PROOF COVER SHEET
Journal acronym: TEJP
Author(s):
Nike Fuchs, Eleonora Scalco, Wiebe H.C.F. Kooistra, Philipp Assmy and Marina Montresor
Article title:
Article no:
Enclosures:
Genetic characterization and life cycle of the diatom Fragilariopsis kerguelensis
849360
1) Query sheet
2) Article proofs
Dear Author,
1. Please check these proofs carefully. It is the responsibility of the corresponding author to check these and approve or
amend them. A second proof is not normally provided. Taylor & Francis cannot be held responsible for uncorrected errors,
even if introduced during the production process. Once your corrections have been added to the article, it will be considered
ready for publication.
Please limit changes at this stage to the correction of errors. You should not make insigniﬁcant changes, improve prose
style, add new material, or delete existing material at this stage. Making a large number of small, non-essential corrections
can lead to errors being introduced. We therefore reserve the right not to make such corrections.
For detailed guidance on how to check your proofs, please see
http://journalauthors.tandf.co.uk/production/checkingproofs.asp.
2. Please review the table of contributors below and conﬁrm that the ﬁrst and last names are structured correctly
and that the authors are listed in the correct order of contribution. This check is to ensure that your name will appear
correctly online and when the article is indexed.
Sequence
Preﬁx
Given name(s)
Surname
1
Nike
Fuchs
2
Eleonora
Scalco
3
Wiebe H.C.F.
Kooistra
4
Philipp
Assmy
5
Marina
Montresor
Sufﬁx
Queries are marked in the margins of the proofs.
AUTHOR QUERIES
General query: You have warranted that you have secured the necessary written permission from the appropriate
copyright owner for the reproduction of any text, illustration, or other material in your article. (Please see
http://journalauthors.tandf.co.uk/preparation/permission.asp.) Please check that any required acknowledgements
have been included to reﬂect this.
No Queries
Eur. J. Phycol. (2013), 00(00): 1–16
Genetic characterization and life cycle of the diatom
Fragilariopsis kerguelensis
NIKE FUCHS1, ELEONORA SCALCO2, WIEBE H.C.F. KOOISTRA2, PHILIPP ASSMY3* AND MARINA
MONTRESOR2*
5
Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven,
Germany
2
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy
3
Norwegian Polar Institute, Fram Centre, 9296 Tromsø, Norway
1
(Received 13 January 2013; revised 1 May 2013; accepted 14 May 2013)
10
15
20
25
The planktonic diatom Fragilariopsis kerguelensis plays an important role in the biogeochemical cycles of the Southern Ocean,
where remains of its frustules form the largest deposit of biogenic silica anywhere in the world. We assessed the genetic identity of
26 strains, from cells collected at various sites in the Southern Ocean, using three molecular markers, LSU and ITS rDNA and
rbcL. The LSU sequences were identical among the tested strains, ITS sequences were highly similar, and only one base pair
difference was detected among the rbcL sequences. These results, together with a large number of successful mating experiments
demonstrated that the strains belong to a single biological species. We investigated the mating system and life cycle traits of F.
kerguelensis. Cell size diminished gradually in clonal strains. Gamete formation only occurred when strains of opposite mating
type – within a cell size range of 7–36 µm – were mixed together. Two binucleate gametes were formed in each gametangium and
gamete conjugation produced a zygote that had four nuclei and was surrounded by thin siliceous scales. Two out of the four nuclei
subsequently degenerated and the zygote expanded to form an auxospore surrounded by a transverse and a longitudinal
perizonium. Staining with the ﬂuorochrome PDMPO provided for the ﬁrst time a clear demonstration that the longitudinal
perizonium is formed after auxospore expansion is complete. Initial cells produced within the mature auxospores were 78–101 µm
in length. Various authors have shown that the average valve size of F. kerguelensis varies in sediment samples collected in regions
and seasons with different primary production regimes and this parameter has thus been proposed as a biological proxy for palaeoproductivity. A better understanding of the life cycle of F. kerguelensis should help the design of future investigations aimed at
testing the link between cell size distribution in the natural environment and the role that environmental factors might have in the
regulation of population cell size.
Key words: auxospores, diatoms, Fragilariopsis kerguelensis, internal transcribed spacer (ITS), life cycle, LSU rDNA, molecular
systematics, plankton, rbcL, sexual reproduction, Southern Ocean
Introduction
30
35
40
The pennate diatom genus Fragilariopsis includes
species that are important primary producers in the
marine plankton and in the sea ice of polar seas
(Hustedt, 1958; Lundholm & Hasle, 2010). In the
Southern Ocean, the heavily siliciﬁed species
Fragilariopsis kerguelensis has been reported as
among the dominant diatoms in the water column of
the ice-free, offshore Antarctic Circumpolar Current
(ACC) (Hart, 1942; Smetacek et al., 2004).
Cells of Fragilariopsis kerguelensis can form long,
ribbon-shaped chains, and they can be distinguished
from other species in the genus by the strong siliciﬁcation of their frustule and coarse pattern of the striae
on the valve face (Hasle, 1965, 1968; Hasle &
Correspondence to: Marina Montresor. E-mail: marina.
[email protected]; Philipp Assmy. E-mail: [email protected]
Syvertsen, 1997; Cefarelli et al., 2010). Mechanical
stress-testing of frustules of live cells of F. kerguelensis have demonstrated that the frustule architecture
provides high mechanical resistance to deformation
and breakage, which is probably an adaptation against
grazing (Hamm et al., 2003). The robust nature of the
F. kerguelensis frustule is further reﬂected in the
exceptionally high Si : N ratios of this species relative
to most other diatoms (Brzezinski, 1985; Hoffmann
et al., 2007). Together, these morphological and physiological characteristics demonstrate why this species has such a high demand for silica.
Fragilariopsis kerguelensis contributes up to 90%
of the diatom frustules in the diatom ooze making up
the Antarctic opal belt (Zielinski & Gersonde, 1997),
which is the largest deposit of biogenic silica in the
world ocean (Treguer et al., 1995). Therefore, F. kerguelensis is one of the most important diatoms in the
ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/13/000001-16 © 2013 British Phycological Society
http://dx.doi.org/10.1080/09670262.2013.849360
45
50
55
60
N. Fuchs et al.
65
70
75
80
85
90
95
100
105
110
115
global silicon cycle (Zielinski & Gersonde, 1997) and
an indicator species of a silica-sinking regime in an
otherwise iron-limited ecosystem (Smetacek et al.,
2004). Valve size measurements of F. kerguelensis
from sediment samples of different sites in the
Atlantic and Paciﬁc sector of the Southern Ocean
and from sediment cores spanning the Holocene and
the last glacial maximum (LGM) showed the presence
of larger frustules close to the highly productive
Antarctic Polar Front (APF) and in the LGM layers,
while smaller frustules were recorded in the less productive zones north and south of the APF and in the
interglacial layers (Cortese & Gersonde, 2007).
Further size distribution measurements of F. kerguelensis in core samples of the Holocene showed the
presence of longer and more abundant valves during
the warmer Mid-Holocene period and of smaller and
less abundant ones during the colder Late-Holocene
period (Crosta, 2009). These results led the authors to
propose F. kerguelensis as a biological proxy for
palaeo-productivity and temperature conditions in
the Southern Ocean. Productivity in this area is
strongly linked to iron availability and an increased
abundance of F. kerguelensis in response to artiﬁcial
iron addition has been recorded in three Southern
Ocean iron-fertilization experiments (Gall et al.,
2001; Assmy et al., 2006, 2007).
Variations in valve size over geological time scales,
as well as short-term changes resulting from iron
addition experiments (Cortese et al., 2012), prompted
us to investigate the life cycle of this important diatom. Changes in cell size frequency distribution (SFD)
in many diatoms are linked to the functioning of the
life cycle. Part of the timing mechanism is related to
the morphology of the cell wall, which consists of two
rigid siliceous thecae that are slightly different in size.
This results in a progressive decrease of the average
population cell size as vegetative division proceeds.
This progressive reduction in cell size – mostly along
the apical axis in pennate diatoms – can be circumvented by the onset of sexual reproduction and the
formation of large-sized cells (Round et al., 1990;
Chepurnov et al., 2004). In situ evidence for sexual
reproduction comes either from direct observation of
sexual stages (e.g. Crawford, 1995; Sarno et al., 2010;
Holtermann et al., 2010) or from estimates of cell size
spectra, where the detection of larger cell size classes
is an indirect evidence for recent sexual events (e.g.
Mann, 1988; Jewson, 1992; D’Alelio et al., 2010).
Despite the ubiquity of F. kerguelensis in the Southern
Ocean, evidence of sexual reproduction – e.g. the
presence of gametangia, auxospores and initial cells
– in ﬁeld samples was reported for the ﬁrst time only
recently (Assmy et al., 2006).
The ﬁrst aim of our study was to test if F. kerguelensis is a genetically homogeneous species or if it
includes genetically different cryptic species. Cryptic
diversity, i.e. morphologically similar or identical but
2
genetically different species, have been detected
within several diatoms (e.g. Amato et al., 2007;
Sarno et al., 2007; Evans et al., 2008; QuijanoScheggia et al., 2009). To this end, we obtained both
nuclear ribosomal (LSU and ITS rDNA) and plastid
(rbcL) sequences from strains isolated in different
locations of the Atlantic sector of the Southern Ocean.
The second aim was to study the life cycle of F.
kerguelensis in laboratory conditions, testing its mating system and the way in which gametes, auxospores
and the large initial cells are produced. Insight into the
mechanisms through which sexual reproduction
occurs is a prerequisite for further investigations
aimed at testing the link between cell size distribution
in the natural environment and the role of environmental factors on the regulation of population cell
size.
120
125
130
135
Materials and methods
Culture material
A total of 51 cultures of Fragilariopsis kerguelensis
(Supplementary Table 1 and Supplementary Fig. 1) were
established during cruises ANT-XXI/3 (EIFEX, European
Iron Fertilization EXperiment: January–February 2004) and
ANT-XXV/3 (LOHAFEX, ‘loha’ is the Hindi word for iron,
Fertilization EXperiment: January–March 2009) carried out
in the Atlantic sector of the Southern Ocean. Single cells or
short chains were isolated with a micropipette from phytoplankton samples collected with a 20 µm-mesh-size phytoplankton hand net. At the Alfred Wegener Institute (AWI),
strains were grown in 70-ml tissue culture bottles ﬁlled with
30 ml of f/2 medium (Guillard & Ryther, 1962) prepared with
0.2 µm-ﬁltered and autoclaved Antarctic seawater (at a salinity of 34.6), modiﬁed to obtain a higher concentration of
silicic acid (210 µmol l–1) and a lower concentration of nitrate
(100 µmol l–1) and phosphate (6.25 µmol l–1). Strains were
maintained at a temperature of 2°C, an irradiance of 50 µmol
photons m–2 s–1 provided by Osram Biolux lamps (Osram
L18W/965 Biolux: OSRAM, Munich, Germany), and a
photocycle of 16 : 8 h light : dark. At the Stazione
Zoologica Anton Dohrn (SZN), strains were grown in standard f/2 culture medium prepared with 0.2 µm-ﬁltered and
autoclaved oligotrophic Mediterranean seawater adjusted to a
salinity of 35. Cultures were maintained at a temperature of
5°C, an irradiance of 50 µmol photons m–2 s–1, provided by
Philips (Royal Philips, Amsterdam, the Netherlands) cool
white lamps, and a photocycle of 12 : 12 h light : dark.
140
145
150
155
160
165
DNA extraction, ampliﬁcation and sequence analysis
Cultures of F. kerguelensis were harvested during the exponential growth phase by ﬁltration on 0.45 µm pore-size
polycarbonate ﬁlters. DNA extraction and puriﬁcation were
performed as described by Kooistra et al. (2003). PCR
ampliﬁcation of the hyper-variable D1–D3 domains in the
nuclear-encoded LSU ribosomal RNA region, the nuclearencoded internal transcribed spacer region (ITS1, 5.8S rDNA
and ITS2), and the plastid-encoded rbcL region were carried
out as described by Amato et al. (2007). Sequence reactions
170
Life cycle of Fragilariopsis kerguelensis
175
180
185
were obtained with Big-Dye Terminator Cycle Sequencing
technology (Applied Biosystems, Foster City, CA, USA) and
puriﬁed using a robotic Biomek FX station (Beckman
Coulter, Fullerton, CA, USA). Products were analysed on
an Automated Capillary Electrophoresis Sequencer 3730
DNA Analyzer (Applied Biosystems). Sequences were
aligned by eye with sequences downloaded from GenBank,
using the sequence alignment editor Se-Al version 2.0a11
(Rambaut, 2002). The alignment of the ITS sequences is
provided as Supplementary ﬁle 1. M-fold (http://mfold.rna.
albany.edu) was used to explore the various possible secondary structure solutions for the ITS-2 sequences of the strains.
Life cycle
190
195
200
205
210
215
Cell size reduction. A ﬁrst estimate of the monthly cell
size reduction rate of F. kerguelensis was obtained using two
time-point measurements for 47 strains with average apical
length spanning 13–76 µm kept in the AWI standard growth
conditions. The apical lengths of ﬁve cells for each strain
were measured at time 0 and after 24 months.
Seven strains of F. kerguelensis (marked with § in
Supplementary Table 1), kept in the SZN growth conditions,
were used to estimate cell size reduction rates. The apical
length of 300 cells for each strain was measured at the
beginning and at the end of the observation period (7
months); the monthly reduction rate of the apical length
was estimated as the difference between the two average
measures. The growth rate of the seven strains was measured
as well to obtain an estimate of cell size reduction at each cell
division. At the beginning of the observation period, an
aliquot (between 1 and 3 ml, depending on cell concentration) of the exponentially growing strains was inoculated into
glass tubes (three tubes for each strain) ﬁlled with 20 ml of
culture medium. Growth was estimated by measuring ﬂuorescence with a Turner Designs ﬂuorometer model 10-005R
(Turner Designs, Sunnyvale, CA, USA) every 2 or 3 days.
Cultures were monitored for 24 days, i.e. until the end of the
exponential growth phase. The growth rate, as divisions·day–
1
, was estimated by calculating the linear regression over all
data points between day 0 and 24. The growth curves of the
seven strains were rather similar and the exponential growth
phase was completed between day 18 and day 23. We have
chosen to estimate growth rates over 24 days, because this
was the average time interval (± 1 day) at which cultures were
transferred during the experiment (lasting 7 months).
Mating behaviour.
220
225
230
Mating-type designation of F. kerguelensis was done on the basis of the results of two sets of
crosses: in the ﬁrst set, seven strains and in the second set 14
strains were mixed in all pairwise combinations (marked with
* in Supplementary Table 1). Crosses were carried out in
12-well tissue culture plates (Corning, Corning, NY, USA)
ﬁlled with 1 ml of modiﬁed f/2 medium. In each well, pairs of
exponentially growing strains were added (c. 0.5 ml of each
strain). Control wells were inoculated with double volume of
a single strain. Culture plates were incubated in the AWI
experimental conditions speciﬁed above and were checked
every 2 days for the presence of sexual stages.
Sexual life cycle. Detailed observations on the life cycle
of F. kerguelensis were carried out at different times and
using different strains as follows: (1) experiment A was
3
carried out in February 2009 with up to six strains mixed in
the same culture plate (strains whose code starts with ‘L’ in
Supplementary Table 1; the average apical length of these
strains was between 10 and 25 µm); (2) experiment B was
carried out in August 2011 with strains PA_P8B1 (mean
apical length: 16 µm) and MM_P13D2 (mean apical length:
15 µm); (3) experiment C was carried out in July 2010 with
strains L2D6 (average apical length: 12.78 ± 1.09 µm, mean
± s.d.) and L9C3 (average apical length: 14.26 ± 1.60 µm).
In Experiment A (carried out on board RV Polarstern), the
wells of a 6-well culture plate were ﬁlled with exponentially
growing cultures – 0.5 ml for each strain – of four or six
different strains isolated during the ANT-XXV/3 cruise and
0.5 ml of growth medium. The culture plate was incubated at
5°C, 50 µmol photons m–2 s–1 and an 18 : 6 h L : D photocycle, and inspected daily for the presence of sexual stages.
During the ﬁrst days in which sexual stages were recorded,
the material of one well was stained with the ﬂuorochrome
PDMPO [2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole: Molecular Probes,
Eugene, OR, USA), following the protocol by Leblanc &
Hutchins (2005), to visualize newly formed silica frustule
elements of the vegetative cells and auxospores.
In Experiment B (carried out at AWI), strains PA_P8B1 and
MM_P13D2 were mixed and dispensed into a 12-well tissue
culture plate (Corning, Corning, NY, USA) and 12 Utermöhl
chambers. All vessels were incubated in the standard AWI
conditions and inspected twice per day for 10 days. At every
inspection, the material from one Utermöhl chamber was ﬁxed
with hexamethylenetetramine-buffered formaldehyde at a ﬁnal
concentration of 2% and stained with DAPI (4′,6-diamidino-2phenylindole; Sigma-Aldrich, St. Louis, MO, USA) at a ﬁnal
concentration of 0.5 µg ml–1 to visualize the nuclei. On day 6,
when sexual stages were detected in the culture, the material
from three culture wells was incubated with PDMPO for 12 h.
In Experiment C (carried out at SZN), a stock culture
was prepared with 150 ml of f/2 culture medium in which
exponentially growing strains L2D6 and L9C3 were
inoculated together to reach a ﬁnal concentration of
about 4000 cells ml–1 (i.e. 2000 cells ml–1 for each
mating type); aliquots of 4 ml were dispensed in two 6well tissue culture plates and incubated at the standard
SZN culture conditions. Four ml of each parental strain
were placed in a plastic vial and ﬁxed with formaldehyde
solution at a ﬁnal concentration of 1.6%; these two samples were used to estimate the percentage composition of
single cells and cells arranged in chains in the parental
strains. On day 7, when cells were observed detaching in
the dual cultures approaching the sexual phase, the material from three wells was ﬁxed with formaldehyde; this
sample was used to estimate the percentage composition
of single cells and cells arranged in chains. On each of
the subsequent 3 days, the material of two wells was
pooled in vials, ﬁxed, and used for the observation of
sexual stages. The ﬁxed samples were stained with DAPI
as described above.
The culture material from all three experiments was examined and photographed with a Zeiss Axiovert 200 epiﬂuorescence microscope (Zeiss, Oberkochen, Germany) equipped
with a Zeiss AxioCam, a long pass DAPI ﬁlter set (EX G 365,
BS FT 395, EM LP 420) and a band pass DAPI ﬁlter set (EX
G365, BS FT 395, EM BP 445/50).
235
240
245
250
255
260
265
270
275
280
285
290
295
N. Fuchs et al.
Scanning electron microscopy
300
305
310
For observation of different life stages by scanning electron
microscopy, the material from experiments B and C was ﬁxed
with glutaraldehyde at a ﬁnal concentration of 2% at 4°C for
1 h. The ﬁxed samples were gently ﬁltered over a membrane
ﬁlter placed in a ﬁlter holder connected to a syringe and
subsequently dehydrated with ethanol at increasing concentrations (10%, 25%, 50%, 75%, 95%, 2 × 100%) for at least
10 min for each step. After using 100% ethanol, the ﬁlter was
placed on an aluminium stub, critical point dried, and sputter
coated with gold-palladium. Samples were observed with a
FEI Quanta 200F (FEI, Hillsboro, OR, USA) SEM at AWI or
a JEOL 6700-F (JEOL, Tokyo, Japan) at SZN. Two natural
samples collected during the Lohafex cruise were also prepared for SEM, using the protocol described above.
Results
Genetic and morphological characterization of
Fragilariopsis kerguelensis
315
320
325
330
335
340
345
350
The LSU sequences of the 26 analysed strains were
identical. The rbcL region was sequenced for 11
strains and sequences were identical with one exception: two strains (MM_E13C5 and MM_E13B2)
showed a C at position 1074 in the gene region
(1055 in our alignment) whereas the remainder
showed an A at that position. The ITS sequences of
the 11 tested strains were also highly similar.
Differences were restricted to 13 positions in the
alignment, exhibiting base changes or ambiguities,
but
without
any
phylogenetic
pattern
(Supplementary Table 2). One of these positions, position 115 from the 5′-end of ITS-2, exhibited either an
A, a T or a W (= ambiguity A and T). The various
alternative RNA folding patterns provided by M-fold
generally located this position against a U at position
249 from the 5′-end of ITS-2 in a stem region, ﬂanking
a small internal loop. Therefore, an A at position 115
in ITS-2 resulted in an A–U bond whereas a U at that
position increased the size of the internal loop.
The apical length of the strains used in this study
ranged from 70 µm (strain PA_P10C3) to less than 10
µm, the average cell length below which strains died.
Cells were single (Fig. 4) or arranged in chains of
different length (Figs 1–3), where cells were joined
together valve to valve by mucilaginous material
(Fig. 5). In smaller cells (up to 15–20 µm long), the
contact between neighbouring cells was along the whole
surface of the two adjacent valves, while longer cells
were in contact along the central portion of the cells; this
suggests that larger cells are slightly inﬂated in their
central portion in girdle view (Fig. 1). The nucleus was
situated in the central part of the cell, with two large
chloroplasts located on either side (Fig. 1).
The frustule included two thecae, each with a valve,
a valvocopula and two open cingular (girdle) bands
(Figs 6–10). The opposite ends of the valvocopula
4
were pointed and joined forming a diagonal suture
on the portion of the cingulum close to the cell apex
(Figs 7–portion of the cingulum close to the cell apex
(Figs 7–10).10). The ﬁrst cingular band was hairpinshaped and its ends were not in direct contact. The
second band was very thin and its ends were almost in
contact on the narrow side of the cingulum; a ligula
was present in its central portion, on the other narrow
side of the cingulum (Figs 8, 10). This ligula was
inserted between the ends of the ﬁrst cingular band.
A thin raphe ran along the margin of each valve and
the raphes of the two valves of each frustule were in
the trans positions (Fig. 6), i.e. the raphes lay diagonally opposite each other in intact frustules (nitzschioid
symmetry). Large cells were lanceolate in valvar view,
with a heteropolar outline (Fig. 11), while smaller
cells were isopolar (Fig. 6). The number of striae in
10 µm ranged between 6 and 8 and the number of
poroids in 10 µm ranged between 8 and 10. The
poroids were 200–300 nm in diameter and were
closed by a hymenate thin layer with minute perforations (Figs 12, 13).
355
360
365
370
Mating behaviour
Gametangia, auxospores and initial cells were never
observed in clonal strains, but were only observed
when mixing strains of compatible mating type. The
average cell lengths of parental strains that were
involved in successful crosses ranged between 12
and 36 µm (Table 1). The results of the ﬁrst set of
crosses (upper matrix in Table 1) involving seven
strains revealed that three of them belonged to one
mating type [deﬁned as Fk1, following the recommendation of Chepurnov et al. (2005)] and the other four
to the opposite mating type Fk2. All combinations
between crosses of the opposite mating type produced
auxospores and viable initial cells. In the second set of
crosses carried out with 14 strains (lower matrix in
Table 1), nine of them belonged to one mating type
and ﬁve to the opposite one. In this case, sexual stages
were recorded in all combinations of opposite mating
types except one (L8B1 × L8B6). Assignment of the
mating type in F. kerguelensis is only based on the
results provided by the matrix of crosses because no
morphological or behavioural differences could be
detected between strains of different mating type
(see the following section). The designation Fk1 and
Fk2 thus denotes only that strains belong to opposite
mating types, without assigning any biological meaning, i.e. ‘male’ vs ‘female’ or ‘active’ vs ‘passive’.
Life cycle
All clonal strains showed a progressive reduction of the
apical axis of the cells over time. Cell size reduction
estimates based on measurements of 47 cultivated
strains over a duration of 24 months showed that the
375
380
385
390
395
400
Life cycle of Fragilariopsis kerguelensis
5
Figs 1–13. Light (Figs 1–4) and SEM (Figs 5–13) micrographs of Fragilariopsis kerguelensis vegetative cells. 1–3. Cells of different
apical length arranged in chains; an arrowhead points to the nucleus (Fig. 1, strain PA_P6C3; Fig. 2, strain MM_P7A4; Fig. 3, strain
MM_E8A6). 4. A single cell in valvar view (strain MM_E13C5). 5. A detail of a chain in which mucous material is visible between
the valves of adjacent cells (large F1 generation cells of cross PA_P8B1 × MM_P13D2, experiment B). 6. An isopolar valve; the
narrow raphe on the valve margin is arrowed (natural sample). 7. Epi- and hypotheca of a dividing cell showing the valvocopula and
the two cingular bands; the diagonal suture in the valvocopula is marked with an arrow and the ligula on the second, thin cingular
band is marked with arrowheads (cross PA_P8B1 × MM_P13D2, experiment B). 8. A detail of Fig. 7. 9. Epitheca showing
valvocopula and two cingular bands; note the diagonal suture in the valvocopula (arrow) and the ligula on the narrow second
cingular band (arrowhead) (cross PA_P8B1 × MM_P13D2, experiment B). 10. Detail of Fig. 9. 11. A heteropolar valve (strain Lynn
5). 12. Detail of the valve with the poroids (strain Lynn 5). 13. Detail of the same valve, showing a single poroid with minute
perforations. Scale bars = 10 µm (Figs 1–5, 7, 11), 5 µm (Figs 6, 8–10), 1 µm (Fig. 12) and 100 nm (Fig. 13).
405
410
415
average reduction in apical length was 0.13–1.77 µm
per month and it was larger for longer cells (≥ 50 µm
long). However, considerable variability in cell size
reduction rates was detected in these longer cells
(regression coefﬁcient for size reduction vs length =
0.11; Fig. 14), whereas for cells ≤ 50 µm a tighter linear
relationship was detected between average cell size and
the monthly reduction rate (regression coefﬁcient: 0.9;
Fig. 14). Seven strains with an average apical length
spanning 29 and 68 µm were measured after a time
interval of 7 months and showed average cell size
reduction rates between 0.45 and 1.06 µm∙month–1
(Table 2, Fig. 14, Supplementary Fig. 2). The average
growth rates estimated over 24 days ranged from 0.076
to 0.121 divisions day–1 and the rate of length reduction
per division was 0.18–0.47 µm (Table 2).
The ﬁrst sign of interaction between strains of
opposite mating type (within a few days of inoculation
in the same culture vessel) was the detachment of the
cells from the chain (Fig. 16). A quantitative estimate
was obtained for the cross L2-D6 × L9-C3. About
90% of the cells were arranged in chains in the
420
425
L9D3
L9D2
L8D4
L2C2
L8C3
L9C3
L8A5
L2A2
L2B2
L2B6
L2C3
L2D3
L8B6
L8C6
L9C6
L9D4
L8B1
L8B5
L8C2
L9A4
L9C2
Strain
Fk1
Fk1
Fk1
Fk2
Fk2
Fk2
Fk2
Fk1
Fk1
Fk1
Fk1
Fk1
Fk1
Fk1
Fk1
Fk1
Fk2
Fk2
Fk2
Fk2
Fk2
14
15
n.a.
13
14
36
18
13
13
13
13
13
13
14
14
14
12
14
12
13
13
Mating Size
type
(µm)
0
0
+
+
+
+
L9D3
0
+
+
+
+
L9D2
+
+
+
+
L8D4
0
0
0
L2C2
0
0
L8C3
0
L9C3
L8A5
0
0
0
0
0
0
0
0
+
+
+
+
+
L2A2
0
0
0
0
0
0
0
+
+
+
+
+
L2B2
0
0
0
0
0
0
+
+
+
+
+
L2B6
0
0
0
0
0
+
+
+
+
+
L2C3
0
0
0
0
+
+
+
+
+
L2D3
0
0
0
0
+
+
+
+
L8B6
0
0
+
+
+
+
+
L8C6
0
+
+
+
+
+
L9C6
+
+
+
+
+
L9D4
0
0
0
0
L8B1
0
0
0
L8B5
0
0
L8C2
0
L9A4
L9C2
Table 1. The results of crossing experiments carried out with different strains of Fragilariopsis kerguelensis; in the ﬁrst column and in the ﬁrst row the strain code, in the second column the mating
type (m.t., see text for further details), in the third column the average apical length of the strains (Size). Grey-ﬁlled squares indicate the absence of intraclonal sexual reproduction; + indicates the
presence of sexual stages; 0 indicates the absence of sexual stages; n.a. = not available.
N. Fuchs et al.
6
Life cycle of Fragilariopsis kerguelensis
7
Table 2. Rate of reduction in cell size (apical length) and growth rate for seven Fragilariopsis kerguelensis strains. The cell size is the
average cell size on day 0 and the growth rate is the average of three measurements.
Strain
MM_P7A4
MM_E13B2
MM_E13C5
MM_P9C3
PA_P6C3
Lynn 1
Lynn 4
Cell size (µm)
Growth rate (divisions∙d–1)
Rate of size decrease (µm month–1)
Size decrease per division (µm)
29.86
31.36
34.05
65.21
67.99
68.00
68.27
0.093
0.083
0.081
0.121
0.076
0.091
0.105
0.84
0.45
0.62
1.09
1.06
0.68
0.74
0.30
0.18
0.26
0.30
0.47
0.25
0.23
Fig. 14. Average monthly reduction rate of the apical length of
Fragilariopsis kerguelensis clonal strains of different cell size;
black circles represent the 47 strains measured after a time
interval of 24 months, white circles represent the seven strains
measured after a time interval of 7 months.
430
435
440
445
450
individual parental strains, but on day 7 after the
mixing of the two parental strains, when the ﬁrst
sexual stages were detected, only 30% of the cells
were arranged in chains (Fig. 15). A similar timing
and behaviour was observed also in the cross
PA_P8B1 × MM_P13D2, where most of the cells
were detached within 3–4 days after inoculation.
After detachment from the chains, single cells
increased mobility and several formed pairs (Fig.
16). The contact point between paired cells was variable. Most of the time cells were in contact at the level
of the cingular bands, but their orientations varied; in
some cases the valves were parallel to each other, in
other cases perpendicular (Figs 16 and 18). The observation in SEM of a sample from cross PA_P8B1 ×
MM_P13D2 containing numerous paired cells
showed the presence of mucous threads joining cells
along the cingular bands (Figs 18, 19). At the stage in
which chains were disassembled, many cells – either
paired or single – showed a considerably enlarged
nucleus (Fig. 17). In these cells, the two chloroplasts
were appressed to the cell wall. These cells expanded
along their pervalvar axis by the deposition of additional cingular bands in the hypotheca (Figs 20, 21,
22) and were interpreted as gametangia. Figure 22
shows the hypovalve of the parental gametangium
still connected to the auxospore.
Fig. 15. Percentage distribution of single cells (grids), cells in
chains (white) and sexual stages (black) in monoclonal parental strains (L2-D6 and L9-C3) of Fragilariopsis kerguelensis
and in three replicate crosses on day 7 after inoculation.
In the gametangia, the two-step meiotic division took
place. Meiosis I was followed by cytokinesis (Figs 23,
24), but the two took place without an accompanying
cell division (acytokinetic division) and, in several cases,
it was not synchronous in the two protoplasts within one
gametangium. After completion of meiosis II, two
binucleate gametes were formed (Figs 23–26). In most
cases, the two paired gametangia remained connected
during meiotic division. The gametangium opened
along the cingular bands and the two gametes became
exposed, but each still lay within one gametangial theca
(Figs 25, 26). We could not follow gamete conjugation
in real time and we cannot state if conjugation occurred
between gametes while they were still situated within the
appressed gametangia, or if the gametes escaped from
the gametangial thecae and moved towards other
gametes. We observed only a few naked binucleate
stages (gametes) in the samples and we thus hypothesize
that conjugation occurred between gametes still associated closely with the appressed gametangia. There
appeared to be no mucous envelopes surrounding the
paired gametangia.
Rounded stages with four nuclei were observed,
almost always attached to the empty valve of one
gametangium (Figs 27–30). These stages were interpreted as zygotes. Support for this conclusion also
derived from the observation of samples incubated
with PDMPO, which showed the presence of thin,
455
460
465
470
475
N. Fuchs et al.
8
Figs 16–22. Light (Figs 16, 17) and SEM (Figs 18–22) micrographs of gametangia of Fragilariopsis kerguelensis. 16. A few days
after the inoculation together of strains of opposite mating type: cells have detached from chains and several cells are in contact
(arrows) (mixture of different strains in experiment A). 17. Picture taken on the same day as Fig. 16, where single cells in girdle view
have an enlarged nucleus (arrowhead) and chloroplasts appressed to the valves (experiment A). 18. Two cells (gametangia) close to
and perpendicular to each other, in contact via the cingulum (cross PA_P8B1 × MM_P13D2, Experiment B). 19. Detail of the same
pair of cells showing the mucous material extruded from the girdle region that keeps the two adjacent cells together. 20. A
gametangium in girdle view with extra cingular bands in the hypotheca (arrows) (cross L2D6 × L9C3, experiment C). 21.
Gametangium with extra cingular bands in the hypotheca (cross PA_P8B1 × MM_P13D2, experiment B). 22. An auxospore and
three gametangial hypothecae with extra cingular bands (cross L2D6 × L9C3, experiment C). Scale bars = 20 µm (Figs 16, 17), 5 µm
(Figs 18, 20–22) and 1 µm (Fig. 19).
480
485
490
495
slightly siliciﬁed scales on the outer membrane of the
rounded zygote (Fig. 30) and on both ends of the
auxospore (Figs 31, 32). SEM preparations showed
that the caps at both ends of the auxospore consisted of
thin, tightly appressed and partially overlapping round
scales with diameters of 2.3–3.7 µm (Fig. 45). The
zygote had four nuclei (Fig. 28) and the elongating
auxospore had two (Figs 33, 34); we thus assume that
the degeneration of the two supernumerary nuclei
occurred in the young auxospore. The vast majority
of auxospores remained attached by a mucous plug to
a valve of one gametangium until they had completed
development. The incubation of samples with
PDMPO allowed visualization of the formation of
the transverse (Figs 35, 36) and longitudinal perizonium (Figs 37, 38). The transverse perizonium, which
allows the bipolar expansion of the auxospore, was
composed of a series of slightly siliciﬁed perizonial
bands (Figs 35, 36, 39, 44). The longitudinal
perizonium was synthesized when the auxospore had
completed elongation and when the cytoplasm started
detaching from the transverse perizonium (Figs
37, 38). Figures 37 and 38 show that, within the period
of incubation with PDMPO (12 h), the auxospore
synthesized the distal bands of the transverse perizonium and part of the longitudinal perizonium, which is
presumably composed by thin longitudinal bands,
though the exact number could not be determined.
Figures 40 and 41 represent an auxospore in which
one valve has been formed during incubation with
PDMPO. This cell has two nuclei and one of them
seems to be degenerating: we interpret this as the
acytokinetic mitotic division that accompanies the
deposition of one valve of the initial cell. While still
wrapped by the perizonium, the initial cell contained a
single nucleus (Figs 42, 43).
The average cell length of parental strains that
produced sexual stages, both in the crosses for testing
500
505
510
515
Life cycle of Fragilariopsis kerguelensis
9
Figs 23–41. Light and epiﬂuorescence micrographs of auxosporulation in Fragilariopsis kerguelensis. 23. Two appressed gametangia in girdle view; the cell on the right shows the cleavage of cytoplasm (cytokinesis) (cross L2D6 × L9C3, experiment C). 24. The
same gametangia with DAPI-stained nuclei; the cell on the left has an expanded nucleus in meiotic prophase, the cell on the right has
undergone the second meiotic division and each protoplast has two nuclei. 25. A gametangium after completion of cytokinesis and the
formation of two gametes (cross L2D6 × L9C3, experiment C). 26. The same gametangium with DAPI-stained nuclei; each gamete
has two nuclei. 27. A zygote still attached to a theca of the gametangium (cross L2D6 × L9C3, experiment C). 28. The same zygote
with four DAPI-stained nuclei (arrowheads). 29. A zygote still attached to a theca of the gametangium (cross PA_P8B1 ×
MM_P13D2, experiment B). 30. The same zygote stained with PDMPO, showing thin siliceous platelets in its wall (arrowheads).
31. A young auxospore still attached to one theca of a gametangium (cross PA_P8B1 × MM_P13D2, experiment B). 32. The same
auxospore stained with PDMPO, showing thin siliceous platelets at both ends of the auxospore (arrowheads). 33. An elongating
auxospore still attached to one theca of a gametangium (mix of different strains in experiment A). 34. The same auxospore with two
(continued )
N. Fuchs et al.
520
525
mating behaviour (Table 1) and in those carried out to
study the life cycle, ranged between 7 and 36 µm. We
also measured the sizes of 57 empty gametangial
thecae still connected to auxospores or initial cells in
crosses carried out within experiment A: their lengths
were 6.9–26.1 µm. Within the same experiment, we
also measured the length of 27 initial cells, i.e. cells
still wrapped in the perizonium but in which the siliceous valves were already visible, and of the nearby
gametangial theca: initial cells measured 78.4–100.8
µm and gametangia 10.2–26.1 µm (Fig. 46).
Discussion
530
535
540
545
550
555
The results obtained with the different genetic markers
showed no evidence for cryptic diversity in
Fragilariopsis kerguelensis. All tested strains shared
the same LSU and their highly similar ITS sequences
differed only in positions showing variation between a
particular base and an ambiguity consisting of that
same base and another base, and in one case, variation
between A, T and ambiguity W. These few variable
positions did not reveal any phylogenetic pattern
among the sequences of F. kerguelensis presented.
Both markers have been used extensively in assessing
phylogenetic relationships amongst closely related
diatom species and for barcoding purposes, and they
have proved to represent reliable markers for species
identiﬁcation, although with slightly different results
amongst the taxa tested (e.g. Moniz & Kaczmarska,
2010; Hamsher et al., 2011). Amato et al. (2007)
demonstrated that strains of Pseudo-nitzschia with
identical ITS sequences were reproductively compatible if crossed with strains of the opposite mating
type. Far more pronounced sequence differences
than the ambiguities observed among the ITSsequences from strains of F. kerguelensis were
observed among strains within Pseudo-nitzschia multistriata, but even these strains were sexually perfectly
compatible as long as they were of the opposite mating type (D’Alelio et al., 2009b). The single base-pair
difference in the rbcL sequences among the tested
strains cannot be considered as a proof of species
difference either, because Levialdi Ghiron et al.
(2008) have shown that strains of Pseudo-nitzschia
exhibiting a total of four base pair differences among
their rbcL sequences mated successfully. Comparison
10
of Fragilariopsis and Pseudo-nitzschia is sound
because these two planktonic genera are close relatives (Lundholm et al. 2002). The results of the molecular analyses, together with the large number of
successful mating experiments carried out in this
study, demonstrate that all the tested F. kerguelensis
strains belong to a single biological species.
The gross morphology of the cell and the valve
ultrastructure of the strains examined in light and
electron microscopy ﬁt the descriptions provided for
F. kerguelensis by different authors (Hasle, 1965,
1968; Hasle & Syvertsen, 1997; Cefarelli et al.,
2010). The ultrastructure of the poroid hymen of our
specimens corresponds perfectly with the TEM
images provided by Hasle (1965), thus further conﬁrming the uniformity of the species. The pioneering
work carried out by Hasle (1965, 1968) did not
include information on the number and ultrastructure
of the girdle bands. Recently, Cefarelli et al. (2010)
reported the presence of one valvocopula and one
girdle band in F. kerguelensis. We here provide evidence for the presence of an additional thin band at the
abvalvar end of the cingulum; this band bears a ligula
that inserts between the open ends of the ﬁrst cingular
band. Similar narrow bands have been detected in
other Bacillariaceae, e.g. some Nitzschia species
(Trobajo et al., 2013).
Gradual cell size reduction was detected in all clonal strains of F. kerguelensis and the results of mating
experiments showed that the sexual phase was
induced only when compatible strains within the
gametangial size window were mixed together.
These facts indicate that F. kerguelensis is heterothallic. Furthermore, we never observed the formation of
auxospores nor detected a sudden increase in cell size
in any of the clonal cultures (which were monitored
for a long time), indicating the absence of homothallic
or uniparental (automictic or apomictic) auxosporulation (Kaczmarska et al., 2013). Fragilariopsis kerguelensis is the only species in the genus Fragilariopsis
for which information on the life cycle is available
(Assmy et al., 2006; this study) and its general pattern
conforms to what has been described for other raphid
pennate diatoms (Chepurnov et al., 2004).
Nevertheless, differences occur between genera and
in the following we discuss the main features of the
life cycle in the frame of the available literature
information.
Figs 23–41. Continued
DAPI-stained nuclei. 35. An elongated auxospore still attached to the theca of a gametangium; mix of different strains in experiment
A). 36. The same auxospore stained with PDMPO, showing the bands of the transverse perizonium. 37. An elongated auxospore still
attached to the theca of a gametangium (mix of different strains in experiment A). 38. The same auxospore stained with PDMPO,
showing the terminal bands of the transverse perizonium (arrowheads) and the longitudinal perizonium. 39. An elongated auxospore;
the sample was incubated with PDMPO and the internal longitudinal perizonium, the thin bands of the transversal perizonium and the
terminal caps (arrowheads) are visible (cross PA_P8B1 × MM_P13D2, experiment B). 40. A mature auxospore within which one
valve of the initial cell has been deposited (mix of different strains in experiment A). 41. The same auxospore with two DAPI-stained
nuclei and PDMPO-stained valve (on the top side). Scale bars = 10 µm.
560
565
570
575
580
585
590
595
600
605
Life cycle of Fragilariopsis kerguelensis
11
Figs 42–45. Auxospores of Fragilariopsis kerguelensis. 42. Light micrograph of a mature auxospore containing the initial cell (mix
of different strains in experiment A). 43. Epiﬂuorescence micrograph of the same auxospore showing the fusion of the two DAPIstained nuclei. 44. SEM micrograph of an auxospore (cross L2D6 × L9C3, experiment C), showing caps at the ends and the transverse
perizonium (the faint rings around the auxospore: cross L2D6 × L9C3, experiment C). 45. A detail of Fig. 44 showing round scales
covering the terminal portion of the auxospore. Scale bars = 10 µm (Figs 42–44) and 1 µm (Fig. 45).
Fig. 46. The lengths of 27 initial cells, still wrapped in their
perizonium, and the corresponding gametangial thecae attached
to them, measured in crosses carried out in experiment A.
Transition from the vegetative to the sexual phase and
formation of gametes
610
615
620
The ﬁrst evidence of interactions between strains of
opposite mating type placed in the same culture vessel
was the detachment of chains into single cells. When
joined in chains, cells of F. kerguelensis in the cell size
range for sexualization are in close contact along the
whole surface of their valves and it is reasonable to
assume that this arrangement would impede the contact between individual cells that precedes the differentiation of gametangia and their subsequent
conjugation. The detachment of cells arranged in
chains has been reported for other chain-forming
planktonic diatoms, such as Pseudo-nitzschia pungens (Chepurnov et al., 2005) and P. multistriata
(E. Scalco, unpublished data). However, in Pseudonitzschia cells are joined together by their tips and the
cell surface available for contacts with other cells is
considerably larger. In fact, the formation of auxospores on gametangia still joined in a chain has been
reported for natural populations of P. cf. delicatissima
and P. cf. calliantha (Sarno et al., 2010) and for
P. pungens in culture (Chepurnov et al., 2005).
In F. kerguelensis, the detachment of chains was
followed by cell pairing, which involved girdle-to
girdle contact in a variable fashion and was mediated
by thin mucilage threads. Pairing modality differs
amongst Bacillariaceae, where pairing of gametangial
cells generally occurs along the adjacent valves in
species of the genus Pseudo-nitzschia (e.g.
Davidovich & Bates, 1998; Chepurnov et al., 2005)
and along the cingular bands in Nitzschia species (e.g.
Mann, 1986; Kaczmarska et al., 2007; Trobajo et al.,
2009). In F. kerguelensis, the paired gametangia
showed the presence of a higher number of cingular
bands in the hypovalve, which allowed their enlargement in the transapical direction. The synthesis of
additional cingular bands was detected only in gametangia. Paired cells of F. kerguelensis showed an
enlarged nucleus, which we interpret as being in
early meiotic I prophase, by analogy with what has
been observed in other pennate diatoms (e.g.
Seminavis robusta, Chepurnov et al., 2002; Navicula
oblonga, Mann & Stickle, 1989). In the gametangia of
F. kerguelensis, the ﬁrst meiotic division is followed
by protoplast division, while the second meiotic division is acytokinetic, i.e. the nuclear division is not
followed by cytokinesis. In this way, two binucleate
gametes are formed by each of the paired gametangia.
This pattern corresponds to ‘type I auxosporulation’,
following the classiﬁcation scheme provided by
Geitler (1973). This sequence of events is similar to
625
630
635
640
645
650
655
N. Fuchs et al.
660
665
670
675
680
what has been reported for some other diatoms,
including the benthic raphid diatoms Seminavis
robusta (Chepurnov et al., 2002) and Pinnularia cf.
gibba (Poulíčková et al., 2007), but different patterns
have been reported within raphid diatoms (reviewed
by Chepurnov et al., 2004). As an example, only one
uninucleate gamete is formed in each gametangium of
the benthic freshwater genus Sellaphora (Mann,
1989), while two uninucleate gametes are produced
in each gametangium of Pseudo-nitzschia pungens
(Chepurnov et al., 2005). Unfortunately, we could
not follow gamete conjugation in F. kerguelensis in
real time. This species grows at low temperature and
observation of live material in light microscopy can be
carried out for only very limited periods to avoid cell
death. Therefore we cannot conﬁrm that conjugation
occurs between the gametes of the two adjoined gametangia or if gametes are released in the medium and
conjugate with gametes produced by different gametangia. Nevertheless, the binucleate gametes were
almost always attached to the gametangial thecae
and very few rounded protoplasts were observed free
in the culture medium. This suggests that conjugation
occurs between gametes of adjoined gametangia.
Auxospore formation and development
685
690
695
700
705
710
715
In samples incubated with PDMPO – a stain that binds
to newly deposited silica – we observed four-nucleate
zygotes surrounded by thin ﬂuorescent ‘platelets’,
which corresponded to the thin scales visible on the
incunabula at the ends of the auxospores in SEM
preparations. The presence of slightly siliciﬁed scales
surrounding the auxospore is a common feature of
centric diatoms (Kaczmarska et al., 2001), but thin,
lightly siliciﬁed scales have been described also in
some pennate diatoms (e.g. Idei et al., 2013). Within
the Bacillariaceae, scale are present in Nitzschia longissima, where they occur on the surface of gametes of
both mating types and, following auxospore elongation, remain at the tip of the caps and/or along the
auxospore surface (Kaczmarska et al., 2007); and in
Nitzschia inconspicua, where circular scales are present in the incunabula surrounding the zygotes and
again persist in the caps at the ends of the expanding
auxospores (Mann et al., 2013). Thin scales have been
recorded also on the walls of passive gametes and on
the auxospore caps of Pseudo-nitzschia multiseries
(Kaczmarska et al., 2000), but have not been reported
in other Pseudo-nitzschia species that have been
investigated in SEM so far (e.g. Amato et al., 2005;
Chepurnov et al., 2005; D'Alelio et al., 2009a).
Information on the ultrastructure of the longitudinal
perizonium and the modality and timing of synthesis
is limited due to the fact that this thin structure is
almost invisible in light microscopy. Incubation of F.
kerguelensis auxospores with PDMPO showed that
the longitudinal perizonium is deposited at the end
12
of auxospore elongation. The fact that during the
incubation period only a part of the perizonium
became visible, suggests that this structure is composed by longitudinal bands that are deposited in
sequence. The longitudinal perizonium can be
observed in SEM preparations in which the outer,
transverse perizonium has become broken. In
Nitzschia inconspicua, it has been possible to determine that the longitudinal perizonium has an asymmetrical structure and is constituted by ﬁve bands
comprising one wider primary band, two narrower
secondary bands on one side of the primary band,
and two extremely narrow ones on the other side
(Mann et al., 2013).
Cardinal points in the life cycle of F. kerguelensis
The ﬁrst evidence for sexual reproduction in F. kerguelensis came from a record of auxospores in phytoplankton samples collected in the Southern Ocean
(Assmy et al., 2006). In the natural samples, auxospores of various sizes (i.e. in different stages of development) were recorded and they were often still
connected to gametangial thecae 10–31 µm in length.
The overall size in the natural population was 12–90
µm (P. Assmy unpublished data) and the fact that the
gametangial thecae detected were only 10–31 µm
long suggests that the cell size window for sexualization is close to that range. In the crosses carried out in
the present study, most of the parental strains we tested
had average lengths of ≤ 25 µm with one exception,
which was strain L9C3 with an average length of 36
µm. This strain underwent sexual reproduction when
crossed with compatible strains, showing that at least
some F. kerguelensis cells can be sexualized up to 36
µm. Our current observations provide additional
information on the size range of initial cells, which –
in culture –were 78.4–100.8 µm in length; this is a
slightly wider range than was recorded in natural
samples by Assmy et al. (2006), who reported 76–90
µm. The fact that initial cells are often attached to the
theca of the gametangia in F. kerguelensis makes it
easy to link the sizes of these two cardinal points or
stages (sensu Geitler, 1932) in the life cycles; our data
suggest that there is no clear relationship between
their size in F. kerguelensis (see Fig. 38). The few
experimental studies addressing this question have
provided contrasting results: a direct correlation
between the size of parental strains and initial cells
has been recorded in some species but not in others
(Davidovich, 1994; Edlund & Bixby, 2001).
Interestingly, different size ranges of gametangial
and initial cells have been recorded between populations of the same species, suggesting that this trait
might be used for their differentiation (Edlund &
Bixby, 2001). Davidovich (1994) investigated the
possible effect of different irradiances and day-lengths
on the size of initial cells but could not detect any
720
725
730
735
740
745
750
755
760
765
770
Life cycle of Fragilariopsis kerguelensis
775
780
785
790
795
800
805
810
815
820
825
signiﬁcant relationship; he also showed that auxospore development was possible when crosses between
opposite mating types were made in darkness. These
results led him to conclude that the elongation capacity of the auxospore depends on increase in the
volume of the vacuole, which is independent from
photosynthesis and thus from the duration and intensity of the irradiance provided.
The length of a species’ sexual cycle is determined
by the size of the initial cells, the rate of cell size
reduction, and the size at which descendant cells
become sexually inducible. In turn, the rate of cell
size reduction depends on both the rate of cell division
and the amount by which cells decrease in size at each
division. In Pseudo-nitzschia delicatissima it has been
shown that larger cells have the highest rates of cell
size decrease per division, and this parameter progressively reduces as the average cell size of the strain
diminishes (Amato et al., 2005). A similar trend has
been detected in P. multistriata (D'Alelio et al.,
2009a). Moreover, both of these Pseudo-nitzschia
species show differences in their maximum growth
rates during progression through their vegetative life
phase, with larger cells showing lower growth rates.
The data obtained for F. kerguelensis in the present
study showed – at least for cells ≤ 50 µm in length – a
direct relationship between cell size and the monthly
reduction rates for strains grown under the same
experimental conditions; this suggests either that
smaller cells have lower division rates or that they
possess a mechanism to limit cell size reduction at
each division. We could estimate growth rates and
thus cell size reduction per division only for seven
strains ranging in size between 30 and 68 µm and no
signiﬁcant differences in cell size reduction per division were observed between the different strains.
These results and the considerable variability detected
in cell size reduction rates of larger strains suggest the
presence of considerable intraspeciﬁc variability in
growth rate, so that it will be necessary to examine a
higher number of strains, covering the whole cell size
range, in order to gain better insights into the mechanism and speed of cell size reduction in F. kerguelensis.
Fragilariopsis kerguelensis as a palaeo-proxy: a life
cycle perspective
Valve morphometrics of Fragilariopsis kerguelensis
have been suggested to have potential for reconstructing palaeo-oceanographic conditions in the Southern
Ocean, based on the fact that the average frustule size
varies amongst sites characterized by different productivity regimes (Cortese & Gersonde, 2007; Crosta,
2009; Cortese et al., 2012). Cortese & Gersonde
(2007) analysed the valve length and valve area of F.
kerguelensis frustules in surface sediments from the
Paciﬁc and Atlantic sectors of the Southern Ocean
(providing a spatial gradient) and one piston core
13
(giving a temporal gradient) near the Antarctic Polar
Front. Average valve size was larger in the proximity
of the APF, characterized by high productivity, while
it was smaller in the less productive zones to the north
and south of it. In sediment cores, larger cells were
found in layers from the glacial period compared with
the interglacials. The authors related the presence of F.
kerguelensis populations with larger average cell size
to alleviation of iron limitation during iron-replete
glacial conditions (Cortese & Gersonde, 2007). The
possible link between glacial–interglacial changes in
the average cell size of F. kerguelensis and iron availability (cells with a larger valvar area occurring in
glacial periods characterized by a higher Fe deposition
from atmospheric dust) was further conﬁrmed by
Cortese et al. (2012). The authors also reported a
larger valvar area for F. kerguelensis inside fertilized
patches of two iron-fertilization experiments.
Furthermore, analysis of Holocene core samples collected in the Eastern Antarctic Continental Shelf, an
area not limited by iron, has shown that F. kerguelensis valves were longer and more abundant during the
warmer Mid-Holocene period and shorter and less
abundant during the colder Late-Holocene (Crosta,
2009). In this case, differences in the average population cell size were linked to changes in the length of
the growth season, sea surface temperature and ice
coverage.
During vegetative growth the average cell size of
diatoms decreases while its variance increases
(according to the MacDonald–Pﬁtzer rule;
Chepurnov et al., 2004). Cell size decrease – in the
absence of sexual reproduction – is proportional to the
number of cell divisions that have occurred. The faster
cells can divide, the faster they become smaller.
Sexual reproduction, however, circumvents the progressive size diminution of the population by producing large initial cells. Thus, the average cell size of a
diatom population is the integrated result of intrinsic
life cycle traits and the response to proximate environmental factors. Iron availability, or the availability
of other nutrients, such as silicate required for the
synthesis of the frustules, might in fact inﬂuence
valve morphometrics or the size of initial cells.
Laboratory experiments have shown that Pseudonitzschia species can change their valve aspect ratio,
increasing their transapical axis, when acclimated to
iron-limited culture conditions (Marchetti & Harrison,
2007). If F. kerguelensis is found to have a similar
response, it might help explain the differences in valve
area recorded under iron-replete and -deplete conditions (Marchetti & Cassar, 2009). However, when
comparing differences in cell size between natural
populations, one should not only consider the average
cell size/valvar area but the whole cell-size distribution. A higher average value can, in fact, be due to a
relatively rapid response to speciﬁc environmental
conditions that prompted the variation of the cell
830
835
840
845
850
855
860
865
870
875
880
885
N. Fuchs et al.
890
895
900
905
910
915
920
925
aspect ratio or to the presence of a higher proportion of
larger cells produced following sexual reproduction.
The plots of frequency distributions of valve length of
F. kerguelensis reported by Crosta (2009) show the
presence of large cells in the same size range as the
initial cells reported in the present study, thus supporting the idea that sexual reproduction may have been
occurring in the natural populations whose frustules
were subsequently deposited in the sediments.
However, the only direct evidence of the occurrence
of sexual reproduction in natural populations of F.
kerguelensis was provided by Assmy et al. (2006),
who found auxospores, accounting for 0.03–0.4% of
the total cell number during an iron-fertilization
experiment. The highest percentages of auxospores
were recorded in the fertilized patch, suggesting that
iron-replete conditions might favour the onset of the
sexual phase and the consequent production of largesized cells. Furthermore, our data show considerable
length variation amongst initial cells, suggesting
genetic differences amongst strains, which opens the
possibility of rapid environmental selection for bigger
or smaller initial cells, and consequently for the average cell size of the whole population over time.
The possibility of manipulating the life cycle of F.
kerguelensis in laboratory conditions, demonstrated
here, opens the possibility of investigating the extent
to which the various phases of the life cycle of this
diatom are regulated by speciﬁc environmental conditions and assessing the plasticity and intraspeciﬁc
diversity of different life cycle traits. This approach,
coupled with a better understanding of the level at
which sediment records reﬂect water column events,
and the analysis of cell size distributions with appropriate measurement strategies, will help in assessing
the possible use of F. kerguelensis parameters as a
proxy for palaeoecological conditions and improve
our understanding of the mechanisms through which
life history traits have evolved in planktonic diatoms
and contributed to their ecological success.
Acknowledgements
930
935
Part of this work was funded by the German Science
Foundation (grant no. DE 1455/2‐1) granted to N.F. and
part from the PhD thesis of E.S. supported by Stazione
Zoologica Anton Dohrn (SZN). The research of P.A. at
SZN was supported by the European Community –
Research Infrastructure Action under the FP7
‘Capacities’ Speciﬁc Programme ASSEMBLE (grant
no. 227799). The authors wish to thank Carmen
Minucci for strain maintenance at SZN.
Supplementary information
The following supplementary material is accessible via
the Supplementary Content tab on the article’s online
page at http://dx.doi.org/10.1080/09670262.2013.849360
14
Supplementary ﬁle 1. ITS alignment, in FASTA
format. Position 1 constitutes the 5′-end of ITS-1 and
position 776 the 3′-end of ITS-2; ITS-1 spans positions 1–309; 5.8S rDNA, 310-481; and ITS-2,
482–776. Note that all ITS sequences, except the
one of strain 4-20, are incomplete at the 5′-end and
that the ITS sequences of strains MM-E13C5 and
MM-E13D2 are incomplete at the 3′-end.
Supplementary Table 1. Fragilariopsis kerguelensis
strains used in this study, including GenBank
accessions.
Supplementary Table 2. Nucleotide differences
between the ITS sequences of F. kerguelensis strain
4-20 (sequence EF660061) and those of the strains
used in this study.
Supplementary Figure legends.
Supplementary Figure 1. Map of the Atlantic Sector of
the Southern Ocean with the sampling stations at
which Fragilariopsis kerguelensis strains were
collected.
Supplementary Figure 2. Size spectra of seven clonal strains of Fragilariopsis kerguelensis measured at
time 0 and after 7 months.
940
945
950
955
960
References
AMATO, A., ORSINI, L., D'ALELIO, D. & MONTRESOR, M. (2005). Life
cycle, size reduction patterns, and ultrastructure of the pennate
planktonic
diatom
Pseudo-nitzschia
delicatissima
(Bacillariophyceae). Journal of Phycology, 41: 542–556.
AMATO, A., KOOISTRA, W.H.C.F., LEVIALDI GHIRON, J.H., MANN, D.
G., PRÖSCHOLD, T. & MONTRESOR, M. (2007). Reproductive isolation among sympatric cryptic species in marine diatoms. Protist,
158: 193–207.
ASSMY, P., HENJES, J., SMETACEK, V. & MONTRESOR, M. (2006).
Auxospore formation in the silica-sinking oceanic diatom
Fragilariopsis kerguelensis (Bacillariophyceae). Journal of
Phycology, 42: 1002–1006.
ASSMY, P., HENJES, J., KLAAS, C. & SMETACEK, V. (2007). Mechanisms
determining species dominance in a phytoplankton bloom induced
by the iron fertilization experiment EisenEx in the Southern
Ocean. Deep Sea Research Part I: Oceanographic Research
Papers, 54: 340–362.
BRZEZINSKI, M.A. (1985). The Si: C: N ratio of marine diatoms:
interespeciﬁc variability and the effect of some environmental
variables. Journal of Phycology, 21: 347–357.
CEFARELLI, A.O., FERRARIO, M.E., ALMANDOZ, G.O., ATENCIO, A.G.,
AKSELMAN, R. & VERNET, M. (2010). Diversity of the diatom
genus Fragilariopsis in the Argentine Sea and Antarctic waters:
morphology, distribution and abundance. Polar Biology, 33:
1463–1484.
CHEPURNOV, V.A., MANN, D.G., VYVERMAN, W., SABBE, K. &
DANIELIDIS, D.B. (2002). Sexual reproduction, mating system,
and protoplast dynamics of Seminavis (Bacillariophyceae).
Journal of Phycology, 38: 1004–1019.
CHEPURNOV, V.A., MANN, D.G., SABBE, K. & VYVERMAN, W. (2004).
Experimental studies on sexual reproduction in diatoms.
International Review of Cytology, 237: 91–154.
CHEPURNOV, V.A., MANN, D.G., SABBE, K., VANNERUM, K., CASTELEYN,
G., VERLEYEN, E., PEPERZAK, L. & VYVERMAN, W. (2005). Sexual
reproduction, mating system, chloroplast dynamics and abrupt
cell size reduction in Pseudo-nitzschia pungens from the North
Sea (Bacillariophyta). European Journal of Phycology, 40:
379–395.
CORTESE, G. & GERSONDE, R. (2007). Morphometric variability in the
diatom Fragilariopsis kerguelensis: implications for Southern
965
970
975
980
985
990
995
1000
Life cycle of Fragilariopsis kerguelensis
1005
1010
1015
1020
1025
1030
1035
1040
1045
1050
1055
1060
1065
1070
1075
Ocean paleoceanography. Earth and Planetary Science Letters,
257: 526–544.
CORTESE, G., GERSONDE, R., MASCHNER, K. & MEDLEY, P. (2012).
Glacial-interglacial size variability in the diatom Fragilariopsis kerguelensis: possible iron/dust controls? Paleoceanography, 27: 1–14.
CRAWFORD, R.M. (1995). The role of sex in the sedimentation of a
marine diatom bloom. Limnology and Oceanography, 40:
200–204.
CROSTA, X. (2009). Holocene size variations in two diatom species
off East Antarctica: productivity vs environmental conditions.
Deep-Sea Research Part I Oceanographic Research Papers, 56:
1983–1993.
D'ALELIO, D., AMATO, A., LUEDEKING, A. & MONTRESOR, M. (2009a).
Sexual and vegetative phases in the planktonic diatom Pseudonitzschia multistriata. Harmful Algae, 8: 225–232.
D’ALELIO, D., AMATO, A., KOOISTRA, W.H.C.F., PROCACCINI, G.,
CASOTTI, R. & MONTRESOR, M. (2009b). Internal Transcribed
Spacer polymorphism in Pseudo-nitzschia multistriata
(Bacillariophyceae) in the Gulf of Naples: recent divergence or
intraspeciﬁc hybridization? Protist, 160: 9–20.
D’ALELIO, D., RIBERA D'ALCALÀ, M., DUBROCA, L., SARNO, D.,
ZINGONE, A. & MONTRESOR, M. (2010). The time for sex: a biennial
life cycle in a marine planktonic diatom. Limnology and
Oceanography, 55: 106–114.
DAVIDOVICH, N.A. (1994). Factors controlling the size of initial cells
in diatoms. Russian Journal of Plant Physiology, 41: 220–224.
DAVIDOVICH, N.A. & BATES, S.S. (1998). Sexual reproduction in the
pennate diatoms Pseudo-nitzschia multiseries and P. pseudodelicatissima (Bacillariophyceae). Journal of Phycology, 34:
126–137.
EDLUND, M.B. & BIXBY, R.J. (2001). Intra- and inter-speciﬁc differences in gametangial and initial cell size in diatoms. In
Proceedings of the 16th International Diatom Symposium 2000
(ECONOMOU-AMILLI, A., editor), 169–190. University of Athens,
Athens.
EVANS, K.M., WORTLEY, A.H., SIMPSON, G.E., CHEPURNOV, V.A. &
MANN, D.G. (2008). A molecular systematic approach to explore
diversity within the Sellaphora pupula species complex
(Bacillariophyta). Journal of Phycology, 44: 215–231.
GALL, M.P., BOYD, P.W., HALL, J., SAFI, K.A. & CHANG, H. (2001).
Phytoplankton processes. Part 1: Community structure during the
Southern Ocean Iron RElease Experiment (SOIREE). Deep Sea
Research Part II: Topical Studies in Oceanography, 48:
2551–2570.
GEITLER, L. (1932). Der Formwechsel der pennaten Diatomeen
(Kieselalgen). Archiv für Protistenkude, 78: 1–226.
GEITLER., L. (1973). Auxosporenbidung und Systematik bei pennaten Diatomeen und die Zytologie von Cocconeis-Sippen.
Österreichische Botanische Zeitschrift, 122: 299–321.
GUILLARD, R.L. & RYTHER, J.H. (1962). Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology, 8:
229–239.
HAMM, C.E., MERKEL, R., SPRINGER, O., JURKOJC, P., MAIER, C.,
PRECHTEL, K. & SMETACEK, V. (2003). Architecture and material
properties of diatom shell provide effective mechanical protection.
Nature, 421: 841–843.
HAMSHER, S.E., EVANS, K.M., MANN, D.G., POULÍCKOVÁ, A. &
SAUNDERS, G.W. (2011). Barcoding diatoms: exploring alternatives
to COI-5P. Protist, 162: 405–422.
HART, T.J. (1942). Phytoplankton periodicity in Antarctic surface
waters. Discovery Reports, 21: 263–348.
HASLE., G.R. (1965). Nitzschia and Fragilariopsis species studied in
the light and electron microscopes. III. The genus Fragilariopsis.
Skrifter utgitt av det Norske Videnskaps-Akademi I Oslo: I
Matematisk-Naturvideskapeling Klasse, 21: 1–49.
HASLE, G.R. (1968). Observations on the marine diatom
Fragilariopsis kerguelensis (O'Meara) Hust. in the scanning electron microscope. Nytt Magasin for Botanikk, 15: 205–208.
HASLE, G.R. & SYVERTSEN, E.E. (1997). Marine diatoms. In
Identifying Marine Phytoplankton (TOMAS, C.R., editor), 5–385.
Academic Press, San Diego, CA.
15
HOFFMANN, L.J., PEEKEN, I. & LOCHTE, K. (2007). Effects of iron on
the elemental stoichiometry during EIFEX and in the diatoms
Fragilariopsis kerguelensis and Chaetoceros dichaeta.
Biogeosciences, 4: 569–579.
HOLTERMANN, K.E., BATES, S.S., TRAINER, V.L., ODELL, A. &
ARMBRUST, E.V. (2010). Mass sexual reproduction in the toxigenic
diatoms Pseudo-nitzschia australis and P. pungens
(Bacillariophyceae) on the Washington coast. Journal of
Phycology, 46: 41–52.
HUSTEDT, F. (1958). Diatomeen aus der Antarktis und dem
Südatlantik. Deutsche Antarktische Expedition 1938/39, 2:
103–191.
IDEI, M., SATO, S., WATANABE, T., NAGUMO, T. & MANN, D.G. (2013).
Sexual reproduction and auxospore structure in Diploneis papula
(Bacillariophyta). Phycologia, 52: 295–308.
JEWSON., D.H. (1992). Size reduction, reproductive strategy and the
life strategy of a centric diatom. Philosophical Transactions of the
Royal Society of London, Series B, 336: 191–213.
KACZMARSKA, I., BATES, S.S., EHRMAN, J.M. & LÉGER, C. (2000). Fine
structure of the gamete, auxospore and initial cell in the pennate
diatom Pseudo-nitzschia multiseries (Bacillariophyta). Nova
Hedwigia, 71: 337–357.
KACZMARSKA, I., EHRMAN, J.M. & BATES, S.S. (2001). A review of
auxospore structure, ontogeny and diatom phylogeny. In
Proceedings of the 16th International Diatom Symposium 2000
(ECONOMOU-AMILLI, A., editor), 153–167. Amvrosius Press,
Athens.
KACZMARSKA, I., DAVIDOVICH, N.A. & EHRMAN, J.M. (2007). Sex cells
and reproduction in the diatom Nitzschia longissima
(Bacillariophyta): discovery of siliceous scales in gamete cell
walls and novel elements of the perizonium. Phycologia, 46:
726–737.
KACZMARSKA, I., POULÍČKOVÁ, A., SATO, S., EDLUND, M.B., IDEI, M.,
WATANABE, T. & MANN, D.G. (2013). Proposals for a terminology
for diatom sexual reproduction, auxospores and resting stages.
Diatom Research, 28: 263–294.
KOOISTRA, W.H.C.F., DE STEFANO, M., MEDLIN, L.K. & MANN, D.G.
(2003). The phylogenetic position of Toxarium, a pennate-like
lineage within centric diatoms (Bacillariophyceae). Journal of
Phycology, 39: 185–197.
LEBLANC, K. & HUTCHINS, D.A. (2005). New applications of a biogenic silica deposition ﬂuorophore in the study of oceanic diatoms.
Limnology and Oceanography – Methods, 3: 462–476.
LEVIALDI GHIRON, J.H., AMATO, A., MONTRESOR, M. & KOOISTRA, W.
C.H.F. (2008). Plastid inheritance in the planktonic raphid pennate
diatom Pseudo-nitzschia delicatissima (Bacillariophyceae).
Protist, 159: 91–98.
LUNDHOLM, N. & HASLE, G.R. (2010). Fragilariopsis
(Bacillariophyceae) of the Northern Hemisphere – morphology,
taxonomy, phylogeny and distribution, with a description of F.
paciﬁca sp. nov. Phycologia, 49: 438–460.
LUNDHOLM, N., DAUGBJERG, N. & MOESTRUP, Ø. (2002). Phylogeny of
the Bacillariaceae with emphasis on the genus Pseudo-nitzschia
(Bacillariophyceae) based on partial LSU rDNA. European
Journal of Phycology, 37: 115–134.
MANN, D.G. (1986). Methods of sexual reproduction in Nitzschia:
systematic and evolutionary implications. Diatom Research, 1:
193–203.
MANN, D.G. (1988). Why didn't Lund see sex in Asterionella? A discussion of the diatom life cycle in nature. In Algae and the Aquatic
Environment (ROUND, F.E., editor), 385–412. Biopress, Bristol.
MANN, D.G. (1989). The diatom genus Sellaphora: separation from
Navicula. British Phycological Journal, 24: 1–20.
MANN, D.G. & STICKLE, A.J. (1989). Meiosis, nuclear cyclosis, and
auxospore
formation
in
Navicula
sensu
stricto
(Bacillariophyceae). British Phycological Journal, 24: 167–181.
MANN, D.G., SATO, S., ROVIRA, L. & TROBAJO, R. (2013). Paedogamy
and auxosporulation in Nitzschia sect. Lanceolatae
(Bacillariophyta). Phycologia, 52: 204–220.
MARCHETTI, A. & CASSAR, N. (2009). Diatom elemental and morphological changes in response to iron limitation: a brief review with
potential paleoceanographic applications. Geobiology, 7: 419–431.
1080
1085
1090
1095
1100
1105
1110
1115
1120
1125
1130
1135
1140
1145
N. Fuchs et al.
1150
1155
1160
1165
1170
MARCHETTI, A. & HARRISON, P.J. (2007). Coupled changes in the cell
morphology and elemental (C, N, and Si) composition of the
pennate diatom Pseudo-nitzschia due to iron deﬁciency.
Limnology and Oceanography, 52: 2270–2284.
MONIZ, M.B.J. & KACZMARSKA, I. (2010). Barcoding of diatoms:
nuclear encoded ITS revisited. Protist, 161: 7–34.
POULÍČKOVÁ, A., MAYAMA, S., CHEPURNOV, V.A. & MANN, D.G. (2007).
Heterothallic auxosporulation, incunabula and perizonium in
Pinnularia (Bacillariophyceae). European Journal of Phycology,
42: 367–390.
QUIJANO-SCHEGGIA, S.I., GARCÉS, E., LUNDHOLM, N., MOESTRUP, O.,
ANDREE, K. & CAMP, J. (2009). Morphology, physiology, molecular
phylogeny and sexual compatibility of the cryptic Pseudonitzschia delicatissima complex (Bacillariophyta), including the
description of P. arenysensis sp. nov. Phycologia, 48: 492–509..
RAMBAUT, A. (2002). Sequence Alignment Editor v2.0a11. Available
at http://tree.bio.ed.ac.uk/software/seal/
ROUND, F.E., CRAWFORD, R.M. & MANN, D.G. (1990). The Diatoms.
Biology and Morphology of the Genera. Cambridge University
Press, Cambridge.
SARNO, D., KOOISTRA, W.C.H.F., BALZANO, S., HARGRAVES, P.E. &
ZINGONE, A. (2007). Diversity in the genus Skeletonema
(Bacillariophyceae): III. Phylogenetic position and morphological
variability of Skeletonema costatum and Skeletonema grevillei,
16
with the description of Skeletonema ardens sp. nov. Journal of
Phycology, 43: 156–170.
SARNO, D., ZINGONE, A. & MONTRESOR, M. (2010). A massive
and simultaneous sex event of two Pseudo-nitzschia species.
Deep Sea Research Part II: Topical Studies in Oceanography,
57: 248–255.
SMETACEK, V., ASSMY, P. & HENJES, J. (2004). The role of grazing in
structuring Southern Ocean pelagic ecosystems and biogeochemical cycles. Antarctic Science, 16: 541–558.
TREGUER, P., NELSON, D.M., BENNEKOM, A.J.V., DEMASTER, D.J.,
LEYNAERT, A. & QUEGUINER, B. (1995). The silica balance in the
world ocean: a reestimate. Science, 268: 375–379.
TROBAJO, R., CLAVERO, E., CHEPURNOV, V.A., SABBE, K., MANN, D.G.,
ISHIHARA, S. & COX, E.J. 2009. Morphological, genetic and mating
diversity within the widespread bioindicator Nitzschia palea
(Bacillariophyceae). Phycologia, 48: 443–459.
TROBAJO, R., ROVIRA, L., ECTOR, L., WETZEL, C.E., KELLY, M. &
MANN, D.G. (2013). Morphology and identity of some ecologically important small Nitzschia species. Diatom Research, 28:
37–59.
ZIELINSKI, U. & GERSONDE, R. (1997). Diatom distribution in
Southern Ocean surface sediments (Atlantic sector): implications
for paleoenvironmental reconstructions. Palaeogeography,
Palaeoclimatology, Palaeoecology, 129: 213–250.
1175
1180
1185
1190
1195