Diatom Research

ISSN: 0269-249X (Print) 2159-8347 (Online) Journal homepage: http://www.tandfonline.com/loi/tdia20

Molecular phylogeny suggests transfer of

Hemidiscus into Actinocyclus (Coscinodiscales,
Coscinodiscophyceae)

Fernando Gmez, Lu Wang, David U. Hernndez-Becerril, Yuliya O. Lisunova,

Rubens M. Lopes & Senjie Lin

To cite this article: Fernando Gmez, Lu Wang, David U. Hernndez-Becerril, Yuliya O. Lisunova,
Rubens M. Lopes & Senjie Lin (2017) Molecular phylogeny suggests transfer of Hemidiscus
into Actinocyclus (Coscinodiscales, Coscinodiscophyceae), Diatom Research, 32:1, 21-28, DOI:
10.1080/0269249X.2017.1313783

To link to this article: http://dx.doi.org/10.1080/0269249X.2017.1313783

Published online: 24 Apr 2017.

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Diatom Research, 2017
Vol. 32, No. 1, 2128, http://dx.doi.org/10.1080/0269249X.2017.1313783

Molecular phylogeny suggests transfer of Hemidiscus into Actinocyclus (Coscinodiscales,

Coscinodiscophyceae)
FERNANDO GMEZ 1 , LU WANG2,3 , DAVID U. HERNNDEZ-BECERRIL4 , YULIYA O. LISUNOVA5 ,
RUBENS M. LOPES6 & SENJIE LIN2,3
1 Carmen Campos Panisse 3, E-11500 Puerto de Santa Mara, Spain
2 Marine Biodiversity and Global Change Research Center and State Key Laboratory of Marine Environmental Science, Xiamen
University, Xiamen, Peoples Republic of China
3 Department of Marine Sciences, University of Connecticut, Groton, CT, USA
4 Instituto de Ciencias del Mar y Limnologa, Universidad Nacional Autnoma de Mxico, Ciudad de Mxico, Mxico
5 Microsystems Laboratory, cole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland
6 Laboratory of Plankton Systems, Oceanographic Institute, University of So Paulo, So Paulo, Brazil

Hemidiscus cuneiformis, type of the family Hemidiscaceae, is a diatom with a distinctive semi-circular shape in valve view. A strain of
H. cuneiformis was established from the coastal region of the tropical southwestern Atlantic Ocean o Brazil. Cells of this strain showed
semi-circular to asymmetrically elliptical valves, indicating morphological plasticity. In the small subunit (SSU) rDNA phylogeny, the
type species of Hemidiscus branched between two clades of Actinocyclus, one of which was a sister group, the other in a basal position.
Beyond cell shape, there are no signicant morphological dierences between Hemidiscus and Actinocyclus. We propose the transfer of
H. cuneiformis and H. kanayanus into Actinocyclus. The family Hemidiscaceae remains for the genus Actinocyclus.
Keywords: Bacillariophyta, DNA barcoding, molecular phylogenetics, new combination, pseudonodule, pseudonodolus

Introduction the characteristic pseudonodulus and reinstated the genus

Hemidiscus cuneiformis G.C. Wallich is a marine plank-
tonic species with distinctive, almost semi-circular, valves
and a widespread distribution, particularly in warmer
oceans, although it can also be carried in warm cur-
rents to temperate seas (Wallich 1860, Romero et al.
2005). Schtt (1896) transferred H. cuneiformis into Euo-
dia Bailey ex Ralfs as Euodia cuneiformis (G.C. Wal-
lich) F. Schtt. The type species of Euodia, E. gibba
(Bailey) Ralfs, as well as E. orbicularis Castracane, E.
recta Castracane and E. ventricosa Castracane are con-
sidered to be heterotypic synonyms of H. cuneiformis
(Hustedt 1930). Euodia J.W. Bailey ex Ralfs is illegiti-
mate because it is a junior homonym of the plant genus,
Euodia J.R. Forster & J.G.A. Forster. In addition to the
almost semi-circular valve outline, the main diagnostic
character of Hemidiscus is the pseudonodulus; a marginal
to sub-marginal structure, with a single open hole or
circular area on each valve covered by smaller densely-
packed areolae (Simonsen 1975). Palmeria Greville is
another genus with a semi-circular valve shape which
Mann (1907) transferred to Hemidiscus G.C. Wallich.
However, Simonsen (1972) noted that these taxa lacked
Palmeria, which Hasle (1995) subsequently transferred to
Palmerina Hasle because Palmeria Greville is a junior
homonym of Palmeria F. von Mueller. Besides the type,
H. cuneiformis, other fossil and extant species have been
placed in Hemidiscus (see Table S1). According to Simon-
sen (1972) and Hasle & Syvertsen (1997) the genus encom-
passes two extant species, the type, H. cuneiformis, and H.
kanayanus Simonsen, which dier in their areolation (size
and arrangement).
Hemidiscus has previously been placed within the fam-
ily Euodieae F. Schtt (Cupp 1943) or within the Eupodis-
caceae Ktzing, together with Eupodiscus J.W. Bailey,
Roperia Grunow ex Pelletan, Actinocyclus Ehrenberg and
Auliscus Ehrenberg, based on the similarity of their ocelli
(Hendey 1974). Currently, H. cuneiformis is accepted as
the type of its own family, Hemidiscaceae Hendey emend
Simonsen ex Hasle. Simonsen (1975) placed genera with
a pseudonodulus (Hemidiscus, Actinocyclus and Roperia)
within the Hemidiscaceae. The genus Azpeitia M. Pra-
gallo, which lacks a pseudonodulus, has also been placed
within the Hemidiscaceae (Fryxell et al. 1986, Round et al.
1990).

*Corresponding author. E-mail: fernando.gomez@toplancton.com

(Received 27 October 2016; accepted 15 March 2017)

2017 The International Society for Diatom Research

22 Gmez et al.
In their taxonomic key, Hasle & Syvertsen (1997) sepa-
rated Actinocyclus and Hemidiscus on valve shape, circular
or semi-circular, respectively. Actinocyclus encompasses
about 400 fossil and extant species (and infraspecic)
names, including species originally described within the
genera Coscinodiscus Ehrenberg, Eupodiscus or Charcotia
M. Pragallo. Currently about 70 species of Actinocy-
clus are considered valid (Hasle 1977, Fryxell & Semina
1981, Villareal & Fryxell 1983, Andersen et al. 1986,
Watkins & Fryxell 1986, Hayashi et al. 2012). Hasle &
Syvertsen (1997) recognised three types of fasciculation
of the areolae within Actinocyclus. The type, A. octonar-
ius Ehrenberg, A. subtilis (Gregory) Ralfs in Pritchard,
A. actinochilus (Ehrenberg) Simonsen and A. normanii
(Juhlin-Dannfelt) Hustedt have radial areolar rows, paral-
lel to a central row. Other species, such as A. curvatulus
Janisch in A. Schmidt, have a radial areolar row parallel
to the edge (side row). Other species, such as A. spiritus
Watkins, have areolar rows parallel to the central and/or
the edge row (Hasle & Syvertsen 1997).
Molecular data for the family Hemidiscaceae is cur-
rently restricted to three species, A. actinochilus, A. curvat-
ulus and A. subtilis (Medlin et al. 1996, Damste et al. 2004,
Theriot et al. 2015), and several sequences that have not
been identied to species level (Theriot et al. 2010, 2015,
Ashworth et al. 2013, Nakov et al. 2015). In this study, we
provide the rst molecular data for the type of the Hemidis-
caceae, H. cuneiformis. We also examine the morphology
and classication of Hemidiscus and Actinocyclus within
the context of these new molecular phylogenetic data.
Material and methods
Isolation and culturing
Cells of H. cuneiformis were gathered from daily sam-
ples from surface waters in December 2014, taken with
a phytoplankton net (20 m mesh size) o Ubatuba, So
Paulo State, Brazil (23 31 27.80 S, 45 04 59.48 W).
Aliquots of the net samples were left to settle in a settling
chamber, examined with an inverted microscope (Nikon
TS100, Nikon, Tokyo, Japan) and photographed with a dig-
ital camera (Cyber-shot DSC-W300, Sony, Tokyo, Japan)
mounted on the microscope eyepiece. The cells were indi-
vidually micropipetted using a ne capillary into a clean
chamber, and washed several times through a series of
drops of 0.2 m-ltered seawater to remove other organ-
isms. The cells were placed in 12-well tissue culture plates
in 0.2 m-ltered and sterilized seawater supplemented
with f/2 medium with silicates, and incubated at 23C,
with 100 mol photons m2 s1 from cool-white tubes,
in a 12:12 h L:D cycle. Individual cells were re-isolated
and placed in 6-well tissue culture plates, and then later
in 50 mL polystyrene tissue culture asks under the same
conditions.
To preserve cells of Hemidiscus, glutaraldehyde (50%
solution) was added to the culture to a nal concen-
tration of 5%. Cells were washed with distilled water,
placed on a glass slide and then dried for 2 h in
an oven at 110C. Samples were mounted on stubs,
sputter-coated with gold and viewed using a MERLIN
eld-emission scanning electron microscope (Zeiss, Jena,
Germany).
DNA extraction, PCR and sequencing
Exponentially growing cultures were harvested by cen-
trifugation and the pellet was placed in a 1.5 mL Eppendorf
tube lled with absolute ethanol. The sample was kept at
room temperature and in darkness until the molecular anal-
ysis could be performed. Prior to PCR, the sample tube
was centrifuged, and ethanol was evaporated by placing
the tube overnight in a desiccator at room temperature.
Four hundred microlitre of DNA lysis buer (10 mM Tris
pH 8.0; 0.1 M EDTA pH 8.0; 0.5% SDS; 200 g mL1
proteinase K) was used to re-suspend the cell pellets; the
resuspension was transferred into a 1.5-mL tube and incu-
bated at 55C for 2 days. Ceramic beads (0.5 mm diameter)
were added to the lysate after incubation and the remain-
ing intact cells were disrupted through a FastPrep-24 bead
mill (MP Biomedicals, Irvine, CA, USA) at 6 m s1 for 1
min. Subsequent DNA extraction steps followed a CTAB
method coupled with DNA Clean-up & Concentration col-
umn (Zhang et al. 2005). At the end of the extraction
process, DNA was eluted in 30 L 10 mM Tris-HCl buer
(pH 8.0). Fragments of SSU rDNA were amplied using
the universal primers 18ScomF1 and 18ScomR1 (Wang
et al. 2016). The PCR conditions were: 94C for 1 min,
35 cycles of 15 s at 95C, 30 s at 56C and 45 s at 72C,
followed by an additional step of extension at 72C for 7
min. The resultant amplicons were puried using Clean
& Concentrator column (Zymo Research, Orange, CA,
USA) and directly sequenced with corresponding primers
(DNA Analysis Facility, Yale University, New Haven,
CT, USA). The PCR products were also cloned into a T-
vector and eight clones were picked up and sequenced.
Sequence reads were aligned and assembled using MEGA
5.05 (Tamura et al. 2013). The newly generated sequence
was deposited in DDBJ/EMBL/GenBank under accession
number KY362440.
Phylogenetic analyses
The SSU rDNA sequences were assembled from the
most similar sequences identied using BLAST (http://
blast.ncbi.nlm.nih.gov/Blast.cgi). All available sequences
of Actinocyclus spp., Actinoptychus Ehrenberg and other
centric diatoms (Ashworth et al. 2013, Theriot et al.
2015, Medlin 2016) were included, and sequences
from the stramenopiles Bolidomonas pacica Guillou &
Chrtiennot-Dinet and Bolidomonas mediterranea Guillou
 Hemidiscus is part of Actinocyclus 23

& Chrtiennot-Dinet were used as outgroups. Alignments Results

were performed using SSU-Align package (Nawrocki Morphology of H. cuneiformis
et al. 2009), which performs secondary structure align-
Hemidiscus cuneiformis is a common member of the phy-
ments based on the covariance model (CM; Cannone
toplankton assemblage in the tropical waters of Brazil,
et al. 2002; for a diatom example see Theriot et al.
although it never reaches high abundances. The cells were
2009). The sequences were aligned to the consensus CM
solitary and rarely observed in division. The valves were
model of Eukarya integrated in the SSU-Align pack-
at and the shape was of a semicircle, resembling orange
age. Alignment columns with low posterior probability
segments (Figs 17). The valve faces lay at acute angles to
(PP 0.95), which generally corresponded to large loops
each other. The dorsal margin of the valve was strongly
for which positional homology and co-varying nucleotides
convex, without constrictions near the valve poles. The
were dicult to assign, were removed using the SSU-
ventral margin was regularly and gently convex, not con-
Mask routine in the SSU-Align package. After mask-
cave near the ends, often with a median ination on the
ing the total alignment was 1559 nucleotides (nt) long;
ventral side. No intercalary bands or septa were present
322 nt were masked. Phylogenetic analysis used max-
in the girdle. The apical axis ranged from 40180 m and
imum likelihood in a PhyML web server (http://www.
the transapical axis 3090 m. Cells in culture were often
atgc-montpellier.fr/phyml/). The Bayesian inference anal-
smaller in size and asymmetrically ellipsoidal in shape
ysis was performed with MrBayes 3.1.2 (Ronquist &
(Figs 6, 7), showing yellowish brown pigmentation with
Huelsenbeck 2003) using the evolutionary model GTR + I
numerous small chloroplasts (Figs 25, 710).
(0.3334) + G (0.6083) selected under the AIC criterion by
In culture conditions, H. cuneiformis grew very slowly
MrModeltest v.2 (Nylander 2004). Markov chain Monte
compared to other planktonic diatoms (Chaetoceros Ehren-
Carlo simulations were run for 500 000 generations, sam-
berg, Thalassiosira Cleve). A clonal culture required about
pling every 500th generation using the settings: Nst = 6,
six weeks to cover the bottom of the culture ask. In
rates = invgamma, statefreqpr = dirichlet (1,1,1,1). Anal-
senescent cultures, cells deprived of nutrients did not show
yses were run until the average standard deviation of split
any modication in valve morphology before dying. Spore
frequencies was 0.004. The rst 1000 trees were discarded
formation was not observed in any stage of the cultures.
as burn-in. All the remaining trees were used in the calcu-
With scanning electron microscopy, the valve surface
lation of posterior probabilities applying the majority rule
was covered with a ne pattern of areolae, which radiated
consensus.
irregularly from the centre of the valve (Figs 1115). The

Figs 110. Light micrographs of Hemidiscus cuneiformis. Figs 17. Note the dierence in size and shape. Figs 35, 710. Cells in
division. Figs 610. Cells with asymmetrical ellipsoidal shape. Scale bars = 20 m.
24 Gmez et al.

valve face was at, the mantle shallow and distinguish-
able by its smaller areolae. The areolae formed irregular
radial fascicles, but short parallel lines towards the valve
centre. Areolae were 7 m in diameter, with 10 are-
olae in 10 m on the valve face, while on the valve
mantle areolae were 5 m in diameter, with 15 are-
olae in 10 m (Figs 1617). A central hyaline area and
rosette were absent. The single pseudonodulus seems to
open by a simple aperture (Fig. 18). Areolae are locu-
late, opening externally through cribra sunk below the
valve surface; internally with round, rimmed foramina
(Figs 1923). Rimoportulae were conned to a row along
the ventral margin. Internally they are shortly stalked auric-
ular structures, the slits more or less parallel to the valve
margin.
Molecular phylogeny
We obtained the SSU rDNA sequence of H. cuneiformis
from direct and clone sequencing. A BLAST search was
conducted on the new sequence to nd related sequences
in GenBank. The closest sequence was the uncultured
eukaryote clone SGYW751 (KJ760721), from the East
Pacic Rise (20 m depth) with 98% sequence identity. The
closest identied sequences were A. curvatulus (X85401,
98% sequence identity) and A. actinochilus (AY485506,
98% sequence identity), followed by several unidenti-
ed species of Actinocyclus, and more distantly, species
of Actinoptychus. In the SSU rDNA phylogenetic tree,
members of the Coscinodiscales were divided into two
clades, the Coscinodiscaceae (Coscinodiscus, Palmerina,

Figs 1123. Scanning electron micrographs of Hemidiscus cuneiformis. Figs 1115. The arrows indicate the pseudonodulus in the
ventral side. Figs 16, 17. Details of the areolae. Fig. 18. Detail of the pseudonodulus. Figs 1923. Details of the wall structure revealed
through breaks. Scale bar = 10 m (Figs 1115) or = 1 m (Figs 1623).
 Hemidiscus is part of Actinocyclus 25

Stellarima Hasle & P.A. Sims) and the Aulacodis-
caceae (Aulacodiscus Ehrenberg), and the Heliopeltaceae
(Actinoptychus) and Hemidiscaceae (Actinocyclus and
Hemidiscus). The Actinocyclus sequences branched into
two strongly supported subclades. One subclade was com-
posed of sequences of A. actinochilus, A. curvatulus and an
unidentied Actinocyclus species that formed a sister clade
with Hemidiscus. Three unidentied Actinocyclus species
formed the other subclade (Fig. 24).
Discussion
Hemidiscus cuneiformis is characterised by its distinc-
tive semi-circular shape, which facilitates its identication.
However, variability in the shape of the valve has led
to the creation of many synonyms for this taxon (see
Table S1). Our cultures conrmed the plasticity of shape,
with valves ranging from semi-circular to asymmetrically
elliptical (Figs 17).
Based on the molecular phylogeny, the Coscinodis-
cales is separated into two clades, one for Aulacodiscus,
Coscinodiscus, Palmerina and Stellarima, and the other
clade for Aulacodiscus, Actinoptychus, Actinocyclus and
Hemidiscus (Fig. 24). Actinoptychus and Aulacodiscus
were placed in the family Heliopeltaceae (Simonsen 1979),
but the molecular data supports the placement of Aula-
codiscus in its own family, the Aulacodiscaceae (Round

Figs 24. SSU rDNA phylogenetic tree based on 1559 aligned positions. Newly sequenced species are shown in bold type. Numbers
after each taxon name are GenBank accession numbers. The posterior probabilities 0.9 (left) from Bayesian analysis, and the bootstrap
values 50 from ML analysis (right), are shown to the left of the branches. Bolidomonas pacica and B. mediterranea were used as
outgroups. Scale bar = 0.05 substitutions per site.
26 Gmez et al.

Table 1. Comparison of the morphological characteristics of Hemidiscacean species.

Areola pattern Areolae Rimoportulae Annulus Mantle and pseudonodule

Hemidiscus Radial Well-dened Rounded, with short Conspicuous, Asymmetrical:

cuneiformis areolae all over neck hyaline high in margin
the valve opposite the
pseudonodule, low
in pseudonodule
margin
Actinocyclus Radial Dened areolae all Truncate, no neck. High
tinydrum over the valve. Labiate.
HK346
California
Actinocyclus sp. Radial, rows of Marginal areolae Rounded, with Hyaline, surrounded
HK347 Guam areolae lead smaller short neck. Main by areolae (up to
to marginal rimoportulae plane nine)
rimoportulae follows valvar plane
Actinocyclus sp. Radial, rows of Irregular areolae, Fairly rounded, with Large foramina,
HK262 Panama areolae lead marginal ones short neck. Main very marginal
to marginal smaller rimoportulae plane pseudonodule
rimoportulae follows valvar plane
Actinocyclus Radial Irregular areolae Truncate, with Surrounded by High mantle,
subtilis HK168 long neck. Main areolae conspicuous
rimoportulae plane pseudonodule
follows valvar plane
Actinocyclus Radial, rows of Well-dened Rounded, with Inconspicuous Very high mantle.
octonarius areolae lead areolae long neck. Main Large conspicuous
to marginal rimoportula plane pseudonodule
rimoportulae follows valvar plane

et al. 1990). The other Coscinodiscalean clade contains
the Heliopeltaceae (for Actinophychus), and the Hemidis-
caceae (Actinocyclus and Hemidiscus), characterised by
the presence of a pseudonodulus.
In our molecular phylogeny, the Hemidiscus sequence
branched between two clades of Actinocyclus species
(Fig. 24). Based on this topology H. cuneiformis should be
transferred into Actinocyclus. If Hemidiscus is retained as
an independent genus, the current placement of Hemidis-
cus in the SSU rDNA phylogenetic tree (Fig. 24) makes
Actinocyclus paraphyletic. Thus, either Hemidiscus is part
of Actinocyclus, or Actinocyclus should be broken up into
dierent genera. Table 1 compares the morphology of
Hemidiscus and species of Actinocyclus for which SSU
rDNA sequences are available. The strains Actinocyclus
HK262, HK345H, HK346 and K347 have LM and/or SEM
photo vouchers available from http://www.protistcentral.
org/Project/get/project_id/79. There are no signicant dif-
ferences between the species of Actinocyclus and Hemidis-
cus. Hasle & Syvertsen (1997) reported valve shape as
semi-circular and circular/elliptical for Hemidiscus and
Actinocyclus, respectively, and thus a diagnostic char-
acter for generic separation. Our observations reveal a
high morphological plasticity in the valve shape of H.
cuneiformis (Figs 17, 1115), yet valve shape is the only
dierentiating character for Hemidiscus and Actinocyclus.
Therefore, there is no morphological or molecular support
for retaining Hemidiscus as an independent genus. Since
Hemidiscus is the later name, Actinocyclus has priority and
new combinations are therefore proposed for the Hemidis-
cus species.
New combinations
Actinocyclus cuneiformis (Wallich) F. Gmez, Lu Wang &
Senjie Lin, comb. nov.
Basionym: Hemidiscus cuneiformis Wallich 1960 (Trans.
Micr. Soc. London, ser. 2, 8: 42, pl. II, gs. 3, 4).
Homotypic synonym: Euodia cuneiformis (Wallich) A.
Mann 1893
Extant heterotypic synonyms: Hemidiscus inornatus (Cas-
tracane 1886) Kuntze 1898, H. orbicularis (Castracane
1886) Kuntze 1898, H. radiatus (Castracane 1886) Kuntze
1898, H. rectus (Castracane 1886) Kuntze 1898, H. ventri-
cosus (Castracane 1886) Kuntze 1898.
Fossil heterotypic synonyms: Hemidiscus barbadensis
(Greville 1861) Kuntze 1898, H. margaritaceus (Brun in
Brun & Tempre 1889) F.W. Mills 1934, H. simplicissimus
Hanna & Grant 1926.
Non Hemidiscus hardmaniana (Grunow 1865) Kuntze
1898 ( = Palmerina hardmaniana Hasle 1996), nec H.
niveus Hanna & Grant 1926 ( = Palmerina hardmaniana
Hasle), nec H. janischii (Grunow in Cleve & Mller 1879)
Kuntze 1898 [ = Leudugeria janischii (Grunow) Tempre
ex Van Heurck 1896], nec H. karstenii Jous 1962 ( = a

species of Cymatodiscus N.I. Hendey), nec H. ovalis
Lohman 1938 ( = a species of Cymatodiscus N.I. Hendey),
nec H. weissogii (Grunow in Van Heurck) Kuntze 1898
[ = Cymatotheca weissogii (Grunow in Van Heurck 1883)
Hendey 1958], nec H. triangularis (Jous 1977) Harwood
& Maruyama 1992 ( = Cosmiodiscus insignis Jous).
Actinocyclus kanayanus (Simonsen) F. Gmez, Lu Wang
& Senjie Lin, comb. nov.
Basionym: Hemidiscus kanayanus Simonsen (1972)
(Ver. Inst. Meeresf. Bremerhaven 13: 265, gs 16).
Supplemental data
Supplemental data for this article can be accessed at http://
dx.doi.org/10.1080/0269249X.2017.1313783
Funding
This work was supported by the Brazilian Conselho Nacional de
Desenvolvimento Cientco e Tecnolgico [grant numbers BJT
370646/201314 to F.G., and 402759/20125 and 311936/2013
0 to R.M.L.]; United States National Science Foundation [grant
number EF-0629624 to S.L.]; Chinese Visiting Scholarship
Council and the National Natural Science Foundation of China
[grant number #41330959 to L.W. and S.L.]. This is contribution
of MEL, Xiamen University.
ORCID
Fernando Gmez http://orcid.org/0000-0002-5886-3488
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