Eur. J. Phycol.

(2015), 114

A new denition of Adenoides eludens, an unusual marine

sand-dwelling dinoagellate without cingulum, and
Pseudadenoides kofoidii gen. & comb. nov. for the species
formerly known as Adenoides eludens

FERNANDO GMEZ1,3, RYO ONUMA2, LUIS F. ARTIGAS3 AND TAKEO HORIGUCHI4

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

Laboratory of Plankton Systems, sala 100, Oceanographic Institute, University of So Paulo, Praa do Oceanogrco 191,
Cidade Universitria, Butant, So Paulo 05508-900, Brazil
2
Department of Natural History Sciences, Graduate School of Science, Hokkaido University, North 10, West 8, 060-0810
Sapporo, Japan
3
Laboratoire dOcanologie et Gosciences, CNRS UMR 8187, Universit du Littoral Cte dOpale, MREN ULCO, 32 av.
Foch, 62930 Wimereux, France
4
Department of Natural History Sciences, Faculty of Science, Hokkaido University, North 10, West 8, 060-0810 Sapporo,
Japan
(Received 9 Jul 2014; revised 24 Sep 2014; accepted 8 Nov 2014)
The species Amphidinium eludens, as described by Herdman (1922; Proc. Trans. Liverpool Biol. Soc. 36: 18) based on her
drawing in g. 1, has been investigated for the rst time by scanning electron microscopy and phylogenetic analysis. The
morphological and molecular data reveal that this species is distantly related to other known dinoagellates. Balech [1956, Rev.
Algol., n. ser. 2(12): 30] cited Amphidinium eludens Herdman (1922, g. 1) as the basionym of the type of Adenoides, while he
described and illustrated Amphidinium kofoidii Herdman (1922, g. 2) as Adenoides eludens. As the nomenclatural rules do not
allow the change of basionym, we have re-dened the genus Adenoides based on the characteristics of Amphidinium eludens
Herdman (1922, g. 1). The thecal plate formula of Adenoides eludens is Po, 5, 6, 0c, 3+s, 5, 3p, 1. This species lacks a
cingulum. The apical pore complex resembles that of peridinioid dinoagellates, while the absence of a cingular groove is
reminiscent of desmokont prorocentroids. We also propose Pseudadenoides kofoidii gen. & comb. nov. based on Herdmans 1922
g. 2 of Amphidinium kofoidii which was described by Balech in 1956 and re-named Adenoides eludens.
Key words: Adenoides kofoidii, Dinophyta, microphytobenthos, molecular phylogeny, new genus, psammophilic Dinophyceae

INTRODUCTION
Dinoagellates are unicellular organisms with two
dimorphic agella. Based on the arrangement of the
agella, the dinokonts (= two agella in grooves) are
dinoagellate cells in which two agella are inserted
ventrally; one agellum is transverse, wrapped around
the cell, and housed in a groove called the cingulum or
girdle (equatorial or transverse) and the other one is a
trailing longitudinal agellum and housed in a sulcus
(longitudinal). The other type, desmokonts (= two
anterior agella) are cells in which two dissimilar
agella emerge from the anterior part of the cell.
They have two leading agella inserted apically,
rather than ventrally. One agellum extends forward
and the other circles its base, and there are no agellar
grooves (cingulum or sulcus). The best known
Correspondence to: Fernando Gmez. (email:
fernando.gomez@toplancton.com)

example of desmokonts is the genus Prorocentrum

C.G. Ehrenberg. However, not all dinoagellates t
into this division. The descriptions of new benthic
species have largely increased in recent years
(Gmez, 2012). The presence of an incomplete cingulum is reported in some sand-dwelling dinokonts
(Amphidiniopsis J. Woloszynska, Cabra Sh. Murray &
D.J. Patterson, Herdmania J.D. Dodge, Rhinodinium
Sh. Murray, Hoppenrath, S. Yoshimatsu, S. Toriumi &
J. Larsen) (Murray & Patterson, 2004; Yamaguchi
et al., 2011). Planktonic dinoagellates such as
Podolampas F. Stein lack a groove (cingulum) to
harbour a transversal agellum that encircles the cell
(Gmez et al., 2010). However, the absence of a cingulum has never been reported in benthic dinokonts.
In one of the earlier studies of sand-dwelling dinoagellates, Herdman (1922) described the species
Amphidinium eludens and A. kofoidii. In her g. 1
she described Amphidinium eludens as oval to ovoid

ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/15/000001-14 2015 British Phycological Society

http://dx.doi.org/10.1080/09670262.2015.1009174

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

F. Gmez et al.
in shape, has a barely visible episome and a hump in
the sulcal area (Herdman, 1922, p. 22, g. 1). In her
g. 2 she described Amphidinium kofoidii as round to
squarish in shape, with a more visible button-like
episome (Herdman, 1922, p. 26, g. 2). Balech
(1956) proposed the new thecate genus Adenoides
based on Herdmans g. 2 of Amphidinium kofoidii.
However, Balech (1956) proposed as basionym
Amphidinium eludens Herdman (1922, g. 1). Later,
Dodge (1982) proposed Adenoides kofoidii for
Amphidinium kofoidii Herdman 1922, g. 2. The taxonomic and nomenclatural history of Adenoides eludens is reported in Hoppenrath et al. (2003).
Adenoides eludens based on Balechs description and
as described by Herdmans g. 2 of Amphidinium
kofoidii was investigated again by Dodge & Lewis
(1986) and Hoppenrath et al. (2003), and molecular
data have been available since Saldarriaga et al.
(2001). To date, there are 45 sequences of different
molecular markers of Adenoides eludens and another
39 sequences labelled as two uncultured Adenoides.
The sequences of Adenoides eludens available in
GenBank are from the cultures CCCM 683 and
CCMP 1891 isolated from the Pacic coasts of
Canada or eld material collected from the same location. Another culture CCMP 2081 was isolated from
Europe. However, there are no molecular data for
Adenoides eludens from the European Atlantic coast
where the species was described. While Adenoides
eludens (= Amphidinium kofoidii) has been subjected
to morphological and molecular studies, little is known
about the species described as Amphidinium eludens
Herdman (1922, g. 1). This organism is one of the
rst described sand-dwelling dinoagellates and
according to Herdman (1922) is responsible for
discolourations in the sands of the European Atlantic
coasts. However, the detailed morphology of this
species remains unknown.
In this study, we provide light microscopy pictures,
the rst scanning electron microscopy pictures and the
rst molecular data for Amphidinium eludens
Herdman (1922, g. 1), and the rst molecular data
for Amphidinium kofoidii Herdman (1922, g. 2) from
the French coasts of the English Channel, where
Balech (1956) described the genus Adenoides. The
morphological and molecular data support the conclusion that the two species described in gs 1 and 2 by
Herdman (1922) belong to independent genera.
Amphidinium eludens is an unusual dinoagellate,
characterized by the lack of a cingular groove similar
to desmokont dinoagellates.

MATERIALS AND METHODS

Source, isolation and microscopy observations
This study was undertaken in the soft sandy sediments of the
shore of Wimereux, France (504612N, 13642E)

2
collected during low tide in June, 2012. Two sites on the
beach in front of the LOG laboratory (MREN ULCO and
Marine Station of Wimereux UL1; Gmez & Artigas, 2014)
were sampled: the moist sands around the border of a large
pool (~50 m diameter, ~1 m depth), and several smaller pools
and moist sands showing a faint brownish discolouration.
The upper centimetre of sand was collected with a spoon
and deposited into a bottle containing seawater collected at
the same location. Then, the sand with seawater was stirred
vigorously and the suspension settled in a composite settling
chamber. The settled material was examined with an inverted
microscope (Nikon Eclipse TE2000-S, Tokyo) and photographed with a Nikon Digital Sight DS-2M camera. The cell
size, described as length (apical to antapical axis) and depth,
i.e. the length along the lateral sides (ventral to dorsal
distance), was measured in 25 specimens. The width
(transdiameter) was measured in ve specimens.
For scanning electron microscopy, the sand samples with
seawater were stirred vigorously, and the suspension was
xed with glutaraldehyde (5%) and ltered onto a 0.8 m
pore size Whatman Nuclepore membrane lter, washed
with distilled water, xed with osmium tetroxide, dehydrated
with a graded series of ethanol and critical point dried with
CO2. Filters were mounted on stubs, sputter-coated with gold
and viewed using a Hitachi S4800 scanning electron microscope. Images were presented on a black background using
Adobe Photoshop CS3.

PCR amplication and DNA sequencing

For molecular analysis, each specimen of Amphidinium eludens and A. kofoidii was micropipetted individually with a
ne capillary into a clean chamber and washed several times
in serial drops of 0.2 m ltered and sterilized seawater.
Finally, 15 specimens of each species were deposited in a
0.2 ml Eppendorf tube lled with several drops of absolute
ethanol. The sample was kept at room temperature and in
darkness until the molecular analysis could be performed.
Prior to DNA extraction, the 0.2 ml Eppendorf tubes were
centrifuged for 10 min at 14462 g in a TOMY MX-201
centrifuge (Tokyo, Japan). Ethanol was then evaporated in a
vacuum desiccator. Cells were resuspended in 10 l of
QuickExtract DNA extraction solution (Epicenter,
Madison) and incubated at 56C for 1 h and 98C for 2 min
in a thermal cycler (GeneAmp PCR System 9700, Applied
Biosystems, Foster City). The product was used as DNA
template for the following polymerase chain reaction
(PCR). In the rst round of PCR, to obtain almost complete
SSU rDNA and partial LSU rDNA sequences, two sets of
primers (SR1-SR12b for SSU rDNA and D1RF1 28-1483R
for LSU rDNA, respectively) were simultaneously applied.
In the second round of PCR, 0.5 l of the rst PCR products
was used as DNA template, and three pairs of primers (SR1SR5, SR4-SR9p and SR8-SR12b; see Yamaguchi
et al., 2006) were used for SSU rDNA amplication and
two pairs of primers (D1RF-25R1 and D3A-28-1483R; see
Daugbjerg et al., 2000) were applied for LSU rDNA amplication. PCR conditions for both rounds of amplication
consisted of one initial cycle of denaturation at 94C for 5
min, followed by 35 cycles of denaturation at 94C for 30 s,
annealing at 55C for 30 s, and extension at 72C for 30 s.
The PCR process was completed by a nal extension cycle

Pseudadenoides gen. & comb. nov. and Adenoides

at 72C for 7 min. The PCR products were directly
sequenced using the ABI PRISM BigDye Terminator
Cycle Sequencing Kit (Applied Biosystems) and a DNA
autosequencer ABI PRISM3100 Genetic Analyzer
(Applied Biosystems). Both forward and reverse strands
were sequenced.

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

Phylogenetic analyses
Both SSU rDNA and LSU rDNA sequences were aligned
based on the secondary structure of the rDNA molecule
(database no longer available), and the alignments were
rened manually. We used sequences from GenBank of
Amoebophrya spp. as outgroups for SSU rDNA and a ciliate
Tetrahymena pyriformis (C.G. Ehrenberg) Lwoff and an apicomplexa Eimeria tenella (Raillet & Lucet) Fantham were
used for outgroups of LSU rDNA analyses, respectively. The
accession numbers of sequences included in the alignment
are indicated in each tree. The aligned sequences were examined using maximum likelihood (ML) analyses with PAUP
version 4.0b10 (Swofford, 2003), and Bayesian analysis with
MrBayes 3.2.1 (Huelsenbeck & Ronquist, 2001). The program Modeltest version 3.04 (Posada & Crandall, 1998),
which employs the hierarchical likelihood ratio test (hLRT)
was used to explore the best ML sequence evolution model
for the dataset. The hRLT model selected for ML analysis of
the dataset was TrN+I+G. In the ML analysis, a heuristic
search was performed with a TBR branch-swapping algorithm, and the starting tree was obtained by the neighborjoining (NJ) method. The parameters in this analysis were:
assumed nucleotide frequencies A = 0.2756, C = 0.1898, G =
0.2392 and T = 0.2954; substitution rate matrix with A<->C
= 1.0000, A<->G = 3.5126, A<->T = 1.0000, C<->G =
1.0000, C<->T = 7.7026 and G<->T = 1.0000; proportion
of sites assumed to follow a gamma distribution with shape
parameter = 0.5633; and number of rate categories = 4.
Bootstrap analysis for ML was calculated for 100 pseudoreplicates. For LSU rDNA analysis, the hLRT model selected
for ML analysis of the dataset was TrN+I+G. In the ML
analysis, a heuristic search was performed with a TBR
branch-swapping algorithm, and the starting tree was
obtained by the NJ method. The parameters in these analyses
were: assumed nucleotide frequencies A = 0.2865, C =
0.1806, G = 0.2701 and T = 0.2628; substitution rate matrix
with A<->C = 1.0000, A<->G = 2.8299, A<->T = 1.0000,
C<->G = 1.0000, C<->T = 6.6145 and G<->T = 1.0000;
proportion of sites assumed to follow a gamma distribution
with shape parameter = 0.6675; and number of rate categories
= 4. Bootstrap analysis for ML was calculated for 100
pseudo-replicates. For Bayesian analysis, GTR+I+G model
was selected by MrModeltest 2.2 (Nylander et al., 2004) as a
suitable evolutionary model. Markov chain Monte Carlo
iterations were carried out until 3 000 000 generations
were attained for SSU rDNA phylogeny, while 5 500
000 generations were required for LSU rDNA phylogeny,
when the average standard deviations of split frequencies
fell below 0.01, indicating a convergence of
the iterations. Our sequences were deposited in DDBJ/
EMBL/GenBank under accession numbers LC002839LC002848.

3
RESULTS
Based on the morphological and molecular data (see
below) the species described as Amphidinium eludens
Herdman (1922, g. 1) and Amphidinium kofoidii
Herdman (1922, g. 2) belong to separate genera. If
we had proposed a new genus name based on
Herdman (1922, g. 1), it would have been illegitimate as a superuous later homotypic synonym of
Adenoides [International Code of Nomenclature for
algae, fungi, and plants (I.C.N.), art. 52.1, McNeill
et al., 2012]. As dened by Balech (1956), the basionym of the type of Adenoides, Adenoides eludens, is
Amphidinium eludens Herdman (1922, g. 1).
According to article 7.3 of I.C.N.: A new combination or a name at new rank (Art. 6.10) is typied by the
type of the basionym even though it may have been
applied erroneously to a taxon now considered not to
include the type (but see Art. 48.1). The characteristics of the genus Adenoides are dened by
Amphidinium eludens Herdman (1922, g. 1), independently of the fact that Balech (1956) provided for
Adenoides eludens the description and illustrations of
the species described as Amphidinium kofoidii by
Herdman (1922, g. 2). Hereafter, we redened the
genus Adenoides based on the morphology of
Amphidinium eludens Herdman (1922, g. 1).
Taxonomic descriptions
Adenoides Balech emended F. Gmez, R. Onuma,
Artigas & Horiguchi
DIAGNOSIS: Armoured cell laterally compressed,
lacking the cingulum and agella inserted ventrally.
Thecal plate formula Po, 5, 6, 0c, 3+s, 5, 3p, 1.
An alternative interpretation is Po, cp, 4, 7, 1c, 1+s,
5, x, 2p, 1.
TYPE SPECIES: Adenoides eludens (Herdman) Balech.
BASIONYM: Amphidinium eludens Herdman 1922,
p. 22, g. 1.
SYNONYM: Adenoides kofoidii sensu Dodge 1982.
Adenoides eludens F. Gmez, R. Onuma, Artigas &
Horiguchi epitype
Given the lack of type material, we have designated
g. 1 in Herdman (1922) as the lectotype for this
species and the cell shown in Fig. 23 of this study as
the epitype under Article 9.7 of the I.C.N. (McNeill
et al., 2012). A detailed description of the epitype is
presented below.
EPITYPE: Fig. 23. A SEM stub was deposited in the
herbarium of the Faculty of Science, Hokkaido
University as SAP114711.

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

F. Gmez et al.
The slightly laterally attened cells are ellipsoidal
in lateral view. The sulcal area is depressed and lies on
the anterior third of the cell, neither extending onto the
epitheca nor reaching the antapex. In lateral view, the
ventral contour of the cell showed a hump (Figs 2, 6).
The cells were 30 2 (2737) m long, 23 2 (1827)
m deep, and 1317 m wide (Figs 115). There is an
intergradation of the size with large and small specimens co-existing in the same sample (Fig. 6). The
epitheca is directly connected to the hypotheca, without any cingulum. We presume that the vestigial cingular groove was placed in the suture between the preand postcingular plates that is at the level of the
agellar insertion.
The height of the epitheca is about one-third the
length of the cell. Some specimens showed a large
pusule in the anterior dorsal part of the cell (Figs 14).
The transversal agellum encircled the cell (Fig. 5).
The cell possesses yellow-brown plastids. The species
Amphidinium kofoidii sensu Herdman is more
pigmented and brownish when compared with
Adenoides eludens (Fig. 7). Some specimens of
Adenoides were found devoid of pigments and with
numerous granules (Figs 810). The cell showed two
pyrenoids with a starch sheath, each pyrenoid placed
in the sides of the mid anterior hypotheca (Figs 6, 11).
The nucleus is oval and situated in the posterior
region, although hardly visible because it is hidden
by plastids. Some specimens experienced ecdysis or
exuviation, releasing empty thecae (Figs 1112).
The empty theca was usually divided into two parts,
which are considered the epitheca and hypotheca
(Figs 1315). One plate that is interpreted as a sulcal
anterior plate is attached to the epitheca (Fig. 13).
The tabulation is illustrated in detail in Figs 1646.
The thecal plate pattern is an apical pore plate (Po),
ve apical plates (5), six precingular plates (6), no
cingular plate (0c), at least three sulcal plates (3+s),
ve postcingular plates (5), three posterior intercalary plates (3p) and one antapical plate (1). The cell
surface is smooth with round pores (approximately
0.15 m). The pores are evenly distributed in the
plates, with a trend to form rows near the sutures
(Fig. 19). There is an accumulation of pores in the
posterior end of the rst and second posterior intercalary plates (1p, 2p) and in the dorsal end of the
antapical plate (Figs 16, 4041). The pores are absent
in the sulcal plates (Figs 22, 2730, 34). The boundary
of the plates shows a thick (~1 m wide) smooth and
depressed margin. These intercalary bands were more
conspicuous in the hypotheca (Figs 19, 21).
The apical pore plate showed a pentagonal to round
shape being bordered by ve apical plates
(Figs 24, 37). The junction of the apical pore plate
and the apical series formed a ridge, except in the rst
apical plate suture which is shorter than in the other
apical plates (Figs 24, 25, 37). The apical pore plate
contains a row of marginal pores (1013 pores) and a

4
round central cover plate (Figs 26, 37). From the
central cover plate emerged a rim that protruded and
extended towards the anterior part of the rst apical
plate (Figs 24, 26, 37).
There are ve apical plates (Figs 20, 23, 37). Plate
3 is the largest and is located in the dorsal side of the
epitheca. Plates 2 and 5 are intermediate in size.
Plates 4 and 1 are the smallest of the apical series.
The apical plates 2 to 5 showed a curved ridge in the
suture with the apical pore plate. Plate 1 did not show
a ridge in the junction with the apical pore plate. Plate
1 is a six-sided irregular smooth-surfaced polygon,
with very few pores when compared with the other
apical plates (Figs 2426). An alternative interpretation of the apical tabulation is to consider plate 1 as a
wide canal plate (cp). Plates 2 and 4 are pentagonal,
especially plate 4 which is a quasi-regular pentagon.
Plates 3 and 5 are six-sided. Plate 5 shows a distinctive undulating ange in the sutures, except in the
anterior end in the suture with plates 5 and 6
(Figs 2526, 37).
At the level of the precingular plate series, there are
six precingular plates and a large smooth-surfaced and
concave plate. This plate is joined to the precingular
plates when the epitheca is separated from the
hypotheca (Fig. 13). Based on the proximity to the
agellar pores, the differences in the shape (concave)
and the absence of ornamentation (smooth-surfaced
lacking pores and intercalary bands) as in the other
sulcal plates, we have considered it as the right anterior
sulcal plate (S.d.a.) (Figs 2122, 34). An alternative
interpretation is to consider this large plate as plate 1.
Following the interpretation as an anterior sulcal plate,
the rst precingular plate (1) is displaced to the left
side in the ventral view. Plates 1 and 5 are the
biggest of the epitheca, plates 2, 3 and 4 are intermediate in size and plate 6 is the smallest of the
precingular series (Figs 1620, 3536). Plate 1 is
polygonal and curved in the suture with the anterior
sulcal plate (Figs 2123). Plates 2, 4 and 5 are
six-sided and plates 3 and 6 are quadrangular
(Figs 1620). Plate 3 is nearly square-shaped
(Fig. 20) and plate 6 is an elongated rectangle oriented
along the antero-posterior axis (Figs 28, 3435).
The cell does not have a cingulum. However,
when the theca is empty, it tended to separate
along the suture between the precingular and postcingular plate series (Figs 1315). This suggests that
this thick suture could be occupying the space of the
cingulum of a hypothetical ancestor. The observations of live specimens revealed that the transversal
agellum encircles this area (Fig. 5). The cell shows
a depression in the sulcal area (Figs 2122, 3235).
The left anterior sulcal (S.s.a.) plate is smooth
and located between the right anterior sulcal plate
(S.d.a.) and a large plate 1. This S.s.a. plate is
elongated and concave and it could be alternatively
interpreted as a rst cingular plate (Figs 2123, 34).

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

Pseudadenoides gen. & comb. nov. and Adenoides

Figs 115. Light microscopy pictures of live specimens and empty thecae of the redened Adenoides eludens (Fig. 7 also includes
Amphidinium kofoidii). Figs 14. Different views of the same specimen. Fig. 5. Note the transversal agellum that encircles the cell.
Fig. 6. Note the different sizes. Fig. 7. Note the different pigmentation between Adenoides eludens (yellowish, on the left) and
Amphidinium kofoidii (brownish, on the right). Figs 810. Specimens lacking pigmentation. Figs 1112. Ecdysis or exuviation from
the theca. Figs 1315. Empty theca with separated epitheca and hypotheca. Fig. 13. Note that the sulcal right (= dexter) anterior plate
(S.d.a.) is attached to the epitheca. LF = longitudinal agellum. TF = transversal agellum. Scale bar = 10 m.

There are two agellar pores in the depressed area of

the sulcus. The longitudinal agellum is inserted in
a pore located in the right side of the sulcus,
adjacent to the suture of plates 6 and 5 (Figs 22,
2730, 34). The right half of this pore is surrounded
by a prominent ridge (Figs 2829). The pore of the
transversal agellum is located in the middle of the
sulcus. It is bordered by a rectangular structure

(Figs 2830). Below the pores, a large sulcal posterior plate with a rounded posterior end is subdivided
into three plates connected by wedge-shaped contours (Figs 2930). The right anterior sulcal (S.d.a.)
plate is in contact with the agellar pores (Fig. 22).
This plate shows an anterior-posterior oriented line,
which suggests the presence of a subdivision of the
plate (Fig. 30).

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

F. Gmez et al.

Figs 1630. Scanning electron micrographs of the re-dened Adenoides eludens. Figs 1617. Right lateral view. Fig. 18. Ventral view.
Fig. 19. Left lateral view. Fig. 20. Dorsal view. Figs 2123. Ventral lateral view. Figs 2425. Detail of the apical plates. Fig. 26. Detail of
the apical pore plate. Figs 2730. Detail of the sulcal plates. Fig. 30. The arrowheads point out tentative sutures in the sulcal plates. S.d.a. =
Sulcal dexter (= right) anterior plate. S.s.a. = Sulcal sinister (= left) anterior plate. S.p. = Sulcal posterior plate. LFP = longitudinal agellar
pore. TFP = transversal agellar pore. Scale bar = 10 m, except Figs 22, 2430 where scale bar = 1 m.

There are ve postcingular plates (Figs 16, 19).

Plate 1 is the longest plate of the cell and extended
for 2/3 the height of the hypotheca. The anterior part

of this ve-sided plate is curved and overhangs at

the anterior end (Figs 18, 22, 34). Plates 2 and 4
are large and broad, plate 3 is intermediate in size

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

Pseudadenoides gen. & comb. nov. and Adenoides

Figs 3141. Scanning electron micrographs of a single specimen of Adenoides eludens. Fig. 31. Ventral view. Figs 3233. Right
lateral view. Fig. 34. Detail of the sulcal area. Fig. 35. Latero-ventral view. Figs 3637. Detail of the epitheca. Figs 3839. Dorsal
view. Figs 4041. Antapical view. Fig. 40. The inset shows the pores of the plates 2p (up) and 1 (down). S.d.a. = Sulcal dexter
(= right) anterior plate. S.s.a. = Sulcal sinister (= left) anterior plate. S.p. = Sulcal posterior plate. Scale bar = 20 m, except Figs 34
and 37 where scale bar = 1 m.

and plate 5 is the smallest of the postcingular

series. Plate 2 is four sided, plate 3 is a regular
pentagon and plate 4 is ve-sided and elongated
posteriorly. Plate 5 is smaller and more anteriorly
placed than the other plates of the postcingular series (Figs 3435). An alternative interpretation is that
plate 5 is equivalent to plate x reported in some
sand-dwelling dinoagellates with incomplete
cingulum.
Four large plates that do not belong to the sulcal and
postcingular series are considered posterior intercalary and antapical plates. Three plates are considered

posterior intercalary plates and the plate situated at the

bottom of the cell is considered the antapical plate 1
(Figs 16, 19). The antapical plate is bordered by plate
1 and three posterior intercalary plates (1p, 2p, 3p).
The rst and second posterior intercalary plates
(1p, 2p) are large and with the shape of an oblique
irregular pentagon (Figs 16, 19). Plates 1p and 2p
have an accumulation of pores in the posterior end,
at the contact with the antapical plate (Figs 3941).
Plate 3p is an elongated pentagon oriented in the
longitudinal axis (Figs 3233). The antapical plate is
hexagonal and it showed, at the dorsal end, the special

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

F. Gmez et al.

Figs 4246. Line drawings of the re-dened Adenoides eludens with the Kofoid system of tabulation. Fig. 42. Right lateral. Fig. 43.
Left lateral. Fig. 44. Apical. Fig. 45. Antapical. Fig. 46. Ventral.

Figs 4756. Light microscopy pictures of live specimens (4750) and empty thecae (5156) of Pseudadenoides kofoidii gen. &
comb. nov. Fig. 47. The arrows point the transversal agella encircling the cingulum. Fig. 50. Megacytic cell. Figs 5156. Empty
thecae. Scale bar = 10 m.

pore eld found in plates 1p and 2p (Fig. 41). An

alternative interpretation of the tabulation of the
hypotheca may appear when plate 5 is considered
to be an x plate (Fig. 48), then plate 3p is considered to
be 5 plate.
A new genus name for Amphidinium kofoidii Herdman
(1922, g. 2)

We had to redene the genus Adenoides due to the

designation of Amphidinium eludens Herdman (1922,
g. 1) as basionym of the type of Adenoides.
Morphological and molecular data reveal that the
species Amphidinium kofoidii Herdman (1922, g. 2)
does not belong to the new denition of the genus
Adenoides or to the other known dinoagellate genus.
Therefore, a new genus name is proposed here. The

Pseudadenoides gen. & comb. nov. and Adenoides

description of the genus and its type species is here

simplied because the morphological characteristics
have been already reported from the English Channel
and North Sea (Balech, 1956; Dodge & Lewis, 1986;
Hoppenrath et al., 2003). For that reason, we have
summarized the description which is similar to that
found, with more detail, in Hoppenrath et al. (2003).

central cover plate. From the central cover plate

emerged a narrow rim that protruded and extended
ventrally towards the anterior part of the rst apical
plate (Fig. 62). The apical plates 1 and 4 are in
contact with the sulcus (Fig. 62). Plate 2 is very
narrow and 4 is relatively large, covering nearly the
entire right half of the epitheca. There are no precingular plates. The shallow cingulum consists of six
plates and there are four small sulcal plates surrounding the agellar pore (s.a., s.s., s.p., s.d.) (Figs 5960).
The hypotheca consists of 11 plates, comprised of ve
postcingular, ve posterior intercalary plates and one
antapical plate. Plates 1 and 2 lie at the left lateral
cell side in the upper fourth of the hypotheca. The
small, pentagonal plate 3 lies dorsally, and the pentagonal, posteriorly pointed plate 4 is relatively
large, lying at the right lateral side. The six-sided
plate 5 is in contact with the sulcus. All ve posterior
intercalary plates are large and cover most of the
hypotheca (Figs 5759). The plates 1p and 5p are in
contact with the sulcus and bordered its posterior
margin. Both are in contact with each other and form
a ventral suture (Figs 5960).

Pseudadenoides F. Gmez, R. Onuma, Artigas &

Horiguchi gen. nov.

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

DIAGNOSIS: Armoured cell laterally compressed, with a

minute epitheca. The plate formula is Po, 4, 0, 6c, 4s,
5, 5p, 1 (or alternatively it can be interpreted as Po
4, 0, 6c, 5s, 5, 3p, 2).
SYNONYM: Adenoides sensu Balech 1956.
ETYMOLOGY: Pseudo-, pseud- (before vowels),
Ancient Greek (pseudes): false, not genuine,
fake. The type species has been confused with
Adenoides eludens. The gender is feminine (art. 62.4
of I.C.N.).
TYPE SPECIES: Pseudadenoides kofoidii (Herdman) F.
Gmez, R. Onuma, Artigas & Horiguchi comb. nov.
BASIONYM: Amphidinium kofoidii Herdman (1922,
p. 26, g. 2)
SYNONYM: Adenoides eludens sensu Balech 1956
EPITYPE: Fig. 60
Pseudadenoides kofoidii (Herdman) F. Gmez, R.
Onuma, Artigas & Horiguchi comb. nov.
Cells are asymmetrical, round to squarish, slightly attened laterally, 2837 m long and 2129 m deep. The
minute epitheca is cup-shaped, depressed and scarcely
visible (Figs 4750). The cingulum lies almost at the
anterior end of the cell, completely encircling the
epitheca and meeting without displacement. The sulcal
area lies on the anterior third of the cell, neither extending onto the epitheca nor reaching the antapex, and is
slightly depressed. The transverse agellum completely
encircles the cell at the cingulum level (Fig. 47). Most of
the cells show one large pusule in the anterior
hypotheca. The cell is full of brown plastids. There are
two conspicuous pyrenoids. The nucleus is oval and
situated in the posterior region, although hardly visible
because it is hidden by plastids (Fig. 4750).
The thecal plate pattern is an apical pore plate (Po),
four apical plates (4), without precingular plates (0),
six cingular plates (6c), four sulcal plates (4s),
ve postcingular plates (5), ve posterior intercalary
plates (5p) and one antapical plate (1) (Figs 5162).
The apical pore plate is an angular square-shape
bordered by four apical plates which form a ridge
running around it (Fig. 60). The apical pore plate
contains few marginal pores (~7 pores) and a round

Molecular phylogeny
We have sequenced specimens of the species
Amphidinium eludens Herdman (1922, p. 22, g. 1,
now Adenoides eludens emended), and the species
Amphidinium kofoidii Herdman (1922, p. 26, g. 2,
now Pseudadenoides kofoidii gen. & comb. nov.). The
SSU- and LSU rDNA sequences of Amphidinium
kofoidii Herdman from specimens of the Pacic
Ocean are available in GenBank (retrieved as
Adenoides eludens). We have also provided the rst
SSU- and LSU rDNA sequences from specimens of
the European Atlantic coasts, where Amphidinium
kofoidii was described. The aligned length for SSUrDNA was 1725 bp and 954 bp for LSU rDNA (D1
D3 region excluding the variable region). The
sequences of the Atlantic and Pacic specimens of
Amphidinium kofoidii were identical (Figs 63, 64).
We provided the rst SSU- and LSU rDNA sequences
of Amphidinium eludens. All the sequences obtained
from different samples were identical (Figs 63, 64).
The species Amphidinium eludens and Amphidinium
kofoidii described in Herdman (1922) are distantly
related in the SSU rDNA (Fig. 63) and LSU rDNA
trees (Fig. 64). Our data strongly support the splitting
of these two species into two distinct genera based on
the considerable evolutionary distance of their respective SSU- and LSU rDNA sequences. Because of the
low resolution of basal positions of the phylogenetic
trees, it is also difcult to establish the accurate relationships between these genera with other dinoagellates (Figs 63, 64).

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

F. Gmez et al.

10

Figs 5762. Scanning electron micrographs of Pseudadenoides kofoidii gen. & comb. nov. Figs 5758. Right lateral view. Fig. 59.
Ventral view. Fig. 60. Apical view. Fig. 61. Dorsal view. Fig. 62. Detail of the pore and apical plates. Scale bar = 20 m, except
Fig. 62 where scale bar = 1 m.

DISCUSSION
In pioneering studies of sand-dwelling dinoagellates, Herdman (1922) described in her g. 1
Amphidinium eludens as forming discolourations in
a beach of the British Isles. Despite the tradition of
taxonomic studies in the European Atlantic coasts
and the relative abundance of A. eludens, to date that
species has remained under investigation. Confusion
has been present since the original description.
Herdman (1922) illustrated Amphidinium eludens
as slightly larger than A. kofoidii in line drawings
lacking any reference to the size and reported a
length of 70 m and 5080 m for A. eludens and
A. kofoidii, respectively. However, in a note at the
end of a subsequent publication (Herdman, 1923, p.
63) she explained that the size measurements were
overestimated and should be 30 m and 2540 m
for Amphidinium eludens and A. kofoidii, respectively. Consequently, the cells of Amphidinium kofoidii tend to be larger than those of A. eludens. This

contrasts with the relative sizes of Herdmans original gures. In our observations, we found an average length of 30 m for Amphidinium eludens,
which is usually slightly less than for A. kofoidii.
The error in the relative size of the original description could have contributed to further confusion in
the identication of both species. When Balech
(1956) proposed Adenoides eludens, he considered
that Herdmans gure 1 and 2 corresponded to the
same species. Dodge & Lewis (1986, p. 224) maintained this view when they reported the cell contents of the two species is very similar it may be that
they are only variants. As a consequence, the species Amphidinium eludens has received little attention, and has been cited as Adenoides eludens sensu
Balech (Paulmier, 1992) a situation that has deprived
us from knowing the unusual morphology of
Amphidinium eludens until now.
Our specimens studied from a sandy beach in the
English Channel correspond to Herdmans g. 1

11

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

Pseudadenoides gen. & comb. nov. and Adenoides

Fig. 63. Maximum likelihood phylogenetic tree of Adenoides eludens and Pseudadenoides kofoidii with other dinoagellates
inferred from SSU rDNA sequences. The species newly sequenced in this study are bold. The numbers at each node represent
bootstrap value (maximum likelihood, ML) and posterior probability (PP). Only values of > 70% (bootstrap) and > 0.9 (PP) are
indicated. Closed circle denotes 100%/1.0 = bootstrap/PP.

12

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

F. Gmez et al.

Fig. 64. Maximum likelihood phylogenetic tree of Adenoides eludens and Pseudadenoides kofoidii with other dinoagellates
inferred from partial LSU rDNA sequences. The species newly sequenced in this study are bold. The numbers at each node represent
bootstrap value (maximum likelihood, ML) and posterior probability (PP). Only values of > 70% (bootstrap) and > 0.9 (PP) are
indicated. Closed circle denotes 100%/1.0 = bootstrap/PP.

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

Pseudadenoides gen. & comb. nov. and Adenoides

reported as Amphidinium eludens. The shared characteristics with the original description including (1)
general cell contour and size; (2) lack of apparent
raised epitheca viewed from the lateral side; (3) the
presence of a hump in the ventral contour of the cell
where agella arise; and (4) the position and shape of
the nucleus and pyrenoids (Figs 16). The main characteristic of the redened Adenoides eludens is the
absence of the cingular groove. With the exception
of Podolampas and allied genera, the absence of a
cingular groove is a rare feature in planktonic species
with a globular shape. Although there is no groove,
the transversal agellum nonetheless encircles the
cell. As a general trend, the cell attening of the
benthic species makes it difcult to interpret the tabulation. This is even more difcult in some sand-dwelling dinoagellates that have an incomplete cingulum.
Adenoides is an extreme case, where the cingulum is
absent or the sulcal left anterior plate could be interpreted as a vestigial rst cingular plate. We can speculate an evolution towards the loss of the cingular
groove as an adaptation to benthic habitats. In fact,
dinoagellates that lack the cingular groove such as
Prorocentrum are widespread and diverse in benthic
habitats. Then, we can speculate that the loss of the
cingular groove may be an advantage in benthic habitats, and tentatively propose that the rotational movement of the transversal agellum is less useful in
interstitial space between the sand grains than in pelagic habitats.
With the molecular phylogeny we can nd a
relationship between the planktonic and benthic
dinoagellates that is useful to interpret their tabulation. For example, as suggested by Saldarriaga et al.
(2003), the sand-dwelling genus Roscofa Balech is
related to Podolampas (Gmez et al., 2010). The genera
Amphidiniopsis or Herdmania are related to planktonic
Protoperidinium-like cells such as Archaeperidinium
minutum (Kofoid) E. Jrgensen (Gmez et al., 2011;
Yamaguchi et al., 2011). The morphology of the apical
pore of Adenoides is reminiscent of peridinioid genera
such as Heterocapsa F. Stein or Azadinium Elbrchter &
Tillmann (Tillmann et al., 2009). The tabulation of the
epitheca of Adenoides is 5 and 6, but it can also be
interpreted as 4 and 6 or 5 and 7. These are common
tabulation features of dinoagellates from different phylogenetic origins.
Taxonomic sampling in the phylogenetic trees
remains incomplete, and for the moment there are
no dinoagellate sequences close to Adenoides or
Pseudadenoides. Numerous sand-dwelling dinoagellates also remain as monotypic genera, lacking
any known relative with which they can be classied, at least at the family level. The lack of the
cingular groove in Adenoides or the absence of
precingular plates in Pseudadenoides makes these
genera particularly interesting for the evolution of
dinoagellates.

13
ACKNOWLEDGEMENTS
We thank Pierre Compre and Gerry Moore for their
useful advice on nomenclature.

FUNDING
F.G. was supported by a UL1 post-doctoral grant and a
CNRS convention of research, and an invited lecturer
grant from Universit Littoral-Cte Opale. F.G. is
currently supported by the Brazilian Conselho Nacional
de Desenvolvimento Cientco e Tecnolgico (grant number BJT 370646/2013-14).

AUTHOR CONTRIBUTIONS
F. Gmez: collection, isolation, light and electron microscopy, drafting and editing manuscript; R. Onuma: molecular analysis; L.F. Artigas: collection and editing
manuscript; T. Horiguchi: phylogenetic analysis and
editing manuscript.

REFERENCES
BALECH, E. (1956). tude des dinoagells du sable de Roscoff.
Revue Algologique, Nouvelle Serie, 2: 2952.
DAUGBJERG, N., HANSEN, G., LARSEN, J. & MOESTRUP, . (2000).
Phylogeny of some of the major genera of dinoagellates based
on ultrastructure and partial LSU rDNA sequence data, including
the erection of three new genera of unarmoured dinoagellates.
Phycologia, 39: 302317.
DODGE, J.D. (1982). Marine Dinoagellates of the British Isles. Her
Majestys Stationery Ofce, London.
DODGE, J.D. & LEWIS, J. (1986). A further SEM study of armoured
sand-dwelling marine dinoagellates. Protistologica, 22:
221230.
GMEZ, F. (2012). A quantitative review of the lifestyle, habitat and
trophic diversity of dinoagellates (Dinoagellata, Alveolata).
Systematics and Biodiversity, 10: 267275.
GMEZ, F. & ARTIGAS, L.F. (2014). High diversity of dinoagellates
in the intertidal sandy sediments of Wimereux (north-east English
Channel, France). Journal of the Marine Biological Association of
the United Kingdom, 94: 443457.
GMEZ, F., MOREIRA, D. & LPEZ-GARCA, P. (2010). Molecular
phylogeny of the dinoagellates Podolampas and Blepharocysta
(Peridiniales, Dinophyceae). Phycologia, 49: 212220.
GMEZ, F., LPEZ-GARCA, P. & MOREIRA, D. (2011). Molecular
phylogeny of the sand-dwelling dinoagellates Amphidiniopsis
hirsuta and A. swedmarkii (Peridiniales, Dinophyceae). Acta
Protozoologica, 50: 255262.
HERDMAN, E.C. (1922). Notes on dinoagellates and other organisms
causing discolouration of sand at Port Erin. II. Proceedings and
Transactions of the Liverpool Biological Society, 36: 1530.
HERDMAN, E.C. (1923). Notes on dinoagellates and other organisms causing discolouration of the sand at Port Erin. III.
Proceedings and Transactions of the Liverpool Biological
Society, 38: 5863.
HOPPENRATH, M., SCHWEIKERT, M. & ELBRCHTER, M. (2003).
Morphological reinvestigation and characterization of the marine,
sand-dwelling dinoagellate Adenoides eludens (Dinophyceae).
European Journal of Phycology, 38: 385394.
HUELSENBECK, J.P. & RONQUIST, F. (2001). MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics, 17: 754755.
MCNEILL, J., BARRIE, F.R., BUCK, W.R., DEMOULIN, V., GREUTER, W.,
HAWKSWORTH, D.L., HERENDEEN, P.S., KNAPP, S., MARHOLD, K.,
PRADO, J., PRUDHOMME VAN REINE, W.F., SMITH, G.F., WIERSEMA,
J.H. & TURLAND, N.J. (2012). International Code of Nomenclature

F. Gmez et al.

Downloaded by [Sistema Integrado de Bibliotecas USP] at 05:09 23 February 2015

for Algae, Fungi, and Plants (Melbourne Code). Regnum

Vegetabile 154. Koeltz, Knigstein.
MURRAY, S. & PATTERSON, D.J. (2004). Cabra matta, gen. nov., sp.
nov., a new benthic, heterotrophic dinoagellate. European
Journal of Phycology, 39: 229234.
NYLANDER, J.A.A., RONQUIST, F., HUELSENBECK, J.P. & NIEVESALDREY, J.L. (2004). Bayesian phylogenetic analysis of combined
data. Systematic Biology, 53: 4767.
PAULMIER, G. (1992). Catalogue illustr des microphytes planctoniques et benthiques des ctes Normandes (Rapport interne
RV-92.007RH). IFREMER, Issy-Les-Moulineaux.
POSADA, M.A. & CRANDALL, K.A. (1998). Modeltest: testing the
model of DNA substitution. Bioinformatics, 14: 817818.
SALDARRIAGA, J.F., TAYLOR, F.J.R., KEELING, P.J. & CAVALIER-SMITH,
T. (2001). Dinoagellate nuclear SSU rRNA phylogeny suggests
multiple plastid losses and replacements. Journal of Molecular
Evolution, 53: 204213.
SALDARRIAGA, J.F., LEANDER, B.S., TAYLOR, F.J.R. & KEELING,
P.J. (2003). Lessardia elongata gen. et sp. nov. (Dinoagellata,

14
Peridinales), Podolampaceae and the taxonomic position of the
genus Roscofa. Journal of Phycology, 39: 368378.
SWOFFORD, D.L. (2003). PAUP*. Phylogenetic Analysis using
Parsimony (*and Other Methods). Version 4. Sinauer Associates,
Sunderland, MA.
TILLMANN, U., ELBRCHTER, M., KROCK, B., JOHN, U. & CEMBELLA, A.
(2009). Azadinium spinosum gen. et sp. nov. (Dinophyceae) identied as a primary producer of azaspiracid toxins. European
Journal of Phycology, 44: 6379.
YAMAGUCHI, A., KAWAMURA, H. & HORIGUCHI, T. (2006). A further
phylogenetic study of the heterotrophic dinoagellate genus
Protoperidinium (Dinophyceae) based on small and large subunit
ribosomal RNA gene sequences. Phycological Research, 54:
317329.
YAMAGUCHI, A., HOPPENRATH, M., POSPELOVA, V., HORIGUCHI, T. &
LEANDER, B.S. (2011). Molecular phylogeny of the marine sanddwelling dinoagellate Herdmania litoralis and an emended description of the closely related planktonic genus Archaeperidinium
Jrgensen. European Journal of Phycology, 46: 98112.