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Absolute abundances (concentrations) of dinoflagellate cysts are often determined through the addition of Lycopodium clavatum marker-grains as a spike to a sample before palynological processing. An inter-laboratory calibration exercise was set up in order to test the comparability of results obtained in different laboratories, each using its own preparation method. Each of the 23 laboratories received the same amount of homogenized splits of four Quaternary sediment samples. The samples originate from different localities and consisted of a variety of lithologies. Dinoflagellate cysts were extracted and counted, and relative and absolute abundances were calculated. The relative abundances proved to be fairly reproducible, notwithstanding a need for taxonomic calibration. By contrast, excessive loss of Lycopodium spores during sample preparation resulted in non-reproducibility of absolute abundances. Use of oxidation, KOH, warm acids, acetolysis, mesh sizes larger than 15 µm and long ultrasonication (> 1 min) must be avoided to determine reproducible absolute abundances. The results of this work therefore indicate that the dinoflagellate cyst worker should make a choice between using the proposed standard method which circumvents critical steps, adding Lycopodium tablets at the end of the preparation and using an alternative method.
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Determining the absolute abundance of dinoagellate cysts in recent marine
sediments: The Lycopodium marker-grain method put to the test
Kenneth Neil Mertens
, Koen Verhoeven
, Thomas Verleye
, Stephen Louwye
, Ana Amorim
Soa Ribeiro
, Amr S. Deaf
, Ian C. Harding
, Stijn De Schepper
, Catalina González
Monika Kodrans-Nsiah
, Anne De Vernal
, Maryse Henry
, Taouk Radi
, Karen Dybkjaer
Niels E. Poulsen
, Susanne Feist-Burkhardt
, Jonah Chitolie
, Claus Heilmann-Clausen
, Laurent Londeix
Jean-Louis Turon
, Fabienne Marret
, Jens Matthiessen
, Francine M.G. McCarthy
, Vandana Prasad
Vera Pospelova
, Jane E. Kyfn Hughes
, James B. Riding
, André Rochon
, Francesca Sangiorgi
Natasja Welters
, Natalie Sinclair
, Christian Thun
, Ali Soliman
, Nicolas Van Nieuwenhove
Annemiek Vink
, Martin Young
Research Unit Palaeontology, Krijgslaan 281 s8, 9000 Gent, Belgium
Instituto de Oceanograa, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
School of Ocean & Earth Science, National Oceanography Centre, Southampton, University of Southampton, European Way, Southampton, SO14 3ZH, UK
University Bremen, Geosciences Department, Historical Geology/Palaeontology, PO Box 330 440, D-28334 Bremen, Germany
GEOTOP, Université du Québec à Montréal, C.P. 8888, succursale "centre ville", Montréal, Qc, Canada H3C 3P8
Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K., Denmark
The Natural History Museum, Palaeontology Department, Cromwell Road, London SW7 5BD, UK
Geologisk Institut, Aarhus Universitet, Høegh-Guldbergs Gade 2, DK-8000 Århus C, Denmark
Université Bordeaux 1, UMR 5805 CNRS OEPOC¹, avenue des Facultés, F-33405, France
Department of Geography, University of Liverpool, Roxby Building, Liverpool, L69 7ZT, UK
Alfred Wegener Institute for Polar and Marine Research, P.O. Box 120161, D-27515 Bremerhaven, Germany
Earth Sciences, Brock University, S. Catharines, Ontario, Canada L2S 3A1
Micropaleontology laboratory, Birbal Sahni Institute of Palaeobotany, 53, University Road, Lucknow-226007, India
School of Earth and Ocean Sciences, University of Victoria, New Science Building (OEASB) A405, P.O. Box 3065 STN CSC, Victoria, B.C., Canada V8W 3V6
British Geological Survey, Kingsley Dunham Centre, Keyworth, Nottingham NG12 5GG, UK
Institut des sciences de la mer de Rimouski (ISMER), Université du Québec à Rimouski, 310, allée des Ursulines, Rimouski, QC, Canada G5L 3A1
Palaeoecology, Institute of Environmental Biology, Faculty of Science, Utrecht University, Laboratory of Palaeobotany and Palynology, Budapestlaan 4, 3584 CD Utrecht, The Netherlands
Geoscience Australia, GPO Box 378, Canberra, ACT, 2601, Australia
Karl-Franzens GRAZ University, Institute of Earth Science, Heinrichstrasse 26, A-8010 Graz, Austria
IFM-GEOMAR, Leibniz-Institute of Marine Sciences, Wischhofstrasse 1-3, 24148 Kiel, Germany
Federal Institute for Geosciences and Natural Resources, Alfred-Bentz-Haus, Stilleweg 2, 30 655 Hannover, Germany
CSIRO Petroleum, 11 Julius Ave, Riverside Corporate Park, North Ryde 2113, NSW, Australia
Research School of Earth Sciences, Australian National University, Bldg 61 Mills Road, Acton, ACT, 0200, Australia
Geology Department, Faculty of Sciences, Tanta University, Tanta 31527, Egypt
abstractarticle info
Article history:
Received 4 September 2008
Received in revised form 28 March 2009
Accepted 8 May 2009
Available online 18 May 2009
dinoagellate cyst
Lycopodium clavatum tablets
inter-laboratory calibration
Absolute abundances (concentrations) of dinoagellate cysts are often determined through the addition of
Lycopodium clavatum marker-grains as a spike to a sample before palynological processing. An inter-
laboratory calibration exercise was set up in order to test the comparability of results obtained in different
laboratories, each using its own preparation method. Each of the 23 laboratories received the same amount of
homogenized splits of four Quaternary sediment samples. The samples originate from different localities and
consisted of a variety of lithologies. Dinoagellate cysts were extracted and counted, and relative and
absolute abundances were calculated. The relative abundances proved to be fairly reproducible,
notwithstanding a need for taxonomic calibration. By contrast, excessive loss of Lycopodium spores during
sample preparation resulted in non-reproducibility of absolute abundances. Use of oxidation, KOH, warm
acids, acetolysis, mesh sizes larger than 15 µm and long ultrasonication (N1 min) must be avoided to
determine reproducible absolute abundances. The results of this work therefore indicate that the
Review of Palaeobotany and Palynology 157 (2009) 238252
Corresponding author.
E-mail address: (K.N. Mertens).
Currently at: Section for Aquatic Biology,Faculty of Sciences, University of Copenhagen, Øster Farimagsgade 2D, DK-1353,Copenhagen K, Denmark and Departamento de Geologia
Marinha, LNEG, Estrada da Portela, Zambujal 2721-866, Alfragide, Portugal.
0034-6667/$ see front matter © 2009 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Review of Palaeobotany and Palynology
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dinoagellate cyst worker should make a choice between using the proposed standard method which
circumvents critical steps, adding Lycopodium tablets at the end of the preparation and using an alternative
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Dinoagellate cyst concentrations are an important component of
paleoceanographical studies (e.g. Pospelova et al., 2006; González
et al., 2008) and can be determined using the volumetric method (e.g.
Dale et al., 2002; Holzwarth et al., 2007). In general, dinoagellate cyst
concentrations are calculated by adding a known amount of exotic
markers or a spiketo every sample according to the method de-
scribed by Stockmarr (1971). The marker commonly used is Lycopo-
dium clavatum Linnaeus (Stag's Horn Clubmoss or Ground Pine).
As noted by Lignum et al. (2008), the so-called standard
palynological processing methods are still very variable in terms of
initial sample sizes, type and concentration of acids, sieve material
and mesh size, sonication time and strength, number of decanting
cycles and use of heavy liquid separation. This is also apparent in
reviews of the preparation techniques for extraction of dinoagellate
cysts given by Wood et al. (1996) and more recently by Riding and
Kyfn-Hughes (2004). However, critical evaluation of the effect of
different laboratory procedures on the marker grain technique for
obtaining dinoagellate cyst concentration has so far never been
attempted. Although it has been reported that several processing
methods such as sonication and chemical treatments can inict da-
mage on organic-walled microfossils to a certain extent (e.g. Schrank,
1988; Hodgkinson, 1991), the effect on palynomorph concentrations
remain unknown.
This study aims to test the reproducibility of the marker-grain
method, in order to understand the discrepancies in the results fol-
lowing different preparation techniques. Similar efforts to test the
reproducibility of specic laboratory techniques have been done for
other microfossil groups: benthic and planktonic foraminifera (Zachar-
iasse et al., 1978), diatoms (Wolfe, 1997 ), nannofossils (Herrle and
Bollman, 2004)andtheirbiomarkers(Rosell-Melé et al., 2001). It is
therefore timely to carry out a similar exercise with dinoagellate cysts.
Surface sediment samples from four localities (North Sea, Celtic
Sea, NW Africa and Benguela) were sent to 23 laboratories. The
samples were processed using the palynological techniques routi-
nely used in these laboratories. An equal amount of Lycopodium
tablets, all from the same batch, were added to each sample. The
reproducibility of both absolute and relative abundances for dino-
agellate cysts is here put to test, and has resulted in a proposal of
recommendations for a standardized method to determine absolute
abundances of Quaternary dinoagellate cysts with the marker-grain
method. Two laboratories used the volumetric method (Dale, 1976)
for comparison purposes. This study focuses additionally on whether
it is necessary to count 300 or 400 dinoagellate cysts and on
taxonomy, since notable interlaboratorial differences in nomencla-
ture were recorded.
2. Material and methods
Late Quaternary surface sediment samples from four sites with
different lithologies were used by the 23 different laboratories in-
volved in the project. The North Sea sample consisted of a homo-
genized surface sediment taken using a Reineck boxcorer (51.47°N,
3.48°E, 10 m water depth). The Celtic Sea sample was assembled
through mixing multi-corer samples from Station 8, collected during
several time slots from the Celtic Sea (51.05°N, 5.83°W, 86 m water
depth) (Marret and Scourse, 2002). The sample from Northwest
Africa was a mixture of multicores GeoB9504-4 (15.87°N, 16.67°W,
43 m water depth) and GeoB9503-3 (16.07°N, 16.65°W, 50 m water
depth). The Benguela sample consists of a mixture of sediment
samples collected offshore Walvis Bay, at a water depth of about
200 m during Meteor cruise M63/2. Sample details are given in
Table 1. Each laboratory was given a number, followed by a letter
when the laboratory used more than one processing method.
Laboratory identication and numbers were kept anonymous. A
brief overview of the methods used is described in Sections 2.12.5.A
special variation of this method is detailed in Section 2.6 and the
volumetric method is detailed in Section 2.7. Details of the methods
used are given in the Supplementary data.
Homogenization was done using the quartile method. The samples
were oven-dried at a temperature of 58 °C for 24 h. The Lycopodium
spore tablets used are produced and distributed by the Subdepart-
ment of Quaternary Geology, University of Lund, Sweden (http:// Ten Lycopodium clavatum tablets of batch
483216, (X= 18.583 per tablet, s=±1708), were dispatched with the
samples, and a xed number of tablets was added by each laboratory
to each sample.
2.1. Chemical treatment
Hydrochloric acid (HCl) with a concentration of 6.536% was
added for the removal of carbonate. Some 20 to 300 ml was used
depending on the intensity of the reaction. Cold HCl was used in most
of the cases, although some laboratories used hot HCl with a tem-
perature ranging between 42 and 80 °C. Afterwards, the residue was
left to settle (15 min to 42 h). Laboratories that used short settle times
at this step, used centrifugation or sieving to concentrate the sample.
For centrifugation, the rotation speed used varied between 1900 and
3500 rpm, and lasted between 5 s to 10 min.
Demineralised or distilled water was used for rinsing until pH
reached more neutral values of 5 to 7. One to 5 decanting cycles with
intervals of 3 to 24 h were needed depending on HCl concentrations
used. To avoid losing residue during decanting, some laboratories
used centrifugation for concentration of the residue. Extensive
rinsing is necessary for the removal of Ca
, to avoid calcium uoride
) precipitation during HF treatment. A few laboratories used
KOH for neutraliz ation (laboratory 2: 1% KOH and l aboratory 18b: 10%
The siliciclastic component of the samples was removed by adding
10 to 250 ml of hydrouoric acid (HF) with a concentration ranging
from 19% to 70%. Commonly a concentration between 40 and 50% was
used. All laboratories used cold HF, except laboratories 12 (42 °C), 2
(50 °C), 6 (60 °C), 10 (70 °C) and 23 (80 °C). Settling times varied
between 12 and 144 h. A few laboratories repeated the HF treatment
up to 3 times before all silicates were removed.
Before neutralisation, about 10 to 300 ml HCl with a concentration
of 6.5 to 36 vol.% was added for the removal of formed uorosilicates.
Table 1
Description of the samples.
Sample Lithology Dry weight Number of
tablets added
# spores
St dev
North Sea Fine-medium sand 10 3 55,749 2959
Celtic Sea Fine silty sand 10 1 18,583 1708
NW Africa Clay 2 2 37,166 2416
Benguela Clay 1 4 74,332 3417
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Mostly cold HCl was used, although some laboratories used hot HCl
with a temperature ranging between 42 and 100 °C. The following
settling time varied between 15 min and 72 h. Again, laboratories that
used short settling times, used centrifugation. The sample was sub-
sequently rinsed with distilled water, until pH reached 57. The
rinsing took 1 to 6 decanting cycles with intervals of 3 to 24 h,
depending on the concentrations used. Again, to avoid losing residue
during decanting, some laboratories used centrifuging for the con-
centration of the samples. One laboratory used KOH for the neutra-
lisation (laboratory 2: 1%). A few laboratories skipped the second HCl
treatment and proceeded directly to the rinsing with distilled water
until pH reached values of 57. Several of these laboratories used
centrifuging and/or sieving for concentration of the samples. During
rinsing toxic HF was decanted and removed.
One laboratory (laboratory 22b) oxidised three of the samples
(excluding the North-West Africa sample) with Schulze's solution
(70% nitric acid saturated with potassium chlorate).
2.2. Mechanical treatment
Heavy liquid separation for the removal of heavy minerals was
carried out by a few laboratories. Labs 10 and 16 used sodium
polytungstate (SPT) at specic densities to isolate the palynological
Between 13 and 1800 s sonication was used to break down organic
matter aggregates by some laboratories. Most laboratories used sonic
baths (Branson, Sonimasse, Sonicor, Eurolab). Laboratory 8
used a standard oscillating sensor.
2.3. Sieving
Some laboratories pre-sieved before the chemical treatment for
the elimination of the coarse fraction (mesh sizes of 100, 106,120 and
150 µm) and/or ne fraction (mesh sizes of 10, 11 and 15 µm). All the
laboratories added the Lycopodium tablets before pre-sieving, except
laboratory 23.
Sieving after the chemical treatment was used to remove the ne
fraction from the residue. Calgon (sodium hexametaphosphate) was
used to disaggregate the material in a few cases. The sieve mesh sizes
used varied from 6 to 20 µm, and meshes were made of nylon,
polyester, polymer or steel. The devices used were hand, mechanical
and water pressure pumps. Some laboratories sieved without using a
2.4. Staining and mounting of the slides
Staining with a colouring agent enhances contrast for optical
microscopy and can be used for the detection of pre-Quaternary
specimens (Stanley, 1966). Safranin-O, Fuchsin or Bismark Brown
was used by a few laboratories. Not every laboratory stained the
residue. Finally a few drops of a copper sulphate solution, thymol or
phenol were often added to the residue for the inhibition of fungal
Slides were mounted on a heated metal plate (65 °C) using a
pipette, by strewing using a spatula or a mix of both methods. The
mounting medium was usually glycerin jelly, but sometimes thymol,
Elvacite, Eukitt, UV adhesive, or Canada balsam was used. Although
sealing is not per se necessary (Poulsen et al., 1990), nail polish or
Plate I. Polykrikos schwartzii extracted from the North Sea sample using different methodologies. Labs are sorted from high (upper lef t corner) to low abundances (lower right
1. Lab 1a.
2. Lab 20a.
3. Lab 13.
4. Lab 12.
5. Lab 19.
6. Lab 2.
7. Lab 11.
8. Lab 21a.
9. Lab 21b.
10. Lab 22a.
11. Lab 10a.
12. Lab 18b.
13. Lab 1b.
14. Lab 16.
15. Lab17.
16. Lab 10b.
17. Lab 18a.
18. Lab 5.
19. Lab 4.
20. Lab 22b, oxidized. All scale bars are 20 µm.
Table 2
Average percentage of the different taxa in the four samples.
Species name North Sea Celtic Sea NW Africa Benguela
Round brown cysts (RBC) 35.8± 16.0 10.0 ±7.7 3.4± 2.3 62.7 ±17.0
Spiny brown cysts (SBC) 15.5± 12.5 1.7± 3.3 2.3±2.4 8.5 ±8.5
cysts of Alexandrium spp. 0.2 ±0.3 0.5 ±0.9 0.1± 0.5
cysts of Gymnodinium spp. 0.3 ±0.6 0.3± 0.6 0.0 ±0.1 0.0 ±0.1
Stelladinium spp. 0.3±0.3 0.2± 0.2 0.3±0.3 0.1± 0.4
Lejeunecysta spp. 9.5± 12.0 1.5±1.6 0.4 ±0.5 1.4 ±1.6
Selenopemphix spp. 5.5±1.7 4.8± 2.1 1.0± 0.6 6.5 ±6.3
Tuberculodinium vancampoae 0.0± 0.1 0.1 ±0.3 0.0 ±0.1
Polykrikos spp. 6.9 ± 3.5 5.7 ± 3.8 1.2± 0.8 1.1 ±0.8
Xandarodinium xanthum 0.2 ±0.3 0.1± 0.1 0.1 ±0.2 0.0 ±0.1
Dalella chathamense ––0.0±0.1
Extremely sensitive cysts (total) 74.3± 7.4 24.8 ±11.2 15.4± 8.2 80.6 ±9.9
Lingulodinium machaerophorum 1.5± 2.5 0.7 ±0.9 86.2± 4.7 0.2 ±0.5
Operculodinium spp. 2.8±1.9 12.3± 3.7 0.5± 0.7 8.4 ± 6.6
Pyxidinopsis reticulata 0.0±0.2 –––
Spiniferites spp. 9.8±3.5 51.8 ±10.7 3.3 ±1.1 5.5±3.2
Quinquecuspis concreta 3.3± 2.1 2.3 ±2.0 0.1± 0.1 1.0±1.5
Trinovantedinium applanatum 0.2± 0.4 1.2 ±1.0 0.2± 0.3 0.3± 0.4
Votadinium spp. 5.8±6.6 0.5± 0.7 0.0±0.1 0.7± 0.7
Moderately sensitive cysts (total) 23.6±7.2 68.9 ±10.7 90.3± 4.2 16.2± 9.7
Nematosphaeropsis labyrinthus 0.0±0.1 0.0± 0.1 0.1± 0.1 2.1 ±2.0
Impagidinium spp. 0.3±0.6 0.15± 0.3 0.0± 0.1 0.0 ± 0.1
Operculodinium israelianum 0.2±0.2 0.0± 0.1 0.4± 0.7 0.4 ±0.7
Pentapharsodinium dalei 0.4± 0.5 2.6 ± 3.5 0.0± 0.1 0.2± 0.5
Polysphaeridium zoharyi 0.4 ±0.6 0.1± 0.3 0.1 ±0.5 0.2 ±0.7
Ataxiodinium choane 0.0±0.1 0.1± 0.1 0.0± 0.0
Bitectatodinium spp. 0.6 ±1.1 3.3± 2.0 0.1 ±0.2 0.2 ±0.6
Resistant cysts (total) 0.5± 0.6 6.2± 3.8 0.7± 0.9 3.1±2.5
240 K.N. Mertens et al. / Review of Palaeobotany and Palynology 157 (2009) 238252
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parafn wax was used to seal the slides to protect the residue from
degradation by dehydration.
2.5. Counting of the palynomorphs and calculation of absolute
Dinoagellate specimens were counted only when they comprised
at least half of a cyst. The same criterion was used for other
palynomorphs, also counted by some of the laboratories. Initially
300 dinoagellate cysts were counted, and subsequently an extra 100
specimens were added. The purpose was to check whether it is
necessary to count 300 or 400 dinoagellate cysts to obtain rep-
resentative relative and absolute abundances. Indeterminate dino-
agellate cysts were grouped as Indeterminate spp., and were not
taken into account for the calculation of the relative abundances, since
every observer had a different concept of what counts as an
indeterminate dinoagellate cyst, and this would introduce observer
bias into the relative abundances. Raw counts together with a sum-
mary of the methodology are available as supplementary data to this
article. Taxonomy follows Fensome and Williams (2004).
Absolute abundances of dinoagellate cysts were calculated
following the equation by Benninghoff (1962):
cconcentration=number of dinoagellate cysts/gram dried
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number of counted dinoagellate cysts
number of Lycopodium spores/tablet
tnumber of tablets added to the sample
number of counted Lycopodium spores
wweight of dried sediment (g)
Maher (1981) devised an algorithm to calculate condence limits
on microfossil concentrations. A slight correction to this algorithm
was made, since the current study used sediment weight instead of
sediment volume. The condence limits calculated based on this
algorithm have a 0.95 probability (Z=1.95). It should be noted that
these condence limits are similar to the total error on concentration
proposed by Stockmarr (1971);(Appendix B). These condence limits
can then be used in a statistical test to check whether microfossil
concentrations are the same in two different samples (Maher, 1981).
To investigate the reproducibility of results from the different
laboratories, the coefcient of variation (or relative standard devia-
tion) of all counts of a particular sample can be compared. Ideally, the
results should fall within the condence limits of Maher (1981), and
thus the coefcient of variation calculated from these condence
limits can be used as a comparison.
2.6. Special methods: the maceration tank method (with HF) and the
washing machine method (without HF)
The maceration tank method (Poulsen et al., 1990; Desezar and
Poulsen, 1994) was used for HF treatment by laboratory 20a. Other
processing steps are similar to those used by the other laboratories
and are detailed in Poulsen et al. (1990) and Desezar and Poulsen
(1994). Each sample is tightly wrapped in lter cloth (25 cm ×25 cm)
with a mesh size of 10 µm, and the lter bags are packed in rubber
foam for protection. The samples are placed inside the maceration
tank and HF is conducted to the tank in PVC tubes. The samples are
treated with cold HF for 78 days, after which the HF is drained out
through a bottom-stop cock and led via PVC tubes directly to a waste-
container for used hydrouoric acid.
With the washing machine method, used by laboratory 20b, no HF
is used. Each sample is tightly wrapped in lter cloth (25 cm× 25 cm)
with a mesh size of 10 µm and the lter bags are packed in rubber
foam for protection. The samples are washed in a standard household
washing machine with a standard household washing powder, after
which carbonates are removed with citric acid at 65 °C. Next the
samples are again given a normal wash with a standard household
washing powder. Finally the remaining minerals are removed by heavy
Plate II. Polykrikos schwartzii extracted from the Celtic Sea sample using different methodologies, sorted from high absolute abundances (upper left corner) to low absolute
abundances (lower right corner).
1. Lab 14.
2. Lab 1a.
3. Lab 13.
4. Lab 3.
5. Lab 19.
6. Lab 12.
7. Lab 1b.
8. Lab 15b.
9. Lab 1c.
10. Lab 21b.
11. Lab 21a.
12. Lab 11.
13. Lab 5.
14. Lab 4.
15. La b 16.
16. Lab 23.
17. Lab 17.
18. Lab 18a.
19. Lab 20a.
20. Lab 2. All scale bars are 20 µm.
Plate III. Lingulodinium machaerophorum extracted from the NW Africa using different methodologies, sorted from high (upper left corner) to low absolute abundances (lower right
corner). (see on page 244)
1. Lab 11.
2. Lab 1a.
3. Lab 14.
4. Lab 13.
5. Lab 19.
6. Lab 10b.
7. Lab 21a.
8. Lab 1b.
9. Lab 12.
10. Lab 17.
11. Lab 21b.
12. Lab 6.
13. Lab18a.
14. Lab 18b.
15. Lab 1c.
16. Lab 15b.
17. Lab 22a.
18. Lab 4.
19. Lab 5.
20. Lab 20b.
21. Lab 16.
22. Lab 8.
23. Lab 23.
24. Lab 3. All scale bars are 20 µm.
242 K.N. Mertens et al. / Review of Palaeobotany and Palynology 157 (2009) 238252
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liquid separation. This method removes the amorphous material very
efciently. Furthermore, since HF is not used, siliceous constituents
(e.g. diatoms) are not destroyed. Heavy liquid separation with zinc
dibromide (ZnBr
) was used at densities of 2.3, 2.0 and 1.8 g/ml to
remove heavy minerals. In order to test the inuence of the specic
density of the ZnBr
, the NW African sample from laboratory 20b, was
separated using heavy liquid densities of 1.8, 2.0 and 2.3 g/ml.
2.7. Volumetric method
For comparison with the marker-grain method, the volume aliquot
method was performed by laboratories 6 and 8, following Dale (1976).
This method was not used for the North Sea sample because of the
difculty associated with counting a xed volume of this sample with
very low abundances.
3. Results
3.1. Relative abundance of dinoagellate cysts
Quantitative and qualitative disparities between assemblages
recorded by the laboratories may be due to the different processing
methods. It is obvious that aggressive agents could destroy the
more sensitive cysts. To check this dependence of preservation on
Plate II.
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Plate III (see caption on page 242).
244 K.N. Mertens et al. / Review of Palaeobotany and Palynology 157 (2009) 238252
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methodology, it is necessary to group the present species according to
their resistance to degradation. It is assumed that both mechanical
and chemical degradation have similar effects on an assemblage. The
grouping proposed here is similar to the grouping described by
Zonneveld et al. (2001). Cysts not referred to by these authors were
added to a particular group based on the assumption that comparable
morphology (e.g. wall thickness, resistance of structures against
folding) is indicative of similar resistance to decay.
Extremely sensitive cysts: cysts of Alexandrium spp., Dalella
chathamense, cysts of Gymnodinium spp., Lejeunecysta spp., Polykri-
kos spp., round brown cysts (RBC), Selenopemphix spp., spiny brown
cysts (SBC), Stelladinium spp., Tuberculodinium vancampoae and Xan-
darodinium xanthum.
Moderately sensitive cysts:Lingulodinium machaerophorum,
Operculodinium spp., Pyxidinopsis reticulata,Quinquecuspis concreta,
Spiniferites spp., Trinovantedinium applanatum and Votadinium spp.
Resistant cysts: Ataxiodinium choane,Impagidinium spp., Nemato-
sphaeropsis labyrinthus,Operculodinium israelianum,Pentapharsodi-
nium dalei,Polysphaeridium zoharyi and Bitectatodinium spp.
It is evident from the dataset that some species were not recorded
by some observers. One obvious example is Dubridinium spp., which
was often counted by some laboratories as RBC or not counted at all.
To partly reduce this observer bias, we decided to group species into
genera or larger groups (Appendix A). Averages of relative abun-
dances were only calculated when at least 300 dinoagellate cysts
were counted. The counts from oxidized samples (laboratory 22b)
were also excluded, since all heterotrophic cysts were destroyed. The
average results of the four samples are shown in Table 2. Representa-
tive cysts from the four samples are shown in Plates IIV.
3.2. Absolute abundances of dinoagellate cysts
The cyst concentration (absolute abundance) in the North Sea
sample ranges from 570 to 3304 cysts/g, excluding the outliers:
laboratory 1a produced a very high number (8342 cysts/g) and lab-
oratory 22b a very low number (278 cysts/g). The average is
1516 cysts/g with a standard deviation of 698 cysts/g (coefcient of
variation, V=46%). The average coefcient of variation from the
condence limits of Maher (1981) is 20%. The volumetric method was
not used for the North Sea sample (Table 3).
The cyst concentration (absolute abundance) in the Celtic Sea
sample ranges from 1240 to 5284 cysts/g, excluding the outliers:
laboratories 14 and 1a produced high numbers of 75,633 and
10,961 cysts/g respectively, while laboratory 20a, 2 and 20b give
respectively low values of 1053, 731 and 501 cysts/g. The average is
2583 cysts/g, with a standard deviation of 1342 cysts/g (V= 52%). The
average coefcient of variation from the condence limits of Maher
(1981) is 25%. Results obtained by the volumetric method give
estimates that are much lower than with the marker grain method. For
the Celtic Sea these values (1160 cysts/g (laboratory 6) and 1167 cysts/
g (laboratory 8)) are even below the lowest value obtained by the
marker grain method (Table 3).
The cyst concentration (absolute abundance) in the NW Africa
sample ranges from 4606 to 38,357 cysts/g, excluding the outliers:
laboratories 11, 1a and 14 produced very high numbers (168,899,
167,651 and 129,236 cysts/g, respectively). The average is
19,441 cysts/g, with a standard deviation of 9148 cysts/g (V= 47%).
The average coefcient of variation from the condence limits of
Maher (1981) is 23%. As before, the volumetric method gave lower
estimates but within the range of the marker grain method
(11,600 cysts/g (laboratory 6) and 9992 cysts/g (laboratory 8))
(Table 3).
The cyst concentration (absolute abundance) in the Benguela
sample ranges from 30,130 to 298,972 cysts/g, excluding the outliers:
Laboratory 1c produced a high number of 1,455,988 cysts/g, while
laboratories 2 0b and 8 give values as low as 18,472 and 15,910 cysts/g,
respectively. The average is 144,299 cysts/g with a standard devi ation
of 84,159 cysts/g (V= 58%). The average coefcient of variation
from the condence limits of Maher (1981) is 21%. The volumetric
method used by laboratory 6 yields 53,200 cysts/g (within the
range above) and 8492 cysts/g by laboratory 8. The volumetric
estimate by laboratory 8 is considered to be an underestimation
caused by the destruction of fragile cysts by sonication (see
Discussion); (Table 3).
3.3. Reworked dinoagellate cysts
About 7% of the recorded dinoagellate cysts in the North Sea
sample were reworked. The pre-Quaternary cysts recorded in the
North Sea sample were Wetzeliella spp. (dominant), Glaphyrocysta
spp., Cordosphaeridium spp., cf. Oligosphaeridium spp. and cf. Cribro-
peridinium spp. In terms of absolute abundances, reworking shows
the same trends as in situ dinoagellate cyst absolute abundances.
Very high absolute abundances were recorded in the sample oxidized
by laboratory 22b. This indicates that the robust pre-Quaternary cysts
are more resistant to oxidation. Reworking is very low (less than 1%)
in the samples from the Celtic Sea, NW Africa and Benguela.
3.4. Other palynomorphs
Chlorophycean palynomorphs such as Cymatiosphaera sp. (not
present in Celtic Sea), Pediastrum sp., Pterospermella sp. (not present
in Benguela), Tas man ite s sp., Botryococcus sp. (not present in
Benguela), Mougeotia sp. (only North Sea), Concentricystes circulus
(only NW Africa), Gelasinicysta sp. indet. (only NW Africa) are
recorded in low numbers in all samples, except the North Sea sample.
Faunal remains such as microforaminiferal linings, scolecodonts,
tintinnids, planktonic crustacean eggs and invertebrate mandibles
were encountered in almost every sample. Planktonic crustacean eggs
are very abundant in the North Sea sample.
Pollen and spores are abundant in the North Sea sample. The
assemblage is dominated by pollen (90%). Non-bisaccate pollen
include Quercus,Corylus, Betula, Alnus, pollen of Poaceae, Cyperaceae
and Chenopodiaceae, whereas bisaccate pollen comprise mainly Pinus
and Picea. Some Cedrus pollen is recorded. Reworked pollen and
spores are present in low numbers.
The Celtic Sea sample is dominated by pollen (94%). Non-bisaccate
pollen comprises mainly pollen of Poaceae, Quercus,pollenof
Ericaceae and Chenopodiaceae. Bisaccate pollen is mainly Pinus
pollen. Reworked pollen and spores are very rare.
The sample from NW Africa is also dominated by pollen (95%).
Non-bisaccate pollen comprise mainly pollen of Poaceae, Quercus,
pollen of Ericaceae and pollen of Chenopodiaceae. The bisaccate
pollen are mainly Pinus pollen. Reworked pollen and spores are very
The Benguela assemblage is dominated by pollen (99%). Non-
bisaccate pollen includes mainly pollen of Poaceae, Asteraceae and
Caryophyllaceae. Bisaccate pollen is mainly Pinus pollen. No reworked
pollen and spores were recorded.
Hyphae and fruiting bodies were counted as fungal remains in
order to check whether the samples were infected by fungi. No
samples showed signicant abundances.
The recorded incertae sedis include Cyclopsiella,Halodinium sp.,
Hexasterias problematica (not present in Northwest Africa), Micrhy-
stridium sp. (Celtic Sea and Benguela), Palaeostomocystis subtilitheca
(North Sea and Celtic Sea), Radiosperma corbiferum (Celtic Sea and
Benguela) and Sigmopollis sp. (NW Africa). These were more
abundant in both North Sea and Celtic Sea samples.
Other organisms occurring are the organic linings of calcareous
dinoagellate cysts, thecamoebians (North Sea, Celtic Sea), chrysomo-
nad cysts (North Sea, Celtic Sea) and diatoms. Diatoms can still be
present when low concentrations of HF are used, possibly combined
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with heavy liquid separation, which enhances the abundance of diatoms
with low densities (laboratories 1c, 9 and 17). Laboratory 20b has good
recovery of diatoms, since the samples are not treated with HF.
4. Discussion
4.1. Is a 300 or 400 dinoagellate cyst count sufcient to reach reliable
diversities and absolute abundances?
There is no general agreement on the number of cysts which
should be counted to obtain reliable data for diversity and absolute
abundance studies. Most palynologists usually count 300 cysts per
sample, which can provide up to 98% condence (Germerad et al.,
1968). To check whether it is necessary to count 300 or 400
dinoagellate cysts, results from counting 300 cysts, plus an
additional 100 cysts are compared using absolute abundances,
species diversity and the ShannonWiener Index for all samples
(Table 3). The com parison shows that the disparities in the results are
insignicant: averages of absolute abundances, species richness and
the ShannonWiener Index show limited changes compared to the
associated standard deviations. The statistical test of Maher (1981)
indicates that all absolute abundances derived from the 300
dinoagellate cyst cou nt statistically produc e the same concentration
as from the 400 dinoagellate cyst count. It can thus be concluded
that a 300 dinoagellate cyst count is sufcient for generating
reliable diversities and absolute abundance data in Quaternary
4.2. Reproducibility of relative abundances
The standard deviations of the relative abundances observed in
the grouping based on cyst preservation are always lower than 11.2%.
These relatively small standard deviations suggest that changes in
the relative abundance counts are caused by observer bias rather
than by differences in methodology. Indeed, the highest standard
deviations in the taxonomical groupings are with the taxa RBC, SBC
and Lejeunecysta s.l. and since it can be assumed that the potential
for preservation of these taxa is similar, it is likely that the disparities
in the counts are the result of observer bias. The high standard
deviation for RBC is probably caused by the high numbers of the
morphologically similar Dubridinium spp. and the unfamiliarity of
many observers with Dubridinium spp. Furthermore, an unambig-
uous denition of a round brown cyst is still lacking. The same is true
for the spiny brown cysts, and several poorly dened species fall
within this group. All other standard deviations are lower than 10%,
which we consider an acceptable range for completely independent
dinoagellate cyst counts. Another possible reason for observer bias
could be related to the use of different illumination techniques
for routine counting of dinoagellate cysts. Comparison of the use of
phase contrast to interference contrast illumination to count
dinoagellate cysts on the same slides by laboratory 15 revealed
that phase contrast emphasizes the transparent cysts (Spiniferites s.l.,
Operculodinium s.l., Nematosphaeropsis labyrinthus,etc.),whilst
interference contrast emphasizes the brown heterotrophic cysts
(RBC, SBC, etc.). Despite the observer bias, there is no doubt that
dinoagellate cyst relative abundance counts by one single observer
are repeatable.
4.3. Explanation of outliers in absolute abundances
The higher numbers can each be explained by examining specic
methodologies employed by particular labs. Labs 1a and 1c lost an
excessive amount of Lycopodium spores due to the use of sieving at
20 µm as shown by Lignum et al. (2008). Labs 11 and 14 experienced
problems with settling after centrifugation and were not condent
that the nal residues were suitable for quantitative analysis.
The lower numbers by laboratory 22b are due to the use of
oxidation, which causes preferential destruction of dinoagellate
cysts. Due to the low amounts of material used in the exercise, the
maceration tank and washing machine method (laboratories 20a
and 20b) did not function optimally and yielded atypical results that
should not be regarded as representative. This might be due to cysts
getting attached to the large lter cloth (25 ×25 cm) used in this
technique (see Discussion,assumption8).Furthermore,oneofthe
samples from NW Africa (laboratory 20b) was separated at specic
gravities of 1.8, 2.0 and 2.3 g/ml. At the specicgravitiesof1.8and
2.3 g/ml, there were almost no dinoagellate cysts in the slides,
whereas ten times more dinocysts were noted at the specic gravity
of 2.0 g/ml. Further investigation is needed to evaluate the effect of
heavy liquid separation at different specic gravities.
For laboratory 8, the use of a sonic oscillator resulted in destruction
of sensitive cysts, again yielding lower numbers.
4.4. Reproducibility and accuracy of absolute abundances, excluding
the outliers
Total cyst count is less dependent on taxonomical expertise, and
thus probably less inuenced by observer bias. The different labo-
ratories participating in the current inter-calibration exercise used
different processing techniques (see Supplementary data). The
Plate IV. Dubridinium spp. extracted from the Benguela sample using differentmethodologies, sorted from high (upper left corner) to lowabsolute abundances (lower right corner).
1. Lab 1c.
2. Lab 3.
3. Lab 19.
4. Lab 11.
5. Lab 13.
6. Lab 1a.
7. Lab 21a.
8. Lab 21b.
9. Lab 6.
10. Lab 16.
11. Lab 18a.
12. Lab 18b.
13. Lab 1b.
14. Lab 23.
15. Lab 10b.
16. Lab 17.
17. Lab 10a.
18. Lab 5.
19. Lab 2.
20. Lab 8. Destructive ultrasonication. All scale bars are 20 µm.
246 K.N. Mertens et al. / Review of Palaeobotany and Palynology 157 (2009) 238252
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reproducibility of estimates of absolute cyst abundances, as
expressed as coefcient of variation in Table 2, shows that there are
differences among the 23 laboratories: the coefcients of variation
are relatively large (4658%)andnearlytwiceashighasthe
coefcients of variations (2025%) which are calculated from
Maher (1981). Our results suggest that the determination of absolute
abundances is mainly dependent on processing methodology. In this
light the accuracy also needs to be considered: a better under-
standing of what is causing the variation can only be achieved when
correct absolute abundances of dinoagellate cysts have been
determined. To estimate whether the absolute abundances give an
accurate picture of the true absolute abundances of the dinoagellate
cysts, results from the marker-grain method are compared with
independent methods. When compared to the volumetric method,
absolute abundances calculated using the marker-grain method, are
4463% higher (Table 2). In a similar study, de Vernal et al. (1987),
noted systematically higher concentrations from the marker-grain
method compared to the results from the volumetric method, and
they suggested that signicant losses of Lycopodium spores (close to
33% on the average) took place during laboratory procedures. On the
other hand, in a study on Paleogene sediments, Heilmann-Clausen
(1985), found marker-grain estimate s varying between 70% and 129%
of volumetric estimates and on average 2% lower concentration was
calculated from the marker-grain method. Our study conrms the
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observation of de Vernal et al. (1987), and even shows larger
deviations. It should also be noted, that counts from strew slides
made from unprocessed samples show much lower abundances than
the average absolute abundances from the marker grain method.
From these observations, it can be concluded that with most
preparation techniques there are signicant losses of Lycopodium
spores, and this is most probably the reason for higher the absolute
abundances using the marker-grain method. Furthermore, there was
no evidence of signicant loss of dinoagellate cysts during the
laboratory preparations, except when oxidation or very long or
destructive sonication was used (see below). Thus, in order to
understand what causes the differences in absolute abundances, one
needs to consider underlying assumptions. Ten assumptions need to
be considered.
(1) Drying samples does not cause decay.
Although drying is often done in palynological preparation, it
should be avoided in organic rich sediments, where drying causes
formation of selenite (gypsum, CaSO
O), by reaction of calcium
carbonate with sulphuric acid, usually derived from pyrite decay. The
formation of sulphuric acid signicantly affects extremely sensitive
dinoagellate cysts. In this case, to calculate the weight of the samples,
wet volumes should be used, corrected with dry bulk densities. In our
samples, gypsum crystals were not observed. The homogenized
samples were oven dried before subdivision into smaller batches
and dispatching to individual laboratories. This was done to avoid
differential drying. However, not all laboratories processed the
samples exactly at the same time. Samples were dispatched in
March 2007, and were processed within the following year. The
possibility exists that samples that were processed at a later stage
dried out more. Clustering of amorphous organic matter around the
cysts seems to occur in more dried out samples (most obvious around
Lingulodinium machaerophorum specimens in Plate III), but there were
no clear signs that this process caused changes in the assemblage. This
assumption is thus acceptable.
(2) Samples are homogenous.
It needed testing if samples processed in a similar manner yielded
reproducible results. All samples were processed twice by laboratory
21 (a and b) with the only difference in preparation the addition of
some soap during sieving (Table 4). Following the test by Maher
(1981), for every studied sample, the microfossil concentration in the
quasi-replicas is the same. It can thus be concluded that the samples
are well-mixed and are homogenous. Furthermore, there are few
differences between both samples in terms of relative abundances.
This assumption is thus acceptable.
(3) A single Lycopodium tablet from batch 483216 contains 18,583 ±
1708 spores.
This reference is given by the supplier (Lund University), and
these numbers were calibrated using a Coulter counter. Lignum et al.
(2008) also used a Coulter counter for verication and obtained
16, 971 ± 12 51 Lycopodium spores. We dissolved one tablet in distilled
water and sieving on a 0.25 µm Millipore lter. The lter was cut into
two pieces, mounted on a slide and counted under a transmitted light
microscope. On this lter, 16,993 Lycopodium spores were counted,
which falls within the range proposed by the supplier and Lignum
et al. (2008). A similar exercise has been done for another batch by
Stabell and Henningsmoen (1981) which found similar results. This
assumption is thus acceptable.
(4) There is no degradation of palynomorphs caused by chemical
treatment such as oxidation or acid treatments by HF and
Since Lycopodium spores are acetolysed during the manufacturing
process, they can withstand acetolysis. Effects of chemicals on Lyco-
podium show that only colour changes are caused by acetolysis or HCl
treatment (Sengupta, 1975). On the other hand, it has been shown
that acetolysis or oxidation selectively destroys the cysts of the
Polykrikaceae and Protoperidiniaceae (Reid, 1977; Marret, 1993). KOH
treatment causes destruction of the Protoperidiniaceae after 5 min (de
Vernal et al., 1996, and Mertens, pers. observations) and causes
swelling of the palynomorphs. Likewise, methods using H
et al., 2007) result in the destruction of protoperidiniacean cysts
(Riding, pers. comm., Hopkins and McCarthy, 2002; Mertens, pers.
obs.). This has also been demonstrated for Late Cretaceous peridinioid
dinoagellate cysts (Schrank, 1988). Oxidation with Schulze's solution
by laboratory 22b resulted in the near complete destruction of the
RBC, SBC and other heterotrophs in all samples, and led to the relative
enrichment of resistant pollen and reworked non-peridinioid dino-
agellate cysts. Cold HF and HCl have never been reported to destroy
dinoagellate cysts. However, hot rinses with HCl after the HF
treatment were particularly harmful to recent peridinoid cysts
(Dale, 1976). Palynomorphs treated with warm HF clearly showed
traces of deterioration: destruction of delicate structures with
fragmentation along sutures and changes in wall texture with a
thickening of the robust structures (Plate I,11,16, Plate III, 6). It can be
concluded that this assumption is acceptable when chemical
degradation is minimized by using only cold hydrochloric and
hydrouoric acid.
(5) Sonication causes no mechanical degradation of the pollen and
spores or dinoagellate cysts.
The extensive use of ultrasound will not harm any dinoagellate
cysts according to Funkhouser and Evitt (1959), however, other
authors report differential damage (e.g. Hodgkinson, 1991). This has
not yet been checked in a quantitative manner for dinoagellate cysts.
The use of a sonic oscillator, although dependent on frequency
(Marceau, 1969) is extremely damaging: the sonication by laboratory
8 resulted in the destruction of RBC and SBC in the Benguela sample
(Plate IV, 20). Laboratory 18a used an ultrasonic bath for 30 min, and
this resulted in extensive damage to the cysts. Many cysts were
fragmented, often with broken or even lost spines and were often
clustered (Plate I,17,Plate III,13,Plate IV, 11). In addition micro-
foraminiferal linings were often fragmented. This assumption is thus
acceptable when an ultrasonic bath is not used for too long. A limit of
60 s is proposed.
(6) Centrifugation causes no mechanical degradation of the
No visible signs were noted that this technique causes degradation
of the cysts. This assumption is thus acceptable.
(7) Sieving causes no loss of palynomorphs.
Lignum et al. (2008) demonstrated that sieving should be done
with a sieve mesh width smaller than 15 µm. Our results conrm this
observation. Laboratories using nylon sieve with widths of 20 µm
(laboratories 1a and 1c) showed extremely high absolute abundances.
This suggests that signicant losses of Lycopodium spores occurred
during the sieving process even larger than the 20% that is proposed
by Lignum et al. (2008). No signicant loss of cysts was documented in
this study. It is possible that cysts of Pentapharsodinium dalei pass
abundances in the studied samples to signicantly affect relative or
absolute abundances. This assumption is thus acceptable when mesh
sizes smaller than 15 µm are used.
(8) Decantation causes no loss of palynomorphs.
An experiment was done to determine how many Lycopodium
spores were lost during decanting and sieving. One gram of the NW
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Africa sample together with one Lycopodium tablet, was processed
with a HCl/HF/HCl cycle, followed by sieving on a nylon mesh of
10 µm. After every decantation, the decanted uid was ltered
through a 0.25 µm Millipore lter. What remained on the lter was
counted under a transmitted light microscope. Only Lycopodium spores
were left on thelters, as well as some amorphous organic matter (Table
5). The number of spores will be dependent of the size of the lter used.
Apparently 24% of the Lycopodium spores were lost during decanting.
This is notsurprising, sinceit is well-known that Lycopodium spores oat
(e.g. Salter et al., 2002). An extra 1.3% was left on the lter and 1% got
stuck to handling material (e.g. spatula, tube). In the slides only 43.4% of
the Lycopodium spores were found. An additional 30.2% spores were
unaccounted for, and could have been lost during sieving and/or could
have been obscured by other material in the slides to some extent.
Because we did not expect any signicant losses to occur during sieving,
we did notcapture sievedmaterial during this experiment. However, we
tested sieving a complete Lycopodium tablet on 10 µm and capture on a
0.25 µm sieve. We found losses to be 0.79% when gently pouring the
dissolved tablet over the sieve and subsequent washing, 0.97% when
using a hand pump to facilitate sieving and 2.01% when using a pipette
tip. Lignum et al. (2006) recorded losses up to 5.8± 1.2% for 15 µm
meshes. It can thus be assumed that only a small part of the missing
spores were pushed through the 10 µm nylon sieve. Presumably, spores
are oftenconcealed by beingobscured by other material, and this plays a
more signicant role in explaining the missing amount of spores. Also, it
is possible that due to the texture of the exines of Lycopodium spores, the
spores get more easily caught in the sieves than smoother palyno-
morphs. However, this loss can be easily checked by the observer. This
assumption is thus not acceptable.
(9) Pre-sieving causes no losses.
It is unclear to what extent presieving causes loss of Lycopodium
spores, although it is evident that it should be avoided in samples from
high productivity areas, where high production of amorphous organic
matter forms large clusters in the sediment, which can be discarded
with the large fraction. However; it can be easily checked whether
Lycopodium spores were lost.
(10) Heavy liquid separation causes no loss of Lycopodium spores.
It has been noted that density separation with heavy liquids
can cause incorporation of mineral particles modifying the density
of the heavy liquid (de Vernal et al., 1996). Litwin and Traverse
(1989) recommend pyrite to be removed prior to density separation.
The results of this study do not show any obvious difculties
with this processing step, although for clarity further study is
From these considerations it can be concluded that a signicant
amount of Lycopodium spores are lost, mainly during decanting and
sieving. There is little evidence that there is loss of dinoagellate cysts
during these manipulations (Table 6).
5. Conclusions and recommendations
(1) This study was designed as a comparative one, where the
degree of variability in preparations could be objectively
assessed. The laboratories concerned agreed to take part on
the basis that the results would be presented anonymously, in
order to ensure maximum participation. The point of this work
was to carefully study the techniques used and to encourage
best practice in the future. This initial work presents a rm
basis for more methodological research.
(2) The exercise demonstrated that relative abundances are re-
producible, but underlined the urgent need for taxonomic
(3) The study also shows that counting 300 dinoagellate
cysts is sufcient both in terms of diversity and absolute
Table 3
Comparison between the marker-grain method and the volumetric method.
Method Variable/sample North
Marker grain
Average (cysts/g) 1516 2583 19,441 144,299
St dev (cysts/g) 698 1342 9148 84,159
Coefcient of variation (%) 46 52 47 58
Coefcient of variation (%)
Maher (1981)
20 25 23 21
Average (cysts/g) 1163 10,796 53,200
St dev (cysts/g) 5 1137 0
Coefcient of variation (%) 0 11 0
Difference Cysts/g 1420 8645 91,099
Table 4
Comparison between the average results after counting 300 dinoagellate cysts, and counting 400 dinoagellate cysts.
Variable/sample North Sea
300 cysts
North Sea
400 cysts
Celtic Sea
300 cysts
Celtic Sea
400 cysts
NW Africa
300 cysts
NW Africa
400 cysts
300 cysts
400 cysts
Average (cysts/g) 1539 1546 2792 2670 33,798 33,684 141,825 142,612
St dev 767 711 1474 1236 43,286 42,193 87,324 88,779
Coefcient of variation (%) 50 46 53 46 128 125 62 62
Species richness 22.00 22.85 24.26 25.26 14.75 16.50 19.13 20.22
St dev 4.67 4.79 5.61 6.02 3.64 4.12 4.94 5.27
ShannonWiener index 2.25 2.25 2.29 2.29 0.70 0.72 1.94 1.92
St dev 0.41 0.41 0.30 0.32 0.22 0.23 0.35 0.33
Table 5
The results of the counts of samples processed and counted by Lab 21, processed with
one processing technique. According to the statistical test by Maher (1981), the results
are reproducible.
Lab number Variable/sample North Sea Celtic Sea NW Africa Benguela
21a Dinoagellate cysts/g 1547 2581 27,851 172,078
95% condence limits
(Maher, 1981)
21b Dinoagellate cysts/g 1447 2723 24,929 170,888
95% condence limits
(Maher, 1981)
116 6
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(4) Absolute abundance calculations of dinoagellate cysts are
dependent on processing methodology, since Lycopodium
spores are being lost during different processing steps.
(5) It is possible that some of the laboratories consistently over- or
underestimate concentrations. The addressed problems in
methodology might partly explain these outliers. Future work
should elucidate possible corrections by detailed investigation
of every different processing step.
(6) At the current state of affairs, there are three possible choices
the Quaternary worker can make to calculate reproducible
absolute abundances:
1. Standardize methodology for the extraction of dinoagellate
Since samples can be reproducible when one xed methodology
is followed (see Section 4.3), a standard methodology is
suggested (Fig. 1). We consider that there are critical steps
that must be avoided in this standard method when preparing
samples for dinoagellate cyst work: the use of oxidation, KOH,
warm acids, acetolysis, mesh sizes larger than 15 µm, decanting
(substituted by sieving) and sonication longer than 1 min.
During sieving, care should be taken to avoid Lycopodium spores
being forced through the sieve. A certain degree of freedom is
allowed in the number of HCl and HF cycles, length of
ultrasonication (060 s), duration of sieving and sieve mesh
size (614 µm), Care should be taken to neutralize HF by dilut-
ing at least ten times before sieving. Further studies are required
to ne-tune the method by focusing on designated issues.
2. Adding Lycopodium tablets at the end of processing.
The marker grain method is based on the assumption that
there is no selective loss of fossil and exotic pollen during the
procedures. However, this assumption has never been checked.
Our study suggests that predominantly Lycopodium spores are
lost, and that losses of dinoagellate cysts are negligible.
Therefore the addition of Lycopodium tablets at the end of the
preparation is suggested, thus limiting the loss of Lycopodium
spores. However, this method is contrary to spiking with an
internal standard before the start of preparation.
3. Alternative methods.
Alternative methods can be used, but may not yield better
results. The use of microbeads was introduced by Ogden
(1986), but often results in much higher abundance estimates,
apparently because of difculty in sustaining an even suspen-
sion of the particles in the stock solution: the higher specic
gravity of microspheres causes them to settle three to four
times more rapidly than pollen grains (McCarthy,1992). Other
marker-grain methods, such as the Eucalyptus globulus
marker-grain method (Matthews, 1969), has also been used
(e.g. de Vernal et al., 1987). However, it is not knownwhether
these methods give more reliable results. The aliquot method
gives more accurate results than the Lycopodium method in
our study, but unfortunately not much is known about the
precision of this method.
André Catrijsse (VLIZ), Karin Zonneveld and James Scourse are
thanked for providing samples. John Lignum (Kingston University)
and Richard Telford (Bjerknes Centre for Climate Research) are
thanked for fruitful discussions. Jane E. KyfnHughes and James B.
Riding publish with the permission of the Executive Director, British
Geological Survey (NERC). Ana Amorim refers to project MICRODYN-
POCTI/CTA/45185/2002. Three anonymous reviewers are thanked for
their constructive comments.
Table 6
Results of an experiment to look into the effects of manipulations on loss of Lycopodium
spores. Shown is the number of Lycopodium spores lost during each manipulation. It is
supposed that one tablet contains 18,583 spores, so the % is calculated by dividing the
number of counted spores by 18,583 spores.
Counted Lycopodium spores %
HCl treatment
First decantation 916 4.9
Second decantation 267 1.4
Third decantation 2485 13.4
HF/HCl treatment
First decantation 6 0.0
Second decantation 143 0.8
Third decantation 650 3.5
Left on lter (not washed off) 242 1.3
Left in tube +stuck on spatula 187 1.0
Found on slides 8067 43.4
Total 12963 69.8
Missing spores 5620 30.2
Fig. 1. Flow-chart of the proposed standardized method. AOM stands for amorphous
organic matter.
250 K.N. Mertens et al. / Review of Palaeobotany and Palynology 157 (2009) 238252
Author's personal copy
Appendix A. Species list.
Species name Grouped under North Sea Celtic Sea NW Africa Benguela
Achomosphaera andalousiensis Jan du Chêne 1977 Spiniferites s.l. x x x
Cysts of Alexandrium afne (Ioue and Fukuyo 1985) Balech 1985 Cyst of Alexandrium spp. x x
Cysts of Alexandrium tamarense (Lebour 1925) Balech 1985 Cyst of Alexandrium spp. x x
Ataxiodinium choane Reid 1974 Ataxiodinium choane xxx
Bitectatodinium spongium Zonneveld 1997 Bitectatodinium spp. x x x
Bitectatodinium tepikiense Wilson 1973 Bitectatodinium spp. x x x x
Tectatodinium pellitum Wall, 1967 emend . Head 1994 Tectatodinium spp. x
cf. Tectatodinium pellitum Wall, 1967 emend. Head 1994 Tectatodinium spp. x
Brigantedinium cariacoense (Wall 1967) Lentin and Williams 1993 Round Brown Cyst x x x x
Brigantedinium majusculum Reid 1977 ex Lentin and Williams 1993 Round Brown Cyst x x
Brigantedinium simplex Wall 1965 ex Lentin and Williams 1993 Round Brown Cyst x x x x
Cyst of Protoperidinium americanum (Gran and Braarud 1935) Balech 1974 Round Brown Cyst x x x x
Dalella chathamense McMinn and Sun 1994 Dalella chathamense x
Diplopelta? symmetrica Pavillard 1993 (Dale et al., 1993) Spiny Brown Cysts x
Dubridinium ulsterum Reid 1977 Round Brown Cyst x x x
Dubridinium caperatum Reid 1977 Round Brown Cyst x x x x
Echinidinium aculeatum Zonneveld 1997 Spiny Brown Cysts x x x x
Echinidinium bispiniformum Zonneveld 1997 Spiny Brown Cysts x x
Echinidinium delicatum Zonneveld 1997 Spiny Brown Cysts x x x x
Echinidinium granulatum Zonneveld 1997 Spiny Brown Cysts x x x x
Echinidinium transparantum Zonneveld 1997 Spiny Brown Cysts x x x
Echinidinium cf. transparantum Zonneveld 1997 Spiny Brown Cysts x x x
Cyst of Gymnodinium catenatum Graham 1943 Cyst of Gymnodinium spp. x x x x
Cyst of Gymnodinium microreticulatum Bolch et al., 1999 Cyst of Gymnodinium spp. x x
Cyst of Gymnodinium nolleri Ellegaard and Moestrup 1999 Cyst of Gymnodinium spp. x x x x
Impagidinium aculeatum (Wall 1967) Lentin and Williams 1981 Impagidinium spp. x
Impagidinium pallidum Bujak 1984 Impagidinium spp. x
Impagidinium paradoxum (Wall 1967) Stover and Evitt 1978 Impagidinium spp. x x x
Impagidinium patulum (Wall 1967) Stover and Evitt 1978 Impagidinium spp. x x x
Impagidinium sphaericum (Wall 1967) Lentin and Williams 1981 Impagidinium spp. x x x
Impagidinium strialatum (Wall 1967) Stover and Evitt 1978 Impagidinium spp. x
Impagidinium velorum Bujak 1984 Impagidinium spp. x x
Islandinium? cezare de Vernal et al., 1989 ex de Vernal in Rochon et al., 1999 Spiny Brown Cysts x
Islandinium minutum Harland and Reid in Harland et al., 1980 Spiny Brown Cysts x x x x
Leipokatium invisitatum Bradford 1975 Lejeunecysta s.l. x
Lejeunecysta diversiforma (Bradford 1977) Artzner and Dörhöfer 1978 Lejeunecysta s.l. x
Lejeunecysta marieae Harland in Harland et al., 1991 ex Lentin and Williams 1993 Lejeunecysta s.l. x
Lejeunecysta oliva (Reid 1977) Turon and Londeix 1988 Lejeunecysta s.l. x x x x
Lejeunecysta paratenella (Benedek 1972) Zonneveld and Marret xxx Lejeunecysta s.l. x x x
Lejeunecysta sabrina (Reid 1977) Bujak 1984 Lejeunecysta s.l. x x x x
Lingulodinium machaerophorum (Deandre and Cookson 1955) Wall 1967 Lingulodinium machaerophorum xxxx
Nematosphaeropsis labyrinthus (Ostenfeld 1903) Reid 1974 Nematosphaeropsis labyrinthus xxxx
Operculodinium centrocarpum sensu Wall and Dale (1966) Operculodinium s.l. x x x x
Operculodinium israelianum (Rossignol 1962) Wall 1967 Operculodinium israelianum xxx x
Operculodinium janduchenei Head et al., 1989 Operculodinium s.l. x x x x
Operculodinium sp. II? Marret, 1994 Operculodinium s.l. x
Operculodinium sp. A of Vink (2000) Operculodinium s.l. x
Cyst of Pentapharsodinium dalei Indelicato and Loeblich III 1986 Cyst of Pentapharsodinium dalei xxxx
Polykrikos kofoidii Chatton 1914 Polykrikos spp. x x x x
Polykrikos schwartzii Bütschli 1873 Polykrikos spp. x x x x
Polysphaeridium zoharyi (Rossignol 1962) Bujak et al., 1980 Polysphaeridium zoharyi xxxx
Pyxidinopsis reticulata (McMinn & Sun 1994) Marret and de Vernal 1997 Pyxidinopsis reticulata x
Quinquecuspis concreta (Reid 1977) Harland, 1977 Quinquecuspis concreta xxxx
Selenopemphix crenata Matsuoka and Bujak, 1988 Selenopemphix s.l. x
Selenopemphix nephroides Benedek 1972; emend. Bujak in Bujak et al.,1980; emend.
Benedek and Sarjeant 1981
Selenopemphix s.l. x x x x
Cyst of Protoperidinium nudum (Meunier 1919) Balech 1974 Selenopemphix s.l. x x x x
Selenopemphix quanta (Bradford 1975) Matsuoka 1985 Selenopemphix s.l. x x x
Spiniferites belerius Reid 1974 Spiniferites s.l. x x x x
Spiniferites bentorii (Rossignol 1964) Wall and Dale 1970 Spiniferites s.l. x x x x
Spiniferites bulloideus (Deandre & Cookson 1955) Sarjeant 1970 Spiniferites s.l. x x x
Spiniferites delicatus Reid 1974 Spiniferites s.l. x x x x
Spiniferites elongatus Reid 1974 Spiniferites s.l. x x x
Spiniferites hyperacanthus (Deandre and Cookson 1955) Cookson and Eisenack 1974 Spiniferites s.l. x x x x
Spiniferites lazus Reid 1974 Spiniferites s.l. x x x
Spiniferites membranaceus (Rossignol 1964) Sarjeant 1970 Spiniferites s.l. x x x x
Spiniferites mirabilis (Rossignol 1964) Sarjeant 1970 Spiniferites s.l. x x x x
Spiniferites pachydermus Rossignol 1964 Spiniferites s.l. x x x
Spiniferites ramosus (Ehrenberg 1838) Loeblich and Loeblich 1966; emend.
Davey and Williams 1966
Spiniferites s.l. x x x x
Stelladinium reidii Bradford 1975 Stelladinium spp. x x x
Stelladinium stellatum (Wall and Dale 1968) Reid 1977 Stelladinium spp. x x x x
Trinovantedinium applanatum (Bradford 1977) Bujak and Davies 1983 Trinovantedinium applanatum xxx x
(continued on next page)
(continued on next page)
251K.N. Mertens et al. / Review of Palaeobotany and Palynology 157 (2009) 238252
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Appendix B. Error calculation according to Stockmarr (1971)
According to Stockmarr (1971) total error is e=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
= error on number of spores in marker tablets
cysts counted
cysts counted = error on dinoagellate cysts counted
spores counted
spores counted = error on the number of spores counted
.Appendix C. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.revpalbo.2009.05.004.
Benninghoff, W.S., 1962. Calculation of pollen and spores density in sediments by
addition of exotic pollen in known quantities. Pollen et Spores 6, 332333.
Dale, B., 1976. Cyst formation, sedimentation, and preservation; factors affecting
dinoagellate assemblages in recent sediments from Trondheimsfjord, Norway.
Review of Palaeobotany and Palynology 22, 3960.
Dale, B., Dale, A.L., Fred Jansen, J.H., 2002. Dinoagellate cysts as environmental
indicators in surface sediments from the Congo deep-sea fan and adjacent regions.
Palaeogeography, Palaeoclimatology, Palaeoecology 185, 309338.
Desezar, Y.B., Poulsen, N.E., 1994. On palynological preparation technique. American
Association of Stratigraphic Palynologists, Newsletter 27 (3), 1213.
de Vernal, A., Larouche, A., Richard, P.J.H., 1987. Evaluation of palynomorph concentra-
tions: do the aliquot and the marker-grain methods yield comparable results?
Pollen et Spores 19 (23), 291304.
de Vernal, A., Henry, M., & Bilodeau, G., 1996. Techniques de préparation et d'analyse en
micropaléontologie. Les cahiers de GEOTOP, 3, Université de Québec a Montréal,
unpublished report, 28 pp.
Fensome, R.A., Williams, G.L., 2004. The Lentin and Williams index of fossil
dinoagellates, 2004 edition. AASP Contributions Series Number 42, 909 pp.
Funkhouser, J.W., Evitt, W.R., 1959. Preparation techniques for acid-insoluble micro-
fossils. Micropalaeontology 5, 369375.
Germerad, J.H., Hopping, C.A., Muller, J., 1968. Palynology of Tertiary sediments from
tropical areas. Review of Palaeobotany and Palynology 6, 189348.
González, C., Dupont, L.M., Mertens, K., Wefer, G., 2008. Reconstructing marine
productivity of the Cariaco Basin during marine isotope stages 3 and 4 using
organic-walled dinoagellate cysts. Paleoceanography 23 (PA3215). doi:10.1029/
Heilmann-Clausen, C., 1985. Dinoagellate stratigraphy of the Uppermost Danian to
Ypresian in the Viborg 1 borehole, Central Jylland, Denmark. Serie A/Danmarks
Geologiske Undersøgelse 7, 169.
Herrle, J.O., Bollman, J., 2004. Accuracy and reproducibility of absolute nannoplankton
abundances using the ltration technique in combination with a rotary splitter.
Marine Micropaleontology 53, 389404.
Hodgkinson, R.I., 1991. Microfossil processing: a damage report. Micropalaeontology 37,
Holzwarth, U., Esper, O., Zonneveld, K., 2007. Distribution of organic-walled dino-
agellate cysts in shelf surface sediments of the Benguela upwelling system in
relationship to environmental conditions. Marine Micropaleontology 64, 91119.
Hopkins, J.A., McCarthy, F.M.G., 2002. Postdepositional palynomorph degradation in
Quaternary shelf sediments: a laboratory experiment studying the effects of
progressive oxidation. Palynology 26, 167184.
Lignum, J., Jarvis, I., Pearce, M., 2008. A critical assessment of standard processing
methods for the preparation of palynological samples. Review of Palaeobotany and
Palynology 149, 133149.
Litwin,R.J., Traverse,A., 1989.Basic guidelinesfor palynomorphextractionand preparation
from sedimentary rocks. In:Feldman, R.M., Chapman, R.E., Hannibal, J.T. (Eds.),Paleo-
techniques: Paleontological Society, Special Publication, vol. 4, pp. 8798.
Maher Jr., L.J., 1981. Statistics for microfossil concentration measurements employing
samples spiked with marker grains. Review of Palaeobotany and Palynology 32,
153 191.
Marceau, L., 1969. Effets, sur le pollen, des ultrasons de basse frequence. Pollen et Spores
11, 14 716 4.
Marret, F., 1993. Les effets de l'acétolyse sur les assemblages de kystes de dinoagellés.
Palynosciences 2, 267272.
Marret, F., Scourse, J., 2002. Control of modern dinoagellate cyst distribution in the
Irish and Celtic seas by seasonal stratication dynamics. Marine Micropaleontology
47, 101116.
Matthews, J.,1969. The assessment of a method for the determination of absolute pollen
frequencies. New Phytologist 68, 161166.
McCarthy, F.M.G., 1992. Quaternary climate change and the evolution of the mid-
latitude western North Atlantic Ocean: palynological, foraminiferal, sedimentolo-
gical, and stable isotope evidence from DSDP sites 604, 607 and 612, unpublished
PhD dissertation, Department of Geology, Dalhousie University, Halifax, 270 pp.
Ogden III, J.G., 1986. An alternative to exotic spore or pollen addition in quantitative
microfossil studies. Canadian Journal of Earth Sciences 23, 102106.
Pospelova, V., Pedersen, T.F., de Vernal, A., 2006. Dinoagellate cysts as indicators of
climaticand oceanographic changes during the past 40 kyr in theSanta Barbara Basin,
southern California. Paleoceanography 21 (PA2010). doi:10.1029/2005PA001251.
Poulsen, N.E., Gudmundsson, L., H ansen, J.M., Husfeldt, Y., 1990. Palynologica l
preparation techniques, a new maceration tank-method and other modications.
Geological Survey of Denmark, Series C 10. 24 pp.
Reid, P.C., 1977. Peridiniacean and glenodinicacean dinoagellate cysts from the British
Isles. Nova Hedwigia 29, 429463.
Riding, J.B., Kyfn-Hughes, J.E., 2004. A review of the laboratory preparation of
palynomorphs with a description of an effective non-acid technique. Revista
Brasileira de Paleontologia 7 (1), 1344.
Riding, J.B., Kyfn-Hughes, J.E., Owens, B., 2007. An effective palynological preparation
procedure using hydrogen peroxide. Palynology 31, 1936.
Rosell-Melé, A., Bard, E., Emeis, K.C., Grimalt, J., Muller, P., Schneider, R., Bouloubassi, I.,
Epstein, B., Fahl, K., Fluegge, A., Freeman, K., Goñi, M., Guntner, U., Hartz, D.,
Hellebust, S., Herbert, T., Ikehara, M., Ishiwatari, R., Kawamura, K., Kenig, F., de
Leeuw, J., Lehman, S., Ohkouchi, N., Pancost, R.D., Prahl, F., Quinn, J., Rontani, J.F.,
Rostek, F., Rullkotter, J., Sachs, J., Sanders, D., Sawada, K., Schultz-Bull, D., Sikes, E.,
Ternois, Y., Versteegh, G., Volkman, J., Wakeham, S., 2001. Precision of the current
methods to measure alkenone proxy UK'37 and absolute alkenone abundance in
sediments: results of an inter-laboratory comparison study. Geochemistry,
Geophysics, Geosystems 2, 128 20 00GC00141.
Salter, J., Murray,B.G., Braggins, J.E., 20 02. Wettableand uns inkable: the hydrodynamics
of saccate pollen grains in relation to the pollination mechanism in the two New
Zealand species of Prumnopitys Phil. (Podocarpaceae). Annals of Botany 89,
Schrank, P., 1988. Effects of chemical processing on the preservation of peridinoid
dinoagellates: a case from the Late Cretaceous of NE Africa. Review of
Palaeobotany and Palynology 56, 123140.
Sengupta, S., 1975. Experimental alterations of the spores of Lycopodium clavatum as
related to diagenesis. Review of Palaeobotany and Palynology 19, 173192.
Stabell, B., Henningsmoen, K.E., 1981. Capsules with Lycopodium spores for absolute
diatom and pollen analysis. Nordic Journal of Botany 1 (5), 701702.
Stanley, E.A., 1966. The problem of reworked pollen and spores in marine sediments.
Marine Geology 4, 397408.
Stockmarr, J.,1971. Tablets with spores used in absolute pollen analysis. Pollen et Spores
13, 615 621.
Wolfe, A.P., 1997. On diatom concentrations in lake sediments: results from an inter-
laboratory comparison and other tests performed on a uniform sample. Journal of
Paleolimnology 18, 261268.
Wood, G.D., Gabriel, A.M., Lawson, J.C.,1996. Palynological techniques processing and
microscopy. In: Jansonius, J., McGregor, D.C. (Eds.), Palynology: Principles and
Applications, vol. 1. American Association of Stratigraphic Palynologists Foundation,
Dallas, TX, pp. 2950.
Zachariasse, W.J., Riedel, W.R., Sanlippo, A., Schmidt, R.R., Brolsma, M.J., Schrader, H.J.,
Gersonde, R., Drooger, M.M., Broekman, J.A., 1978. Micropaleontological counting
methods and techniques; an exercise on an eight metres section of the lower
Pliocene of Capo Rossello, Sicily. Utrecht Micropaleontological Bulletins 265 pp.
Zonneveld, K.A.F., Versteegh, G.J.M., de Lange, G.J., 2001. Palaeoproductivity and post-
depositional aerobic organic matter decay reected by dinoagellate cyst
assemblages of the Eastern Mediterranean S1 sapropel. Marine Geology 172,
181 195.
Appendix A (continued)
Species name Grouped under North Sea Celtic Sea NW Africa Benguela
Tuberculodinium vancampoae (Rossignol 1962) Wall 1967 Tuberculodinium vancampoae xxx
Votadinium calvum Reid 1977 Votadinium spp. x x x x
Votadinium spinosum Reid 1977 Votadinium spp. x x x
Xandarodinium xanthum Reid 1977 Xandarodinium xanthum xxxx
Appendix A (continued)
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... 3862 with n = 9666AE671 spores/tablet) were added to the samples to estimate palynomorph concentration (e.g. Mertens et al. 2009). The palynological analyses consisted of identification and counts of all palynomorphs on the slides, including pollen grains, spores, dinocysts, Halodinium and foraminifer organic linings. ...
... Pollen grains and spores were used to quantify terrestrial inputs, and reworked palynomorphs as indicators of erosion of old sedimentary material, subsequent transport and re-sedimentation. The concentration of palynomorphs is expressed as the number of specimens per gram of sediment, with an approximate error of AE10% for a confidence interval of 0.95 (de Vernal et al. 1987;Mertens et al. 2009). Fluxes were estimated using sedimentation rates based on 14 C dating and the density calculated from multisensor core logger measurements. ...
Full-text available
Palynological and sedimentological analyses were performed on the sediment core HH16‐1205‐GC retrieved from the central Isfjorden, West Spitsbergen. The sequence, which spans the last 7000 years, revealed an overall cooling trend with an important climate shift between 4.4 and 3.8 cal. ka BP, in addition to millennial‐scale oscillations. Sea‐surface reconstruction from dinocyst assemblages indicates a decrease in summer sea‐surface temperature, from 2.5 to 1.5 °C, and primary productivity, from 750 to 650 gC m−2 a−1 over the last 7000 years. From around 6.8 to 5.8 cal. ka BP, the sedimentological and palynological data suggest a predominant sediment supply from the inner part of the fjord, ice rafting, dense sea ice cover, strongly stratified water masses and high primary productivity. The interval from 4.4 to 3.8 cal. ka BP is marked by a layer of coarser material and a significant decrease in the grain‐size mode. Our geochemical data show large‐amplitude fluctuations after 2.0 cal. ka BP, while an increase in the dinocysts Impagidinium pallidum and Spiniferites elongatus from 2.0 to 1.2 cal. ka BP suggests enhanced Atlantic Water inflow. The dinocyst‐based reconstructions also reveal large‐amplitude millennial fluctuations in sea ice cover, summer sea‐surface temperature and salinity. Wavelet analysis and cross‐wavelet analysis on K/Ti ratio coupled with sea‐ice estimates confirm a strong signal with a periodicity of 1200–1500 years.
... Palynomorph concentrations (number of specimens/cm3 of dry sediments) were calculated through the marker grain method (Stockmarr, 1971;de Vernal et al., 1999;Mertens et al., 2009), consisting in adding aliquot volumes of Lycopodium spores before the palynological treatment in each sample, with these exotic spores being counted in parallel with studied marine and continental palynomorphs. ...
... Core MD04 Absolute concentrations of palynomorphs were calculated using the marker grain method (Stockmarr, 1971;de Vernal et al., 1999;Mertens et al., 2009). For such, aliquot volumes of Lycopodium spores were added to each sample before chemical treatments to allow the calculation of palynomorph concentrations as a number of palynomorphs/cm3 of dried sediments (i.e. ...
Ce travail de doctorat a pour objectif de comprendre les mécanismes et les réponses régionales de la variabilité climatique pendant la dernière déglaciation et l’Holocène en Méditerranée occidentale. Les environnements méditerranéens sont particulièrement vulnérables face aux aléas climatiques et à la pression anthropique. Il s'agit ainsi de discuter les forçages naturels et anthropiques à partir de signaux paléoenvironnementaux marins et continentaux couvrant les derniers 14 000 ans. Pour ce faire, deux séquences sédimentaires (Marge Algérienne et Golfe du Lion) ont fait l'objet d’études multiproxies principalement basées sur les études palynologiques (pollen, kystes de dinoflagellés et palynomorphes non-pollmiques), associées à des études sédimentologiques (MSCL, XRF, XRD), isotopiques, biomarqueurs moléculaires (alcénones et n-alcanes) ainsi que sur des quantifications climatiques et hydrologiques. L'analyse croisée de ces signaux, acquis avec une résolution moyenne de 150 ans sur l'Holocène et 30 ans sur l'évènement extrême du 4.2 ka BP, a permis de discuter des interactions océan-atmosphère-surfaces continentales, en questionnant l’évolution des biosphères marine et continentale au cours de l'Holocène. Ces études paléoenvironnementales s’appuient en amont sur l'étude des assemblages dinokystes et pollen reconstruits dans les sédiments modernes des deux zones d'étude. En synthèse, ce travail a permis de mettre en évidence de fortes disparités régionalisées à l’échelle orbitale et infra-orbitale selon des transects ouest-est et nord-sud. L’impact croissant des sociétés humaines, lié à une ouverture de plus en plus marquée des paysages, est discuté sur la marge algérienne depuis 5 000 ans BP, et dans le Golfe du Lion sur les derniers 1000 ans BP, avec une plus forte transmission des marqueurs d'anthropisation sur le plateau à partir de la mise en place des conditions hydrographiques modernes à partir d’environ 3 000 ans BP.
... In addition, heavy liquid separation in a solution of sodium polytungstate calibrated for a specific density of 2 was used (Munsterman and Kerstholt, 1996) for the removal of the remaining silicates and heavy minerals. According to Mertens et al. (2009), heavy liquid separation does not bias the results. Residues were mounted on microscope slides with glycerin jelly. ...
... Reworked palynomorphs were distinguished based on the preservation state and/or their known extinct range. The concentrations of palynomorphs were calculated using the marker grain method of Stockmarr (1971) and are reported as specimens per gram (g) of dry sediment with an approximate error of ±10 % for a confidence interval of 0.95 (de Vernal et al., 1987;Mertens et al., 2009). The stratigraphic occurrence of selected dinocyst and acritarch taxa is reported in Fig. 3 with reference to absolute abundance as follows: occasional (< 10 cysts g −1 ), few (10-100 cysts g −1 ), common (100-1000 cysts g −1 ) and abundant (> 1000 cysts g −1 ). ...
Full-text available
We have analyzed marine palynomorphs (mainly dinocysts and acritarchs) from the Integrated Ocean Drilling Program Site U1307 in the Labrador Sea in order to establish a detailed biostratigraphy for the Late Pliocene to Early Pleistocene. We have defined three magnetostratigraphically calibrated dinocyst and acritarch biozones in the Late Pliocene to Early Pleistocene. Zone LS1 is defined based on the highest occurrence of Barssidinium graminosum and covers the later Pliocene from 3.21 to 2.75 Ma. Zone LS2 is marked by the acme of Pyxidinopsis braboi which occurs between 2.75 and 2.57 Ma, thus encompassing the Plio–Pleistocene transition. Finally, zone LS3 extends from 2.57 to 2.23 Ma in the Early Pleistocene. The palynostratigraphic record of IODP Site U1307 is difficult to correlate to other North Atlantic and Nordic Seas sites mainly because of a different temporal resolution and a lack of well-defined biostratigraphic marker species at the basin scale. The low abundance, discontinuous occurrence and asynchronous events of warm-water Pliocene taxa such as Invertocysta lacrymosa, Impagidinium solidum, Ataxiodinium confusum, Melitasphaeridium choanophorum and Operculodinium? eirikianum suggest cooler conditions in the Labrador Sea than elsewhere in the North Atlantic, reflecting a strong regionalism. Nevertheless, as recorded at other locations in the North Atlantic, the disappearance of many dinocyst and acritarch taxa around 2.75 Ma at Site U1307 reflects a strong ecological response accompanying the intensification of the Northern Hemisphere glaciation.
... Dinoflagellate cyst slides from the sea ice, water and sediment trap samples were prepared at the Marine Biology Laboratory, University of Helsinki, after first settling samples in an Utermöhl chamber for 24 h. The settled material was rinsed into a glass beaker and Lycopodium clavatum marker grains [80][81][82] were added to each sample in order to estimate cyst concentrations and fluxes. Samples were treated with 10% hydrochloric acid to remove inorganic carbon and subsequently rinsed with deionized water. ...
Full-text available
Despite their wide use in past sea-ice reconstructions, the seasonal, habitat and species-based sources of sedimentary sea-ice proxies are poorly understood. Here, we conduct direct observations of the community composition of diatoms, dinoflagellate cysts and highly branched isoprenoid lipids within the sea ice, water column, sediment traps and sediment surface in the Belcher Islands Archipelago, Hudson Bay throughout spring 2019. We find that Arctic diatom and dinoflagellate cysts species commonly used as sea-ice proxies appear to be only indirectly linked to sea-ice conditions, and that the sediment assemblages of these groups overrepresent summertime pelagic blooms. Species contributing to the diverse sea-ice diatom communities are rare in the sediment. Dinoflagellate cysts form a typical Arctic assemblage in the sediment, although they are virtually absent in the sea ice and water column in spring. We also find that certain highly branched isoprenoid lipids that were previously considered indicators of open water, can be produced in sea-ice. We conclude that contextual knowledge and a multiproxy approach are necessary in reconstruction, encouraging further studies on the sources and controls of sea-ice proxy production in different geographic areas.
... Two calibrated tablets of Lycopodium clavatum spores (Batch no. 3862 produced by the Department of Quaternary Geology, University of Lund, Sweden; 9666 spore grains per tablet) were added to each sample to estimate absolute dinoflagellate cyst concentrations (Stockmarr, 1971;Mertens et al., 2009Mertens et al., , 2012. Samples were treated with room temperature 10% HCl to remove carbonates, rinsed with reversed osmosis water, and sieved through 120 μm and 15 μm Nitex nylon meshes to eliminate coarse and fine material. ...
Diatom and dinoflagellate cyst analyses were performed on 22 surface sediment samples from the Chukchi Sea to document their geographical distributions in one of the most understudied sections of the Arctic Ocean and to examine the influence of upper water masses on these two major groups of phytoplankton. Total concentrations vary from 0.9 to 5.9 × 10⁶ valves g⁻¹ for diatoms and from 0.8 to 12.5 × 10³ cysts g⁻¹ for organic-walled dinoflagellate cysts, with the highest values for both groups observed in the southern part of the Chukchi Sea and away from the Bering Strait. Well-preserved microfossils were recovered, with a total of 35 and 88 taxa of dinoflagellate cysts and diatoms, respectively. The most abundant diatoms are Paralia sulcata, Thalassiosira antarctica, Thalassiosira nordenskioeldii, and resting spores of Chaetoceros spp., whereas cysts of phototrophic Alexandrium spp., Operculodinium centrocarpum sensu Wall and Dale (1966), and heterotrophic Islandinium minutum and Brigantedinium spp. were most common in the dinoflagellate cyst assemblages. Cysts of HAB-causing Alexandrium spp. were found in most of the samples, with the highest abundances in Herald Canyon where they contribute ~56.6% to the cyst assemblage. As expected, cysts produced by heterotrophic dinoflagellates were more abundant where sedimentary diatom concentrations were the highest. Statistical analysis identified three major diatom and dinoflagellate cyst clusters: 1. Sites influenced by the Alaska Coastal Current in the eastern part of the Chukchi Sea are characterized by high abundances of P. sulcata and O. centrocarpum sensu Wall and Dale (1966); 2. The western part and Herald Canyon in the northern part of the Chukchi Sea are distinguished by diatoms Chaetoceros spp., T. antarctica and dinoflagellate cysts of Alexandrium spp. and affected by the Siberian Coastal Current and Bering Shelf Water; and 3. Assemblages in the southern part of the Chukchi Sea are recognized by noticeable abundances of T. nordenskioeldii and cryophilic diatom taxa, and dinoflagellate cysts I. minutum, as well as by overall lower percentages of cysts of Pentapharsodinium dalei and Brigantedinium spp. This work revealed the potential applicability of the combined use of diatoms and dinoflagellate cysts for reconstructions of past dynamic water mass changes in the Chukchi Sea.
... Данный вид является космополитным и его распространение чаще всего связано с повышенными концентрациями диатомей как источником пищи [Zonneveld et al., 2013]. Особенностью цист данного вида является их относительная неустойчивость к растворению при попадании в осадок [Mertens et al., 2009]. Высокие концентрации диатомей и цист динофлагеллат, а также максимальные значения их потоков в осадочном веществе придонного слоя вод связаны с взмучиванием верхнего слоя донных осадков течениями и, как следствие, накоплением не только горизонтальных потоков вещества, но и латеральных. ...
... The palynological residues were mounted on glass slides using glycerine jelly, sealed with nail varnish and counted (under 400× magnification) using a Leica DM2500 LED transmitted light optical microscope. When possible, at least 200 dinocyst specimens were counted (Mertens et al., 2009). Slides containing less than 50 dinocyst specimens were excluded from further analysis. ...
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Improvements in our capability to reconstruct ancient surface-ocean conditions based on organic-walled dinoflagellate cyst (dinocyst) assemblages from the Southern Ocean provide an opportunity to better establish past position, strength and oceanography of the subtropical front (STF). Here, we aim to reconstruct the late Eocene to early Miocene (37–20 Ma) depositional and palaeoceanographic history of the STF in the context of the evolving Tasmanian Gateway as well as the potential influence of Antarctic circumpolar flow and intense waxing and waning of ice. We approach this by combining information from seismic lines (revisiting existing data and generating new marine palynological data from Ocean Drilling Program (ODP) Hole 1168A) in the western Tasmanian continental slope. We apply improved taxonomic insights and palaeoecological models to reconstruct the sea surface palaeoenvironmental evolution. Late Eocene–early Oligocene (37–30.5 Ma) assemblages show a progressive transition from dominant terrestrial palynomorphs and inner-neritic dinocyst taxa as well as cysts produced by heterotrophic dinoflagellates to predominantly outer-neritic/oceanic autotrophic taxa. This transition reflects the progressive deepening of the western Tasmanian continental margin, an interpretation supported by our new seismic investigations. The dominance of autotrophic species like Spiniferites spp. and Operculodinium spp. reflects relatively oligotrophic conditions, like those of regions north of the modern-day STF. The increased abundance in the earliest Miocene of Nematosphaeropsis labyrinthus, typical for modern subantarctic zone (frontal) conditions, indicates a cooling and/or closer proximity of the STF to the site . The absence of major shifts in dinocyst assemblages contrasts with other records in the region and suggests that small changes in surface oceanographic conditions occurred during the Oligocene. Despite the relatively southerly (63–55∘ S) location of Site 1168, the rather stable oceanographic conditions reflect the continued influence of the proto-Leeuwin Current along the southern Australian coast as Australia continued to drift northward. The relatively “warm” dinocyst assemblages at ODP Site 1168, compared with the cold assemblages at Antarctic Integrated Ocean Drilling Program (IODP) Site U1356, testify to the establishment of a pronounced latitudinal temperature gradient in the Oligocene Southern Ocean.
... Palynological analyses were performed at 8 cm intervals with a volume of~5 cm 3 of wet sediment following the protocol described by Rochon et al. (1999). Marker grains (Lycopodium clavatum spore tablets, batch n°414831, Lund University) were added to each sample to estimate palynomorph concentrations following the method described by Matthews (1969) and Mertens et al. (2009). The concentrations (cysts cm -3 ) were multiplied by the sedimentation rate (cm yr -1 ) in each sample in order to describe the abundance as influxes (cysts cm -2 yr -1 ). ...
... Only in the upper part (130e50 cm) were larger intervals (4e8 cm) used. A total of 63 samples were processed using standard methods (Moore et al., 1991;Mertens et al., 2009), which included oven drying (80 C), treatment with HCl and HF at room temperature, addition of exotic markers (Lycopodium spores) for concentration estimates and sieving to remove coarse (>120 mm) and fine (<10 mm) materials. ...
A 322-cm-long sedimentary sequence obtained in the shallow marine basin of the Ría de Arousa—a submerged unglaciated river valley on the Atlantic margin of northwestern Iberia—was analysed using a multi-proxy approach to study how climatic and sea level changes affected the coastal ecosystems during the Last Glacial–Interglacial Transition. Past sedimentation, vegetation and marine productivities were inferred from palynological, radiocarbon, seismic and lithological data. A substantial reduction in the pollen and dinoflagellate cyst accumulation rates is observed at ∼12,700 to 11,700 cal a BP, suggesting lower marine and vegetation productivities likely as a response to the Younger Dryas cooling event. Overall, the regional vegetation changed from cold-tolerant open woodlands (Pinus sylvestris/P. nigra and Betula) dominating before ∼10,200 cal a BP to coastal wetlands and the regional spread of Quercus-dominated forests after ∼9800 cal a BP. Cluster analysis and principal component analysis allowed the identification of several small environmental oscillations, such as the 11.4 ka and 10.5 ka cooling events. After that, a conspicuous heath expansion was likely favoured by the palaeotopography, the increased precipitation and the relative sea level rise, which might have caused a profound change in the coastal configuration. Concurrently, both the dinoflagellate cyst and non-pollen palynomorph records reveal variations in the marine productivity and coastal hydrodynamics that also agree with a period of marked marine transgression, warming and increasing river flow. New sedimentary data highlight the high sensitivity of the ria's ecosystems to environmental oscillations and show a close temporal correspondence between terrestrial and marine responses to climate change.
Freitas, A.S.; Aguiar, V.M.C., and Baptista Neto, J.A., 2021. Modern dinoflagellate cyst abundance and trace metals as biomonitoring tools in a tropical bay in Brazil. Journal of Coastal Research, 37(6), 12471259. Coconut Creek (Florida), ISSN 0749-0208. The spatial distribution of the dinoflagellate cyst assemblages in 11 surface sediment samples collected in Guanabara Bay embayments and associated trace metals revealed the applicability of dinocysts as bioindicators of environmental conditions in estuarine systems. The surface sediment samples presented coarse to fine granulometry. The dinoflagellate cyst analysis followed the standard methodology through the elimination of carbonates and silica. Trace metals were extracted by the evaluation of 1 g of sediment plus a mixture of HNO3 and HCl in a microwave digestion system. Dinoflagellate cyst assemblages were dominated by Operculodinium centrocarpum, Spiniferites spp., and Lingulodinium machaerophorum. Trace metals (Cu, Zn, Pb, Cr, and Ni) showed variations at the different sampling points. The values obtained were Cu (6.782.8 mg kg1), Zn (29.5355.4 mg kg1), Pb (18.386.2 mg kg1), Cr (5.1196.2 mg kg1), and Ni (19.6286.0 mg kg1). Several samples showed high pseudototal concentrations of trace metals, which were above the natural concentrations found in nature. Statistical analysis showed a strong correlation between dinocysts species and some trace metals. Lingulodinium machaerophorum showed a high correlation with 4/5 analysed trace metals and is a eutrophication-sensitive species. However, Brigantedinium sp. presented a positive correlation with all trace metals analysed. The high frequency of L. machaerophorum cysts in all analysed samples associated with several trace metals is indicative of environmental eutrophication. The high availability of trace metals in the surface sediments of Guanabara Bay may be related to the untreated industrial wastes that are dumped directly into the bay.
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A safe technique useful in the preparation of spores, pollen, dinoflagellate cysts, acritarchs and other acid-insoluble microfossils is described. The technique utilizes a macerationtank for hydrofluoric acid treatment of palynological samples. Some techniques in subsequent preparation are mentioned. These include heavy-liquid separation, oxidation, ethanol water separation (modified), swirling (modified), ultrasonic treatment, filtration. -from Authors
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Dinoflagellate cysts in the uppermost Danian to Lower Eocene section in the cored Viborg 1 borehole are described. The stratigraphical distribution of the dinoflagellates is shown; eight informal dinoflagellate zones are suggested. The dinoflagellate assemblages in sediments from various Danish localities are correlated with the Viborg 1 cores. A correlation with assemblages from other European Paleocene-Lower Eocene deposits is proposed. Two new species are described, and two new combinations are proposed.
The term palynomorph generally includes the spores of lower vascular plants (isospores/homospores, and megaspores), prepollen, the pollen of gymnosperms and angiosperms, dinoflagellates, acritarchs, fungal spores, and algal remains. Chitinozoans, scolecodonts, and foraminiferal inner tests also are concentrated by palynological processing techniques, and some palynologists therefore include them as palynomorphs. These processes isolate any silt-sized or sand-sized acid-resistant organic-walled particles, however, and also will concentrate foraminiferal inner tests leaf cuticle, vascular debris from plants, chitinous insect parts, etc., which usually are not considered palynomorphs. All of the above are recovered, definition notwithstanding.
This article deals with the most important aspects of nearly twenty years of intensive study of the pollen-and-spore content of Tertiary sediments in some parts of tropical South America, Africa and Asia. For a proper evaluation, the character of the data, including the selection and preparation of the samples, the diversity of previous recording and the statistically achieved uniformity in presentation of these basic data needs a full exposition, given in the introduction. This is directly followed by an explanation of the process of elimination of all stratigraphically unimportant species. The resulting interpretation of climatic and topographical influences on the dispersal of pollen and spores is illustrated with examples from the fossil record. The disturbing effect of redeposition forms a problem, which in some cases can be solved. Now that the main ways of dispersal of pollen and spores are understood, the characteristics of the three major depositional environments can be distinguished by purely statistical analysis, without necessarily having any botanical information from probably related Recent plant species. Additionally botany and palaeontology may bring supporting evidence. This many-sided approach leads to the discrimination between local and regional features of environmental or time-stratigraphical significance which is needed for the evaluation of long-distance correlation. As a result the marker species can be classified into: (1) a restricted number of pantropical marker species; (2) a larger number of marker species which occurred in both the South American and west African regions, tropical today (transatlantic distribution); and (3) a still greater quantity of species which are of significance only within a single botanical province (intracontinental distribution). Thus a broad stratigraphical framework on a pantropical scale is established, which may be further subdivided regionally. These three systems of subzonation are compared with independent zoopalaeontological time-stratigraphical correlation and discussed in great detail, with special emphasis on the Carribean data. The major palynological changes marking the boundaries of the pantropical subzonation are thought to reflect the evolution of new groups of plants. They are mostly marked by a gradual incoming of pollen types. Extinction of plants is stratigraphically of less value, since they may have survived longer in one area than in another. Climatic boundaries are next in importance, but in general they are more restricted to specific regions. Similarly the immigration of plants, although producing sharp and useful boundaries, is only of regional value. Of least significance for regional correlation are the locally restricted boundaries which are caused by changes in habitat or dispersal. They may still be valuable for studies within one basin. An intriguing aspect of the palynological studies is formed by the possible affinity of the fossil type with Recent botanical species. Such affinities are obviously present in many fossil types. Whereas most are restricted to the level of family relationship, some interesting cases of much closer affinity are recorded here. In exceptional cases the morphogenetic development and migration of a restricted group of related pollen types can be traced. In the final section of this paper the species selected for this study have been formally described and illustrated; they include several new ones. The study is further documented by distribution charts and sections showing the stratigraphical significance of the marker types, as discussed in detail in the stratigraphical section.
Several techniques useful in the preparation of spores, pollen, hystrichospherids, and similar acid-insoluble microfossils are described. These include oxidation treatments to remove unwanted organic matter; four aids in the mechanical separation of organic and mineral matter; and two methods for making permanent slides using cover-glass smears. A feature of all of these techniques is that the residues are retained in a water-base medium throughout processing.
Students of micropaleontology need to be aware of the dangers to their specimens inherent in some preparation techniques. This article summarizes much of the published work on the damage caused to calcareous, phosphatic and siliceous material, both fossil and Recent, during mechanical and chemical processing in the laboratory, and includes the unpublished results of my own work. It stresses the importance of keeping records of procedures but does not attempt to explain the chemical reactions that must occur. Subject matter is arranged alphabetically under the headings "Mechanical and Chemical Methods and Miscellaneous Items".