Cryptogamie, Algal.

, 1999, 20 (3): 223-234 223

Entomoneis vertebralis sp. nov. (Bacillariophyceae);

a new species from hypersaline environments

Ester CLAVERO”, Joan 0. GRIMALP & Mariona HERNANDEZ-MARINk?‘*

a Dept. Quimica Ambiental. CID - CSIC. Jordi Girona 18. 08034 Barcelona, Spain.
b Seccid de Bot&nica. Fat. Farmkia. Univ. de Barcelona. Av. Joan XXIII, s/n.
08028 Barcelona, Spain; hernande@farmacia.fal:ub.es

(Received 16 October 1998, accepted 31 May 1999)

Abstract-A hyaline Entomoneis species was isolated during a study of microbial mats from
hypersaline environments in the Exportadora de Sal salt works, Guerrero Negro (Mexico).
Morphological, ultrastructural and ecophysiological characteristics of the species were investigated.
Live cells contained two plastids. In girdle view the fmstules appeared bilobate, deeply constricted,
with a junction line visible only near the central nodule. The margin of the wings was fibulate with
19-23 fibulae in 10 pm, some of which, at irregular intervals, appeared longer and ended in small
puncta. After mild cleaning, neither valve body nor the sigmoid connecting bands appeared
ornamented under the light microscope. Scanning electron microscopy revealed membranaceous
valves without ornamentation and a strongly silicified fibulate keel. Furthermore, keels were the
only part of the cell which could be found and recognized in cleaned field samples. This diatom
species was able to grow over a salinity range of 15-50 %O and hence could be considered a
euryhaline marine species. 0 ADAC / Elsevier, Paris

Bacillariophyceae I diatom / Entomoneis vertebralis I fine structure I hypersaline environments

I new species I plastids

RCsumC - Une espece hyaline d’Entomoneis a Cte isolee lors d’une etude de biofilms microbiens
de milieux hypersahns, dam une exploitation de se1 <<Exportadora de Sal B, Guerrero Negro
(Mexique). Les caracteristiques morphologiques, ultrastructurales et Ccophysiologiques de l’espece
ont CtC Ctudiees. Les cellules vivantes possedent deux plastes et les frustules, en vue connective,
appraissent bilobes, fortement contract& au centre et avec une ligne de jonction seulement visible
p&s du nodule central. Les bords des ailes se caracterisent par 19 a 23 fibules par 10 pm ; certaines,
a intervalles irreguliers, sont plus longues et terminees par un petit point. Apres un nettoyage leger,
ni le corps de la valve, ni les bandes connectives sigmdides n’apparaissent ornementes au
microscope optique. Le microscope Clectronique a balayage revele des valves membraneuses sans
omementation et une car&e fibulee fortement silicifiee. De plus, les carenes constituent la seule
partie de la cellule qui peut &tre trouvee et reconnaissable dam les Cchantillons de terrain nettoyes.
Cette diatomee est capable de croitre dam une gamme de salinite de 15-50 %O et peut done &tre
consideree comme une espece euryhaline marine. 0 ADAC / Elsevier, Paris

Bacillariophyceae I diatomkes I Entomoneis vertebralis I structure fine I environnements

hypersalines I nouvelle espike I plastes

* correspondence and reprints

224 E. Clavero, J.O. Grimalt & M. Hemandez-Marine

INTRODUCTION

The genus Entomoneis (Entomoneidaceae) was first described by Ehrenberg in

1845 (Ehrenberg, 1845) with Nuviculu data Ehrenberg as the only species. Although the
characteristics of the new genus were different from Amphiproru Ehrenberg, species with
features similar to Entomoneis data (Ehrenberg) Ehrenberg were later included in
Amphiproru Ehr. (Cleve, 1894). In 1975, Reimer (in Patrick & Reimer 1975) revised both
genera, and proposed that the name Entomoneis should be used instead of Amphiprora,
transferring four species of Amphiprora to Entomoneis. Since then, some other species
have been transferred (Czarnecki & Reinke, 1982; Osada & Kobayasi, 1985; Osada &
Kobayasi, 1990~; Osada & Kobayasi, 1990b; Poulin et al., 1987) and this more familiar
name has been abandoned in favour of Entomoneis (Round et al., 1990).
The genus Entomoneis has been characterized by the apically oriented sigmoid
keel which is elevated from the valve body, the panduriform shape of the cell in girdle
view, and the complex girdle (Patrick & Reimer, 1975; Round et aZ., 1990). Valves are
usually striated with biseriate or multiseriate rows of puncta (Round et al, 1990).These
features enclose a group of species that presents a variety of raphe structures. Raphe
fissure always opens to a duct (raphe canal) separated from the cavity of the frustule by
one row of fibulae (raphe fibulae). In some taxa fibulae occur at many levels beneath the
raphe, forming a ladder-like structure in transverse section (Osada & Kobayasi, 1985;
Osada & Kobayasi, 1990a; Osada & Kobayasi, 1990b) which was called perforate fibulate
plate by Paddock and Sims (1977, diagram D) in their revision of fibulate raphe systems.
The distal row of fibulae (basal fibulae) separates the winged keel from the valve body
constituting the junction-line (Osada & Kobayasi, 1985), one of the characteristics of the
genus obvious in light microscopical observations. This feature, though common in the
genus, is restricted to the comer of the wing in Entomoneis punctulatu (Gmn.) Osada &
Kobayasi (Osada & Kobayasi, 1990~) or absent in Entomoneis aequabilis Osada and
Kobayasi (Osada & Kobayasi, 1991). In other taxa, raphe fissure opens to a double raphe
canal which is linked to the valve body by comb-like structures (Osada & Kobayasi,
199Oc).
Specimens presenting characters of the genus Entomoneis were found and
isolated from microbial mats in a hypersaline environment. These specimens, which have
neither apparent junction-lines nor a striated valve surface, are described as a new species.

MATERIAL AND METHODS

Field material was collected from the upper layer of a microbial mat in a pond
(salinity 51 %o) of Exportadora de Sal salt works, Guerrero Negro (south Baja California,
Mexico) in April 1997. Field samples were cleaned with HCl and H,O, (Merino et al.,
1995).
The diatom was isolated by placing the upper layers of a piece of microbial mat
onto a petri dish with agar enriched with f/2 (Guillard & Ryther, 1962) at a salinity of
50 %o. Cells that had moved into the agar were transferred to new agar using the streak
plating method (Hoshaw & Rosowski, 1973). The process was repeated until a unialgal
axenic culture was obtained, then a colony was transferred into liquid medium. The
cultures were maintained at 20 “C, under daylight fluorescent light, with a 16/8 h
light/dark cycle and 75 pE mm2s-l irradiance.
In order to examine the salinity tolerance of this diatom, liquid cultures with
brines of various concentrations (5, 15, 30, 50, 75 o/oO)enriched with f/2 were carried out.
 Entomoneis vertebralis sp. nov. (Bacilltiophyceae) 225

The lowest salinities were attained by diluting seawater with an appropriate amount of
distilled water. Growth was observed as the increase of colour in the cultures, and survival
at each salinity was assessed visually under the light microscope.
Four methods for removing organic matter from the culture material were
applied. Method A was the same as that applied to field samples. Method B used the same
reagents but for a shorter time. Method C was a combination of acetone extraction and
enzymatic digestion with pancreatin, used by Reimann et al. (1965) to observe weakly
silicified diatoms. Method D was mild oxidation with peroxodisulphate (Ma & Jeffrey,
1978). Cleaned samples were either mounted in Naphrax for light microscopy (LM) or
allowed to settle and dry on polycarbonate filters which were placed onto aluminium stubs
and coated with gold for SEM, or allowed to dry on formvar-coated grids and stained with
uranyl acetate for TEM. Observations were made with a Nikon Optiphot-2 (LM),
Leica/Cambridge Stereoscan 360 (SEM), Hitachi-SOOMT and Philips 200 (TEM).
The terminology used is that suggested by Ross et al. (1979) and Paddock &
Sims (1977, 1981).

RESULTS

Entomoneis vertebralis sp. nov.

Cellulae solitariae vel catenas for-mantes.
Frustula tenuiter silicea, in facie connectivali valde constricta, 28-32 pm, 17-18 pm lata.
Valvae lineares lanceolatae 33-58 pm longae, l&l1 pm latae. Carina valde sigmoidea et
sine juncturae lineis. Carinae margines Jibulatae, 19.4-22 fibulae in 10 pm. Circa
quaeque quarta3bula longior et puncta terminata. Alae elevatae, superjicies alarum net
omata net striata. Super-ties valvarum non striata. Cingula complexa, cum pleuris
quatuor, 2.6-2.8 pm latis, non ornatis.
Cells are solitary or united to form short straight chains, the cells adhering to
each other by their keels. Frustules very weakly siliceous and strongly constricted in
girdle view, 28-32 pm at the widest part of the keels, 17-18 pm at narrowest (Figs 1, 2).
Valves linear lanceolate 33-58 em long, lo-11 urn wide (Figs 3, 4). Keel
strongly sigmoid in valvar view and strongly elevated in girdle view. Margin of the keel
fibulate, fibulae 19.4-22 in 10 pm. Approximately every fourth fibula is longer and ends
in a small dot (LM). Valve surface membranous, neither costate nor striate. Each
cingulum with four unornamented bands (2.6-2.8 pm wide).
Type slide: BCF-A 12237 in Facultatis Pharmaciae Universitatis Barcinonensis
Herbarium. Type slide prepared from strain E20 1.
Holotypus: Coordinate N34 (located with an England Finder) on slide BCF-A
12237.
Type locality: Upper layer of a microbial mat in pond 3 (salinity, 51 X0) of the
Exportadora de Sal S. A., south Baja California, Mexico.
Strain E201 in culture collection E. Clavero.
The election of culture material for a type was made after verifying that the
features of species did not change in culture specimens. Since the slide was prepared with
mildly oxidized material, it contains whole frustules. Hence it gives more information
about the species than the slides with field material, where the membranous cell wall is
disorganized.
 E. Clavero, J.O. Grimalt & M. Hematrdez-Marine

8
Figs I-11. Enfomoneis vertebralis, LM. Scale bar = 10 pm. Figs 1, 2. Girdle view of whole cell.
Fig. 1. Intact (untreated) dead cell. Fig. 2. Holotype, from culture E201, cleaned with peroxodi-
sulphate. Figs 3,4. Valve view, keel in the focus plane. Fig. 3. Type slide BCF-A 12237. Fig. 4. Live
cell. Figs 5-7. Live cells. Fig. 5. Girdle view, two plastids present, one at each side of the transapical
plane. Fig. 6. Same valve as Fig. 4, plastids in the focus plane, aligned in the apical axis. Fig. 7. Two
unusual cells with more than two plastids; note the internal valve (arrow). Figs 8-11. Changes in
plastid shape and position during the cell division. Fig. 8. Plastids elongated apically at either side
of the apical axis. Fig. 9. Cell cleaves apically. Fig. 10. After cleavage, the new valves are not yet
formed. Fig. 11. Transapical division of the plastid.
 Entomoneis vertebralis sp. nov. (Bacillariophyceae) 227

Live appearance and behaviour

In girdle view, live cells have a central nucleus and two plate-like plastids
arranged symmetrically about the median transapical plane (Figs 5, 6). In cultures, cells
with 3,4 or more plastids have been observed,but such cells are often anomalousshapes
and sometimeshave internal valves (Fig. 7).
Plastid movementsduring the cell cycle hasbeenfollowed. Before cell division,
each plastid disposeunder one valve in an apically elongatedshape(Fig. 8). Then the cell
cleaves axially, eachdaughter cell receiving one plastid (Figs 9, 10). After the new valves
are formed, the plastids move back to the girdle and are cleaved transversely (Fig. 11).
The two plastids remain one on each side of the transapical plane.
Salinity tolerance:
Cultures at less than 15 %Oand over 50 %Ofailed to grow after 15 days. Most
cells were dead but the others had poorly developed plastids. This preliminary salinity
tolerance test revealed that growth was possible over a salinity range of 15-50 %o,
showing that E. vertebralis is a euryhaline species.

Frustule structure

With LM, intact cells in girdle view presenta line at the centre of the cell caused
by inflexion at the junction between valve body and winged keel (Fig. 1).
In EM observations, the siliceous part of the frustule of E. vertebralis appears
to be reduced to two strips running along the margin of the keels which constitute the
raphe system. The rest of the cell wall consistsof a very thin, soft membrane with a
fibrillar structure and presumably organic composition, that resistsenzymatic digestion.
The only part of the frustule that remained after a conventional oxidizing treatment
(Method A) was the raphe system (Fig. 12) which appearedas a bilobed comb. A milder
oxidation (Methods B and D), which may not remove all the organic matter, did not
totally degradethe cell wall (Figs 13, 14). Enzymatic digestion degradedthe cell contents
but not the cell wall (Fig. 15). The cell wall appearedto be composedof a fibrillar net
structure (Fig. 16), that is sensitive to oxidation treatment (Method B) (Fig. 17).
This thin membranecovers the raphe canal and extends laterally, and seemsto
comprise valves and girdle like a normal siliceous frustule (Fig. 15).
In intact cells, the girdle region usually occupies one third of the breadth of the
frustule in girdle view, and comprisesfour to six bands. When the tbecae are isolated, a
maximum of four bands can be observed per cingulum (Fig. 15), suggestingthat the
epicingulum overlaps most of the hypocingulum.
Raphe system:
The raphe slit is located along the apex of the keel and opensto a tubular raphe
canal, the cavity of which is delimited by arched fibulae (Fig. 18). At irregular intervals,
two silica rods are borne on either side of the arch of one fibula (Fig. 19). About 0.7 pm
from the fibula both rods fuse discontinuously forming a small ladder-like structure but
remaining separateat their tips (Figs 20, 21). The set of ladder-like structuresis the distal
row of marginal puncta seenin LM (Figs 1, 2).
At the apices some of the fibulae are sometimesfused (Fig. 22) and beyond
them the silica strip expands laterally, acquiring the shape of an apically-folded
arrowhead(Fig. 23). This arrowhead-like structure is fused to either sideof the arch of the
most apical fibula. On either side of the central nodule the silica strip that enclosesthe
raphe is also expanded transapically (Fig. 24).
228 E. Clavero, J.O. Grimalt & M. Hemartdez-Marine

Figs 12-17. Entomoneis vertebralis, EM. The effect of different treatments on identical structures.
Figs 12-1.5. valves in girdle view, SEM. Scale bars = 10 pm. Fig. 12. Silica part of half a keel
remaining after treating cells with a conventional treatment with H,O,; arrow = central nodule.
Fig. 13. Valve, mild treatment with H,O,. Fig. 14. Valve, mild oxidation with peroxodisulphate.
Fig. 15. Theta, enzymatic digestion. Figs 16, 17. Unsilicified part of the cell wall, TEM. Scale
bars = 0.1 pm. Fig. 16. The cell wall appears to comprise a fibrilar net; enzymatic digestion. Fig. 17.
The fibrilar structure is still visible though it has been eroded by the oxidation treatment; mild
treatment with H,O,.

The raphe slit is narrow and plicate in transverse section (Fig. 25). The central
endings are simple and straight on both external and internal faces(Figs 26,27). On either
side of the internal polar raphe endings the basal siliceouslayer is thicker but does not
form a distinct helictoglossa (Fig. 23).
 Entomoneis vertebralis sp. nov. (Bacillariophyceae) 229

DISCUSSION

The panduriform frustule, valve shape, the complex girdle and the position of
the sigmoid raphe at the apex of a winged keel are characteristc features of the genus
Entomoneis (Patrick & Reimer, 1975) and are present in our new species.
It is not possible to compare E. vertebralis with other species of the genus on the
basis of commonly used features of valve structure like striation since the only silified
structure of its frustule is the raphe system. Though some members of the genus are
weakly silicified (Round et al., 1990), the posession of a cell wall composed of a thin
fibrillar membrane with only the raphe system silicified has not been reported previously.
Similar cell wall organization was described for Cylindrotheca jksiformis Reimann et
Lewin by Reimann et al. (1965). In C. jksiformis the siliceous part of the valve is reduced
to four strips (raphe system and girdle) which are tightly enclosed in an envelope of
organic material. The organic components of the valve are continuous with the organic
material in the girdle region, but do not extend across the raphe fissure. Thus the organic
casing is a replica (a mould) of the frustule. In Entomoneis vertebralis the fibrillar
membrane also covers the siliceous parts (raphe system) and occupies the unsilicified part
of the valve and the girdle.
The raphe system of E. vertebralis is equivalent to the one described for E. alata
(Ehr.) Ehr. var. japonica (Cl.) Osada & Kobayasi, a variety of the type species of the
genus. In E. alata var. japonica the keel is supported by fibulae which link costae on
opposite walls of the wing, forming a ladder-like structure (Osada & Kobayasi, 1985).
This structure is also common to other species like E. decussata (Grunow) Osada &
Kobayasi (Osada & Kobayasi 199Oa), E. centrospinosa Osada & Kobayasi (Osada &
Kobayasi 1990b) or E. pseudodupzex Osada & Kobayasi (Osada 8z Kobayasi, 1985) (Tab.
1). In E. vertebralis the ladder-like structure is formed by the arched raphe fibulae and the
rods that extend from them, which are fused at different levels. Considering that
E. vertebralis has an unsilified wing, intermediate and basal fibulae cannot originate from
wing costa as in the previously mentioned species. Although this has not been observed,
it is possible that the ladder-like structure in E. vertebraEis is joined to the opposite walls
of the keel (Fig. 28). In that case, rods could be equivalent to the transapical costa and the
several fusions between two rods to the fibulae, the distal fusion being the basal fibulae.
Since the junction line visible with LM is a row of basal fibulae (Osada & Kobayasi
1985) the set of ladder-like structures would correspond to the junction-line. Neverthe-
less, since these structures are irregularly spaced, they do not form a conspicuous line in
LM, but a row of distant puncta.

Live appearance and behaviour

Two interphase configurations of plastids have been described for Entomoneis

species, an axial plate-like plastid or two plastids arranged symmetrically on either side
of the median transapical plane (Round et al., 1990). E. vertebralis corresponds to the
latter configuration (Figs 5, 6).
The only report on plastid movements for an Entomoneis species of which the
authors are aware is the description of cell division in Entomoneis paludosa (W. Smith)
Reimer by Geitler (1970), which describes the axial plastid interphase configuration. The
single axial plastid divides transversely before mitosis and the two new plastids shift to
opposite valves. Following mitosis, cytokinesis and new valve formation occur (Geitler,
1970). Besides, plastid division in E. vertebralis occurs following cytokinesis, before the
cell enters into interphase. The configuration of one plastid on each side of the median
230 E. Clavero, J.O. Grimalt & M. Hemtidez-Marin

Figs 18-23. Raphe system of E. vertebralis, SEM except Fig. 19. Scale bars = 1 pm except Fig. 19.
Fig. 18. Arched fibulae delimit the raphe canal. Field material. Fig. 19. Valve in girdle view, at
irregular intervals two silica rods are born at either side of the same fibula (arrow) and are fused
between each other at their ends. TEM, mild treatment with H,O,, scale bar = 10 pm. Fig. 20. Both
rods fuse discontinuously forming a small ladder-like structure and separate at their tips, mild
treatment with peroxodisulphate. Fig. 21. Enlargement of the ladder-like structure. Fig. 22. Fibulae
fused at the apex of the keel. Fig. 23. Apical arrowhead-shaped expansion of the silica strip, fused
to the last fibula (arrowhead) which is broken; note that the fusion among fibulae is also broken but
the fibulae keep pieces of the silicified junction (arrow).

transapical plane persists until the beginning of the next cell division, when each plastid
is arranged under each valve. This pattern of plastid movement is different from that
 Entomoneis vertebralis sp. nov. (Bacillariophyceae) 231

Figs 24-27. Raphe system of E. vertebralis,SEM. Scale bars = 1 pm except Fig. 25. Fig. 24. Keel
centre, transapical expansion of the silica strip. Fig. 25. Broken keel showing the raphe canal, and
the plicate raphe fissure (arrow), scale bar = 0.5 pm. Fig. 26. External view of the central nodule,
the transapical expansion is broken. Fig. 27. Internal view of the central nodule.

observed for E. paludosa in respect to the lapseof time between cytokinesis and plastid
division, and the time between plastid division and plastid movement which are
respectively much shorter and longer in E. vertebralis.
Cells with internal valves were observed in E. vertebralis cultures. The presence
of internal valves is associatedwith an irregular cell division which involves mitosis
followed by unequal cytokinesis (Round et aZ., 1990). In E. paludosa (Geitler, 1970), the
formation of internal valves follows an acytokinetic mitosis, which results in a new cell
containing one plastid, one nucleus and one internal valve. This contrasts with
E. vertebra&, in which the presenceof more than two plastidsin cells containing internal
valves probably indicates that cytokinesis, or at least plastid division, takes place.
Such differences in live cell morphology suggesthigh variability within the
genus, which has also been found for the raphe system structure (Osada & Kobayasi,
1991; Paddock & Sims, 1977; Paddock & Sims, 1981).
The poorly silicified cell wall and the raphe system are characteristic for this
new species,which has been called E. vertebralis becauseof the similarity of its raphe
system to a spinal column.
Acknowledgements. The authors wish to thank the Scientific and Technical Services of
the University of Barcelona. E. C. was supported by a Ministerio de Educacidn y Ciencia fellowship
(FP194) during the present work. We are grateful to Prof. Dr Pierre Compbre for correcting the latin
diagnosis and to two anonymous referees for their helpful comments, which improved the
manuscript.
Tab. 1. Comparison of E. vertebralis with other Entomoneis species with a similar raphe system.

E. vertebralis E. alata var. japonica E. decussata E. centrospinosa E. pseudoduplex E. aequabilis

references this study (Osada & Kobayasi, (Osada & Kobayasi, (Osada & Kobayasi, (Osada & Kobayasi, (Osada & Kobayasi,
1985) 1990b) 199Oa) 199%) 1991)
silification only the raphe system f strong + + + +
girdle view frustule shape panduriform panduriform panduriform pandurifonn panduriform panduriform and
longitudinally
twisted
valve view valve shape linear-lanceolate linear -1anceolate linear -1anceolate broadly linear broadly linear linear slightly
sigmoid
valve ends acute acuminate acute acute acute broad scalpellifurm
keel shape sigmoid strongly sigmoid sigmoid strongly sigmoid strongly sigmoid strongly sigmoid
basal fibulae d&ult close close close close
junction line sinuous arcuate arcuate curved
wing striation transapical radiate decussate decussate decussate oblique
valve striation transapical parallel transapical parallel transapical parallel transapical parallel oblique
plastids two nd nd nd nd nd

SEM features

raphe fissure plicate plicate nd nd nd plicate

raphe system’ ladder like structure ladder-like structure ladder like strocture ladder like structure ladder like structure only raphe fibulae
raphe fibulae + + + + + +
intermediate fibulae several several several several one
basal fibulae + + fused with adjacent fused with adjacent occasionally fused
basal fibulae basal fibulae with adjacent fibulae
fibulae borne on rods that extend from keel costae keel costae keel costae keel costae keel costae
raphe fibulae

+ indicates presence; - indicates absence; nd indicates no data.

’ raphe system in transverse section
 Entomoneis vertebralis sp. nov. (Bacillariophyceae) 233

Fig. 28. Diagrammatic transverse section of the keel of Entomoneis vertebralis at the ladder-like
structure level. The hatched area represents the silicified parts.

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