22 0.05 lm, respectively. Higher areolae sizes
Sinophysis canaliculata Endosymbiont I
Sinophysis canaliculata Endosymbiont II
AB546860 Uncultured cyanobacterium clone SC1
AB294942 Unc. cyanobacterium clone pltb-vmat-28
HM799026 Unc. cyanobacterium clone PRTBB8617
EF160009 Unc. Chroococcales cyanobacterium clone 5ad/556a
JQ726906 Unc. bacterium clone EDWFEBC04
FR666955 Unc. bacterium clone S Aug7
JF261761 Unc. bacterium clone Del0209G03
KM020010 Synechocystis sp. SAG 37.92
EU259177 Unidentified cyanobacterium CLg1
JF261756 Unc. bacterium clone Del0209F09
KJ719259 Cyanobiont of Cymbastela stipitata clone B1
EF160008 Unc. Chroococcales cyanobacterium clone p3b5b/566g
FJ358912 Unc. cyanobacterium clone AO26
EF160007 Unc. Chroococcales cyanobacterium clone 3b5a/p2e4
DQ289923 Unc. cyanobacterium clone SC1-42
JQ580183 Unc. cyanobacterium clone RII-OX096
DQ072915 Unc. cyanobacterium clone ThN-P17
KC298754 Unc. bacterium clone 988
KC298319 Unc. bacterium clone 553
AJ292193 Cyanobacterial symbiont MY3 strain MY3
0.05
AY711919 Unc. cyanobacterium clone SIMO-382
KJ546671 Synechocystis aquatilis ISB32
JF966679 Cyanobacterium IHB-410
KJ546667 Chroococcus minimus ISB36
AM238427 Cyanobacterium aponinum ETS-03 strain ETS-03
AB058249 Cyanobacterium sp. MBIC10216
KC875240 Pseudanabaena sp. NTDP02
KC859033 Synechococcus sp. NTDP03
DQ786164 Cyanobacterium sp. LLi5
KF246492 Geminocystis sp. CENA526
KC621874 Cyanobacterium sp. THH
AM258981 C. stanieri PCC 7202 strain SAG 88.79
KJ654307 Geminocystis sp. 1.1
LN997855 Cyanobacterium sp. KSU-AQIQ-3
HM573462 Nosctoc muscorum Ind33
AB045958 Planktothrix agardhii NIES-596
AF013030 Trichodesmium erythraeum
AF001466 Prochlorococcus sp. CCMP1378
AY224195 Synechocystis sp. PCC6803
AB003164 Oscillatoria rosea IAM M-220
AF32939 Spirulina subsalsa
OUTGROUP
FIG. 2. Maximum likelihood (ML) phylogenetic tree of cyanobacterial 16S rRNA gene sequences. Numbers on the major nodes repre-
sent, from left to right, posterior probabilities based on Bayesian inference and ML (1,000 pseudoreplicates) bootstrap values. Only boot-
strap values >60% are shown. The tree was rooted using Prochlorococcus sp. (AF001466).
SINOPHYSIS IN THE CANARY ISLANDS 3

were located near the cingulum and were not
included to calculate the average of the diameters
length. The narrow thecal cut (Fig. S3, C and E)
located on the left side of the cell was smaller than
in the original description of S. canaliculata
(7.2 0.98 lm vs. 11 1lm).
The 18S rRNA phylogeny placed S. canaliculata
(isolated in Tajao, Tenerife; Acc. No. KX139004;
Fig. S5 in the Supporting Information), in a sub-
clade with S. microcephalus. These species shared
high similarity (95.6%), with only 24 differences
along a 570 nt alignment. Those consisted in 13
transitions (being A/G the most usual), 10 transver-
sions (being C/G, G/T, A/C and T/A the most
usual ones) and one deletion (-/A). In addition,
S. canaliculata sequence contained an ambiguous
nucleotide. Slightly higher genetic distances were
found between the S. canaliculata/S. macrocephalus
subclade and S. grandis (0.130) than to the S. steno-
soma group (0.110). Two incongruities between ML
and BI were observed in the branch location of
sequences belonging to S. grandis (JN587291) and
S. ebriola (JN587292). Two endosymbiont sequences
from two S. canaliculata cells (endosymbionts I, acc.
no. KX139005 and II, acc. no. KX139006) from
Tajao (Tenerife) were used to build the 16S rRNA
phylogeny (Fig. 2). Pair-wise analyses revealed that
endosymbionts I and II from the Canary Islands
were identical (100% of similarity) and shared
99.76% and 99.24% of similarity ,respectively, with
those reported by Escalera et al. (2011). Both
sequences formed a subclade associated to a cluster,
where Synechocystis sp. (acc. no. KM020010, T. Friedl,
D. Hepperle, A. Marrero-Callico, R. Jahn, W. H. Kus-
ber, C. Hallmann unpublished data) and a Chroococ-
cales cyanobacterium (acc. no. EF160009, EF160007)
were the only records with any taxonomic informa-
tion. That cluster also contained a cyanobiont of
the sponge Cymbastela stipitata (Luter et al. 2014).
Sequences from cyanobacteria found in other Dino-
physiales (Foster et al. 2006a,b, Qiu et al. 2011)
were not included in the phylogeny given their
shorter length. Although, we examined the genetic
distances (P-values on a 280 nt long fragment),
between cyanobionts in Sinophysis and 60 sequences
retrieved from Dinophysiales). Our results indicated
that closest cyanobionts (P=0.129–0.155) belonged
to a few sequences retrieved from Histioneis
(AY444938, AY444947, AY444948), Ornithocercus
(AY444959, AY444958), and Amphisolenia (AY444916;
Foster et al. 2006a,b). Nevertheless, these P-values
were similar to those between the cyanobionts in
Sinophysis and other organisms included in our
phylogeny such as Spirulina subsalsa (AF32939,
P=0.142) or Oscillatoria rosea (AB003164, P=
0.147). Our phylogenetic results confirmed that the
Sinophysis cyanobionts from Canary Islands matched
those from Japanese waters (Escalera et al. 2011).
We suggest that they might belong to the order
Chroococcales and their kidney-shape would be
consistent with those collected from epilithic
Chroococcales cyanobacteria in marine tropical areas
(D
ıez et al. 2007). One of the diatom-diazotroph
association, involves a Chroococcales cyanobacteria liv-
ing in association with the diatom Climacodium
frauenfeldianum. (Carpenter and Janson 2000, Foster
et al. 2011). The order Dinophysiales represents the
unique case where cyanobacteria become associated
either as epibionts or endocytobionts. Histioneis
spp., Ornithocercus quadratus, and Citharistes regius are
associated with cyanobacterial ectosymbionts (Gor-
don et al. 1994). TEM and nitrogenase labeling
indicated the presence of nitrogen fixative sym-
bionts in Histioneis depressa (Foster et al. 2006a,b)
and the loss of these symbionts would be lethal for
the host (Stoecker et al. 2009). Future studies
should determine if N fixing takes place in Sinoph-
ysis, associated either with bacteria or cyanobionts,
using, for example, nifH sequencing or in vivo fluo-
rescence techniques (nanoSIMS; Foster et al. 2011).
The potential toxicity of Sinophysis spp. is
unknown and has not been considered in previous
studies. LC-HRMS analyses on Lugol’s fixed
S. canaliculata cells (n=400) from the Canary
Islands were negative for lipophylic toxins (P.
Riob
o, pers. comm.). These results were not conclu-
sive given the potential loss of toxins due to sample
fixation and the limited amount of biomass. Their
potential toxicity could be evaluated alternatively by
biological assays and it should not be discarded that
cyanobacterial endosymbionts could themselves pro-
duce toxins, as it is known to occur in several mar-
ine cyanobacteria (e.g., Trichodesmium erythraeum,
Narayana et al. 2014). Complementary analyses
searching for cyanobacterial toxins would be advis-
able in the future to determine the hypothetical
presence of these compounds in S. canaliculata.
We thank to Isabel Ramilo for helping with Sinophysis cells
identification in different sampling sites, to Pablo Salgado for
microscopy images assistance and to the Centre for Scientific
and Technological Support to Research (University of Vigo)
for SEM analysis. This study was funded by CICAN and
DINOMA projects (CGL2013-40671-R; CGL2013-48861-R,
MINECO, Spain). This is a contribution of Unidad Asociada
IEO-CSIC Microalgas Nocivas. The experimental part of this
study was carried out at the Instituto Espa~
nol de
Oceanograf
ıa (IEO) in Vigo. This research note is going to
be part of M. Garc
ıa-Portela PhD appended to “Marine
Science, Technology and Management” (DO*MAR) doctoral
program at the University of Vigo.
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Supporting Information
Additional Supporting Information may be
found in the online version of this article at the
publisher’s web site:
Figure S1. Phytoplankton sampling sites loca-
tion in the Canary Islands where Sinophysis canalic-
ulata were collected.
Figure S2. LM micrograph of in vivo Sinophysis
canaliculata cell showing its characteristic pale
pink color, SB =10 lm.
Figure S3. Light microscopy micrographs of dif-
ferent positions and numbers semicentral bodies
in Sinophysis canaliculata cells.
Figure S4. SEM micrographs of different
S. canaliculata cells showing (a and b) strong areo-
lated surface, (c) the narrow thecal slit, (d) a ran-
domly distribution of pores (white arrows
indicating different positions, inside and among
the areolae) and (e) the narrow thecal cut
located on the left side of the cell. SB: A–
C=20 lm and D, E =5lm.
Figure S5. Maximum likelihood (ML) phyloge-
netic tree of genus Sinophysis based on the 18S
rRNA gene sequences.
Video S1. Sinophysis canaliculata cell swimming
in a seawater natural sample.
SINOPHYSIS IN THE CANARY ISLANDS 5