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Diatom Research, 2017

Vol. 32, No. 1, 21–28,

Molecular phylogeny suggests transfer of Hemidiscus into Actinocyclus (Coscinodiscales,

1 Carmen Campos Panisse 3, E-11500 Puerto de Santa María, Spain
2 Marine Biodiversity and Global Change Research Center and State Key Laboratory of Marine Environmental Science, Xiamen
University, Xiamen, People’s Republic of China
3 Department of Marine Sciences, University of Connecticut, Groton, CT, USA
4 Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Ciudad de México, México
5 Microsystems Laboratory, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
6 Laboratory of Plankton Systems, Oceanographic Institute, University of São Paulo, São 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 off 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 significant morphological differences 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- Palmeria, which Hasle (1995) subsequently transferred to
tonic species with distinctive, almost semi-circular, valves Palmerina Hasle because Palmeria Greville is a junior
and a widespread distribution, particularly in warmer homonym of Palmeria F. von Mueller. Besides the type,
oceans, although it can also be carried in warm cur- H. cuneiformis, other fossil and extant species have been
rents to temperate seas (Wallich 1860, Romero et al. placed in Hemidiscus (see Table S1). According to Simon-
2005). Schütt (1896) transferred H. cuneiformis into Euo- sen (1972) and Hasle & Syvertsen (1997) the genus encom-
dia Bailey ex Ralfs as Euodia cuneiformis (G.C. Wal- passes two extant species, the type, H. cuneiformis, and H.
lich) F. Schütt. The type species of Euodia, E. gibba kanayanus Simonsen, which differ in their areolation (size
(Bailey) Ralfs, as well as E. orbicularis Castracane, E. and arrangement).
recta Castracane and E. ventricosa Castracane are con- Hemidiscus has previously been placed within the fam-
sidered to be heterotypic synonyms of H. cuneiformis ily Euodieae F. Schütt (Cupp 1943) or within the Eupodis-
(Hustedt 1930). Euodia J.W. Bailey ex Ralfs is illegiti- caceae Kützing, together with Eupodiscus J.W. Bailey,
mate because it is a junior homonym of the plant genus, Roperia Grunow ex Pelletan, Actinocyclus Ehrenberg and
Euodia J.R. Forster & J.G.A. Forster. In addition to the Auliscus Ehrenberg, based on the similarity of their ocelli
almost semi-circular valve outline, the main diagnostic (Hendey 1974). Currently, H. cuneiformis is accepted as
character of Hemidiscus is the pseudonodulus; a marginal the type of its own family, Hemidiscaceae Hendey emend
to sub-marginal structure, with a single open hole or Simonsen ex Hasle. Simonsen (1975) placed genera with
circular area on each valve covered by smaller densely- a pseudonodulus (Hemidiscus, Actinocyclus and Roperia)
packed areolae (Simonsen 1975). Palmeria Greville is within the Hemidiscaceae. The genus Azpeitia M. Péra-
another genus with a semi-circular valve shape which gallo, which lacks a pseudonodulus, has also been placed
Mann (1907) transferred to Hemidiscus G.C. Wallich. within the Hemidiscaceae (Fryxell et al. 1986, Round et al.
However, Simonsen (1972) noted that these taxa lacked 1990).

*Corresponding author. E-mail:

(Received 27 October 2016; accepted 15 March 2017)

© 2017 The International Society for Diatom Research

22 Gómez et al.

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

& Chrétiennot-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-
flat and the shape was of a semicircle, resembling orange
age. Alignment columns with low posterior probability
segments (Figs 1–7). 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 difficult 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 inflation 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 40–180 μm and
imum likelihood in a PhyML web server (http://www.
the transapical axis 30–90 μm. Cells in culture were often 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 2–5, 7–10).
(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 flask. 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 modification in valve morphology before dying. Spore
frequencies was 0.004. The first 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 fine pattern of areolae, which radiated
irregularly from the centre of the valve (Figs 11–15). The

Figs 1–10. Light micrographs of Hemidiscus cuneiformis. Figs 1–7. Note the difference in size and shape. Figs 3–5, 7–10. Cells in
division. Figs 6–10. Cells with asymmetrical ellipsoidal shape. Scale bars = 20 μm.
24 Gómez et al.

valve face was flat, the mantle shallow and distinguish- Molecular phylogeny
able by its smaller areolae. The areolae formed irregular We obtained the SSU rDNA sequence of H. cuneiformis
radial fascicles, but short parallel lines towards the valve from direct and clone sequencing. A BLAST search was
centre. Areolae were ∼ 7 μm in diameter, with ∼ 10 are- conducted on the new sequence to find related sequences
olae in 10 μm on the valve face, while on the valve in GenBank. The closest sequence was the uncultured
mantle areolae were ∼ 5 μm in diameter, with ∼ 15 are- eukaryote clone SGYW751 (KJ760721), from the East
olae in 10 μm (Figs 16–17). A central hyaline area and Pacific Rise (20 m depth) with 98% sequence identity. The
rosette were absent. The single pseudonodulus seems to closest identified sequences were A. curvatulus (X85401,
open by a simple aperture (Fig. 18). Areolae are locu- 98% sequence identity) and A. actinochilus (AY485506,
late, opening externally through cribra sunk below the 98% sequence identity), followed by several unidenti-
valve surface; internally with round, rimmed foramina fied species of Actinocyclus, and more distantly, species
(Figs 19–23). Rimoportulae were confined to a row along of Actinoptychus. In the SSU rDNA phylogenetic tree,
the ventral margin. Internally they are shortly stalked auric- members of the Coscinodiscales were divided into two
ular structures, the slits more or less parallel to the valve clades, the Coscinodiscaceae (Coscinodiscus, Palmerina,

Figs 11–23. Scanning electron micrographs of Hemidiscus cuneiformis. Figs 11–15. The arrows indicate the pseudonodulus in the
ventral side. Figs 16, 17. Details of the areolae. Fig. 18. Detail of the pseudonodulus. Figs 19–23. Details of the wall structure revealed
through breaks. Scale bar = 10 μm (Figs 11–15) or = 1 μm (Figs 16–23).
Hemidiscus is part of Actinocyclus 25

Stellarima Hasle & P.A. Sims) and the Aulacodis- However, variability in the shape of the valve has led
caceae (Aulacodiscus Ehrenberg), and the Heliopeltaceae to the creation of many synonyms for this taxon (see
(Actinoptychus) and Hemidiscaceae (Actinocyclus and Table S1). Our cultures confirmed the plasticity of shape,
Hemidiscus). The Actinocyclus sequences branched into with valves ranging from semi-circular to asymmetrically
two strongly supported subclades. One subclade was com- elliptical (Figs 1–7).
posed of sequences of A. actinochilus, A. curvatulus and an Based on the molecular phylogeny, the Coscinodis-
unidentified Actinocyclus species that formed a sister clade cales is separated into two clades, one for Aulacodiscus,
with Hemidiscus. Three unidentified Actinocyclus species Coscinodiscus, Palmerina and Stellarima, and the other
formed the other subclade (Fig. 24). clade for Aulacodiscus, Actinoptychus, Actinocyclus and
Hemidiscus (Fig. 24). Actinoptychus and Aulacodiscus
Discussion were placed in the family Heliopeltaceae (Simonsen 1979),
Hemidiscus cuneiformis is characterised by its distinc- but the molecular data supports the placement of Aula-
tive semi-circular shape, which facilitates its identification. 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 pacifica and B. mediterranea were used as
outgroups. Scale bar = 0.05 substitutions per site.
26 Gómez et al.

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

Areola pattern Areolae Rimoportulae Annulus Mantle and pseudonodule

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

cuneiformis areolae all over neck hyaline high in margin
the valve opposite the
pseudonodule, low
in pseudonodule
Actinocyclus Radial Defined areolae all Truncate, no neck. – High
tinydrum over the valve. Labiate.
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-defined 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 new combinations are therefore proposed for the Hemidis-
the Heliopeltaceae (for Actinophychus), and the Hemidis- cus species.
caceae (Actinocyclus and Hemidiscus), characterised by
the presence of a pseudonodulus.
In our molecular phylogeny, the Hemidiscus sequence
New combinations
branched between two clades of Actinocyclus species
(Fig. 24). Based on this topology H. cuneiformis should be Actinocyclus cuneiformis (Wallich) F. Gómez, Lu Wang &
transferred into Actinocyclus. If Hemidiscus is retained as Senjie Lin, comb. nov.
an independent genus, the current placement of Hemidis- Basionym: Hemidiscus cuneiformis Wallich 1960 (Trans.
cus in the SSU rDNA phylogenetic tree (Fig. 24) makes Micr. Soc. London, ser. 2, 8: 42, pl. II, figs. 3, 4).
Actinocyclus paraphyletic. Thus, either Hemidiscus is part
of Actinocyclus, or Actinocyclus should be broken up into Homotypic synonym: Euodia cuneiformis (Wallich) A.
different genera. Table 1 compares the morphology of Mann 1893
Hemidiscus and species of Actinocyclus for which SSU Extant heterotypic synonyms: Hemidiscus inornatus (Cas-
rDNA sequences are available. The strains Actinocyclus tracane 1886) Kuntze 1898, H. orbicularis (Castracane
HK262, HK345H, HK346 and K347 have LM and/or SEM 1886) Kuntze 1898, H. radiatus (Castracane 1886) Kuntze
photo vouchers available from http://www.protistcentral. 1898, H. rectus (Castracane 1886) Kuntze 1898, H. ventri-
org/Project/get/project_id/79. There are no significant dif- cosus (Castracane 1886) Kuntze 1898.
ferences between the species of Actinocyclus and Hemidis-
cus. Hasle & Syvertsen (1997) reported valve shape as Fossil heterotypic synonyms: Hemidiscus barbadensis
semi-circular and circular/elliptical for Hemidiscus and (Greville 1861) Kuntze 1898, H. margaritaceus (Brun in
Actinocyclus, respectively, and thus a diagnostic char- Brun & Tempère 1889) F.W. Mills 1934, H. simplicissimus
acter for generic separation. Our observations reveal a Hanna & Grant 1926.
high morphological plasticity in the valve shape of H. Non Hemidiscus hardmaniana (Grunow 1865) Kuntze
cuneiformis (Figs 1–7, 11–15), yet valve shape is the only 1898 ( = Palmerina hardmaniana Hasle 1996), nec H.
differentiating character for Hemidiscus and Actinocyclus. niveus Hanna & Grant 1926 ( = Palmerina hardmaniana
Therefore, there is no morphological or molecular support Hasle), nec H. janischii (Grunow in Cleve & Möller 1879)
for retaining Hemidiscus as an independent genus. Since Kuntze 1898 [ = Leudugeria janischii (Grunow) Tempère
Hemidiscus is the later name, Actinocyclus has priority and ex Van Heurck 1896], nec H. karstenii Jousé 1962 ( = a
Hemidiscus is part of Actinocyclus 27

species of Cymatodiscus N.I. Hendey), nec H. ovalis F RYXELL G.A. & S EMINA H.J. 1981. Actinocyclus exiguus
Lohman 1938 ( = a species of Cymatodiscus N.I. Hendey), sp. nov. from the southern parts of the Indian and Atlantic
nec H. weissflogii (Grunow in Van Heurck) Kuntze 1898 Oceans. British Phycological Journal 16: 441–448.
[ = Cymatotheca weissflogii (Grunow in Van Heurck 1883) H AYASHI T., S AITO -K ATO M. & TANIMURA Y. 2012.
Hendey 1958], nec H. triangularis (Jousé 1977) Harwood Actinocyclus nipponicus sp. nov. and A. bradburyii sp. nov.
(Bacillariophyta) from Miocene lacustrine sediments of the
& Maruyama 1992 ( = Cosmiodiscus insignis Jousé).
proto-Japan Sea. Phycologia 51: 98–112.
H ASLE G.R. 1977. Morphology and taxonomy of Actinocyclus
Actinocyclus kanayanus (Simonsen) F. Gómez, Lu Wang normanii f. subsalsa (Bacillariophyceae). Phycologia 16:
& Senjie Lin, comb. nov. 321–328.
Basionym: Hemidiscus kanayanus Simonsen (1972) H ASLE G.R. 1995. Nomenclatural notes on Palmerina nom.
nov.: Pseudo-nitzschia turgiduloides sp. nov. Diatom
(Veröff. Inst. Meeresf. Bremerhaven 13: 265, figs 1–6).
Research 10: 357–358.
H ASLE G.R. & S YVERTSEN E.E. 1997. Marine diatoms. In:
Supplemental data Identifying marine phytoplankton (Ed. by C.R. T OMAS), pp.
Supplemental data for this article can be accessed at http:// 5–385. Academic Press, San Diego, CA. H ENDEY N.I. 1974. A revised check-list of the British marine
diatoms. Journal of the Marine Biological Association of the
United Kingdom 54: 277–300.
Funding H USTEDT F. 1930. Die Kieselalgen Deutschlands, Österre-
This work was supported by the Brazilian Conselho Nacional de ichs und der Schweiz. In: Kryptogamen-Flora (Ed. by L.
Desenvolvimento Científico e Tecnológico [grant numbers BJT R ABENHORST), pp. 1–920. Band VII, Teil 1. Akademische
370646/2013–14 to F.G., and 402759/2012–5 and 311936/2013–
Verlagsgesellschaft, Leipzig.
0 to R.M.L.]; United States National Science Foundation [grant
number EF-0629624 to S.L.]; Chinese Visiting Scholarship M ANN A. 1907. Report on the diatoms of the Albatross
Council and the National Natural Science Foundation of China Voyages in the Pacific Ocean, 1888–1904. Contributions
[grant number #41330959 to L.W. and S.L.]. This is contribution from the United States National Herbarium 10: 221–
of MEL, Xiamen University. 419.
BROCK U. 1996. Evolution of the diatoms (Bacillariophyta).
Fernando Gómez II. Nuclear-encoded small-subunit rRNA sequence compar-
isons confirm a paraphyletic origin for the centric diatoms.
Molecular Biology and Evolution 13: 67–75.
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