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Acid-free palynological processing: a Permian case study
Journal Pre-proof Acid-free palynological processing: A Permian case study
Alexander Wheeler, Patrick T. Moss, Annette E. Götz, Joan S. Esterle, Daniel Mantle PII:
S0034-6667(20)30247-5
DOI:
https://doi.org/10.1016/j.revpalbo.2020.104343
Reference:
PALBO 104343
To appear in:
Review of Palaeobotany and Palynology
Received date:
26 July 2020
Revised date:
13 October 2020
Accepted date:
27 October 2020
Please cite this article as: A. Wheeler, P.T. Moss, A.E. Götz, et al., Acid-free palynological processing: A Permian case study, Review of Palaeobotany and Palynology (2020), https://doi.org/10.1016/j.revpalbo.2020.104343
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Acid-free palynological processing: a Permian case study Alexander Wheelera,* [email protected], Patrick T. Mossa, Annette E. Götzb, , Joan S. Esterlea, Daniel Mantlec a
School of Earth and Environmental Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia
b
State Authority for Mining, Energy and Geology, Stilleweg 2, 30655 Hannover, Germany
c
MGPalaeo PTY LTD, 5 Arvida Street, Malaga, WA 6090, Australia
*
Corresponding Author.
Abstract: Acid-free palynological processing was performed on Permian material from a borehole of
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the southern Galilee Basin, eastern Australia, to test the efficacy compared to standard
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processing techniques. Both techniques yielded well-preserved assemblages of terrestrial and aquatic palynomorphs. The proportion of phytoclasts was much higher than in samples that
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underwent standard acid processing, while spores and pollen grains occur in lower abundances. A slightly higher species count was gained from samples with acid-free
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treatment but biostratigraphical index taxa were present in both sets of samples and, in
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general, the assemblages appeared to be quantitatively comparable. To improve the efficacy for future application, in particular more accurate statistical analyses with higher counts,
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absolute abundance calculations using a Lycopodium spike and refinement of the acid-free technique using hydrogen peroxide treatments, multiple density separation steps or sonication during sieving are suggested to remove phytoclasts. Further work is required to determine if
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the higher abundances of phytoplankton are influenced by the processing technique or if they better reflect a true proportion within a given sample.
Keywords: palynology; palynofacies; preparation techniques; Galilee Basin; late Permian; Australia
1. Introduction and aims The process of liberating organic material, particularly microfossils, from sedimentary successions is necessary to interpret biostratigraphy as well as environments of deposition. Many papers examining pollen and spores use the term “standard palynological processing” when referring to their processing technique. This generally concerns a series of acidtreatments (usually hydrochloric and hydrofluoric acid) that remove the mineral component and liberate the organic material within a sample. This is sometimes followed by a float-sink 1
Journal Pre-proof using heavy liquid (zinc bromide or zinc chloride, and more recently LST®) and/or sieving. However, many researchers and commercial laboratories have developed their variations on this technique that works for them and the particular type of samples they examine (some of these techniques are summarised in Wood et al., 1996; Traverse, 2007; Brown, 2008; Lignum et al., 2008). This is undertaken because sediment and rock samples containing palynomorphs vary broadly in their mineral composition, thermal maturity, grain size and organic content. The palynomorphs themselves may also be preserved in better or poorer condition in certain samples and may require acetolysis or oxidation in nitric acid for accurate examination and identification. Thus, techniques need to be tailored to the type of sample
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processed to ensure efficient extraction of the palynomorphs contained within. For example,
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Burgess et al. (2020) used sodium polytungstate (SPT; 2.2 specific gravity) to separate finer and heavier organic material to concentrate palynomorphs for examination. In similar
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fashion, Caffrey & Horn (2013) employed LST® to palynomorphs from low-yielding
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sediments.
Because of the efficacy of acid treatments, acid-free techniques are restricted to a few labs
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and are usually only applied to relatively young, i.e. Palaeogene, Neogene, Quaternary and modern samples. Acid treatments can be problematic however due to the inherent danger of
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using strong acids such as hydrofluoric acid. Safety equipment and laboratories equipped with fume hoods are required when using these acids. Stricter safety regulations applied to
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the storage, use and disposal of these have made it more difficult for palynological processing to be undertaken in many laboratories or in the field. Facing these challenges, it has become necessary to develop more effective acid-free palynological processing techniques (O‟Keefe & Eble, 2012). However, acid-free palynological techniques have only been employed on Palaeozoic-aged material on very few occasions and never before on Permian-aged samples (Riding & Kyffin-Hughes, 2006; 2011; Riding et al., 2007).
This study investigated the efficacy of a processing technique previously used only on Cenozoic-aged samples (Van der Kaars, 1991; Moss et al., 2005; Moss & Kershaw, 2007; Moss, 2013; Moss et al., 2016) to Permian-aged material. Simple biostratigraphical and palynofacies counts were used to quantify and compare yields from the standard and acidfree approaches and to see if any technique was preferential towards, or removed any component of the assemblage.
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Journal Pre-proof 2. Materials and methods 2.1. Samples Ten samples were selected from the borehole Tambo 1-1A located on the Springsure Shelf in the southern part of the Galilee Basin (Fig. 1). Samples were collected from the Mantuan Productus Beds, Black Alley Shale and „Burngrove Formation‟ equivalent. These formations were chosen to represent a progradational deltaic sequence (Phillips et al., 2017). The samples represent a consistent biostratigraphical zone but have a variety of terrestrial and aquatic (freshwater and marine) palynomorphs. In terms of thermal maturity, coeval coal measures in the Galilee Basin show low R0 values of between 0.4% to 0.8% with values of
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around 0.6% on the Springsure Shelf (Coffey et al., 2017). Approximately 3 to 5 cm thick
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half-core samples were collected preferentially selecting siltstones and mudstones. Samples
2.2.1. Standard processing (MGPalaeo)
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2.2. Palynological Processing techniques
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were then split and half were sent for standard and the other half for acid-free processing.
Samples were sent to MGPalaeo, a commercial laboratory based in Perth, Western Australia
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for standard acid processing (Wood et al., 1996). The core samples were washed to remove any drilling mud additives or modern pollen contaminants. Fifteen to 23 g of each sample
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was carefully crushed to 2–4 mm diameter pieces before being treated with 32% hydrochloric acid (HCl) to digest any carbonate minerals. The residues were then decanted and treated
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with 48% hydrofluoric acid (HF) to remove silicate minerals. When the material had been sufficiently digested (after 24 hours for these preparations), the remaining HF was decanted and the samples underwent repeated water washes with the centrifuge to reduce the concentration of any remaining acid to non-toxic levels.. A small volume (10–15 ml) of LST® (2.1 specific gravity) was added to each sample and thoroughly mixed with the residue prior to further centrifuging. This heavy liquid separation technique concentrated the organic „float‟ with the inorganic residue settling to the base of the test tube or remaining in solution. The organic float was then washed through a 10 µm polycarbonate filter to remove the unwanted fine fraction of palynodebris.
2.2.2. Acid-free processing (University of Queensland) This technique is a modification of a process used by Van der Kaars (1991) to extract palynomorphs from deep marine sediments. Samples undergoing the acid-free processing are crushed down to a fine sand to silt size fraction (<250 μm). Between 7 and 10 g of material is 3
Journal Pre-proof generally sufficient for processing. 40 ml of 10% sodium hexametaphosphate (commercially known as Calgon) is added to the samples which are heated and left to settle overnight to deflocculate the clays. Van der Kaars (1991) originally employed tetrasodium pyrophosphate as a deflocculant but sodium hexametaphosphate is an adequate replacement, the merits of which are discussed in Riding & Kyffin-Hughes (2004). Next, the material is sieved through a 250 μm sieve to remove sand and gravel particles and through an 8 μm mesh to remove clay particles. Then, the 8 to 250 μm size fraction undergoes a series of water washes using the centrifuge. 6 ml of sodium polytungstate (SPT; 1.9 specific gravity) is then added to the remaining material, which is centrifuged for 30 minutes at 2500rpm. Lastly, the organic
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material on the surface is collected, water-washed again and mounted on a slide using Eukitt,
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a resin-based mounting medium.
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2.3. Microscopy
Both sets of samples were examined on a Zeiss Photomicroscope III with an attached Leica
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MC190HD camera and photographed using the LAS® software package. Biostratigraphical counts were made up to 200 specimens. Palynofacies counts of at least 500 particles were
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done and classified according to a modification of the scheme used by Feist-Burkhardt et al. (2008) (Fig. 2). Biostratigraphical counts were made and plotted using the Tilia palynological
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software package (Grimm, 2004). Identification of key index taxa for the late Permian was
3. Results 3.1. Palynofacies
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based on the scheme of Price (1997).
All samples processed using the acid-free method apart from TAMP28 show a much higher proportion of opaque phytoclasts than the standard processing. The abundance of opaque phytoclasts in the standard samples is between 14.4% and 52.87% while the abundance in the acid-free samples is between 38.08% and 66.28%. The total relative abundance of phytoclasts (opaque and translucent) is higher in the acid-free samples. This can be seen in the counts (Fig. 3) and by visual inspection (Plates I - III). Larger opaque particles tend to clump and may obscure palynomorphs and degraded phytoclasts, refers to clumps of fragmented phytoclast material. These are particularly abundant in the standard samples TAMP23, TAMP28 and TAMP30. High abundances of degraded phytoclasts in these samples made identification and counting of palynomorphs challenging and time consuming, potentially obscuring some key taxa. Spore and pollen grain abundances are also relatively low in the 4
Journal Pre-proof acid-free samples but yields and preservation are generally good enough for sporomorph counts. The standard samples have pollen grain and spore yields that tend to vary but are highest in samples TAMP26 and TAMP27. TAMP23 produced very low yields of palynomorphs using both processing techniques. While some pollen grains and spores are observed in the standard sample, they are too damaged or obscured by degraded phytoclasts to count and thus both samples were regarded as barren. Freshwater and brackish algal abundances tend to be relatively low in all samples apart from a small peak in Botryococcus in the acid-free sample TAMP27. The standard sample also features some Botryococcus, but
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these specimens are not as large or as abundant.
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3.2. Palynology
The acid-free processing yielded 85 different taxa in all 10 samples while the standard
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processing yielded only 77 species (Fig. 4). The species counts also show some differences between the processing techniques. All of the major expected pollen and spore taxa appear in
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both sample sets. The common spore taxon Leiotriletes directus appears to occur at a higher relative abundance in the standard samples, particularly in the lower part of the studied
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interval. Striate bisaccate pollen grains (Protohaploxypinus sp., Alisporites sp.) appear to occur at slightly higher abundances in the acid-free samples but this can vary between
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different taxa. Protohaploxypinus limpidus is the most commonly observed bisaccate pollen taxon and tends to have very similar abundances for each processing technique in most
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samples. Dulhuntyispora parvithola, a key index taxon for the late Permian in Australia (Price, 1997), appears in eight samples in the acid-free samples compared to six in the standard samples. It appears at a particularly high abundance in the acid-free sample TAMP28 (17%). Another key index taxon for the late Permian in Australia is Microreticulatisporites bitriangularis. It appears in four of the acid-free samples compared to just one of the standard samples. The abundance and diversity of algae appears to be different for each type of processing. The acid-free samples appear to yield a more diverse assemblage of algae and comparatively higher abundances of acritarchs in samples TAMP31 and TAMP32 as well as higher abundances of algae, particularly Botryococcus in TAMP27 (38%). However, the Micrhystridium evansii acritarch acme is well represented in both techniques. M. evansii makes up 76% and 83% in the acid-free samples TAMP31 and TAMP32 respectively and 68.5% and 75.5% in the standard samples. In samples TAMP28 and TAMP29, the abundance of Micrhystridium sp. is higher in the standard samples (3.5% and 4.5% respectively) compared to the acid-free samples (2% and 0.5% respectively). 5
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4. Discussion 4.1. Is the acid-free process effective? The acid-free process effectively yields a diverse range of well-preserved palynomorphs though there are some caveats. While there are apparent differences in species diversity between the two processing techniques, these extra species that appear in the acid-free processing all occur in very rare abundances and thus do not suggest a significant difference in species diversity. However, even without measuring concentrations using a marker species such as Lycopodium (Mertens et al., 2009), it is clear that palynomorph yields are generally
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higher when employing the standard processing. This is partly due to the numerous large
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phytoclast fragments overcrowding and covering palynomorphs but also potentially because palynomorphs may struggle to work through the sediments during centrifuging and density
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separation. This is most apparent in samples which contain a high abundance of clay even after significant sieving. The clays overlie the sand and silt particles during centrifuging
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forming a cap that may potentially block palynomorphs from rising to the top of the sodium polytungstate. This problem can usually be alleviated with multiple treatments of sodium
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polytungstate to increase yields. But even with this issue, yields were suitable in the processed samples to easily count to 200 palynomorphs in every sample apart from TAMP23,
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which was barren of palynomorphs in both instances.
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4.2. How does the acid-free process compare to the standard process? Even small differences in processing techniques can have strong effects on the palynomorph yield and composition of the assemblage. Lignum et al. (2008) demonstrated that just changing the type of mesh used for sieving can greatly influence the dinoflagellate cyst assemblage. In this work, each technique uses meshes of slightly different sizes (8 and 10 µm), but more importantly of different construction (nylon vs polycarbonate). Sieving is also done at different stages of the process for different purposes. Sieving is done in the acid-free technique before density separation to remove clay particles, while it is used post-density separation in the standard technique to remove fine organic material. Having said that, the palynological data presented herein show comparable results even though some differences in the composition of the assemblages could potentially be attributed to the different processing techniques. Any differences noted are also not surprising based on the relatively low number of palynomorphs counted, and the biostratigraphically important late Permian taxa for eastern Australia are represented in both assemblages. Sieving of the deflocculated clay particles 6
Journal Pre-proof potentially results in lower palynomorph yields than the standard processing technique (Riding & Kyffin-Hughes, 2011) and must be done carefully to remove as many fine clay particles as possible that can obscure the palynomorphs. Sodium polytungstate (SPT) has previously been used instead of the more toxic zinc bromide to density separate a variety of living and fossilised microorganisms (Savage, 1988; Munsterman & Kerstholt, 1996; Bolsch, 1997). While the initial costs of purchasing sodium polytungstate can be high, it can also be recycled and reused multiple times (Six et al., 1999). Tests on modern lake and peat samples using more statistically rigorous counts show different but relatively comparable pollen yields when comparing HF and sodium polytungstate (Leipe et al., 2019). Moss et al. (2005)
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achieved pollen yields from the mid Eocene Republic Formation in Washington, USA using
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the acid-free techniques, which had been previously described as barren using the standard acid technique. The high abundance of degraded phytoclasts in several samples processed
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using the standard technique and the comparatively low abundance in their acid-free counterparts suggests that these are an artefact of the processing technique. They are easy to
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recognise and differentiate from amorphous organic matter (AOM) and true plant detritus (Tyson, 1995); however, their presence can be problematic as they can affect palynofacies
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counts and obscure palynomorphs. This is potentially the reason for the large difference in the abundance of the relatively fragile Brazilea scissa in TAMP30, whereas Leiotriletes
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directus is robust and easily identifiable.
methods?
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4.3. How does this acid-free technique compare with other non-standard processing
Riding & Kyffin-Hughes (2004; 2006) and Riding et al. (2007) developed and refined a technique employing sodium hexametaphosphate and treatments of hydrogen peroxide to initially deflocculate the clays and then oxidise the phytoclast material to concentrate palynomorphs. This technique was tested on samples of a variety of ages (Ordovician, Carboniferous, Jurassic and Palaeogene) with variable results. Hydrogen peroxide is a powerful oxidising agent and can also damage palynomorph assemblages (Hopkins & McCarthy, 2002). It has also been noted to selectively damage dinoflagellate cysts (Hopkins & McCarthy, 2002). Algal bodies such as Botryococcus have relatively low preservation potential and are easily broken up during sieving and centrifuging (Tyson, 1995; FeistBurkhardt et al., 2008). While the acid-free technique appears to favour preservation of Botryococcus, further work is required to determine if this is due to the processing technique, and if it accurately reflects the true assemblage. 7
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5. Conclusions and outlook The standard processing technique using HF and HCl remains an effective way to liberate palynomorphs from most types of pre-Cenozoic rock and sediments. However, this study shows that the acid-free treatment is also effective at liberating palynomorphs from Permianaged samples. The advantages of this acid-free palynological processing technique lie in its safety relative to HF treatments and the simplicity of the technique, which offers wide applicability. However, further testing and refinement of this technique would be useful to improve its efficacy and to better understand how it compares to the standard technique. For
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more accurate statistical analysis higher counts of up to 1000 specimens would be more
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suitable particularly in the case of Permian samples where common taxa such as Protohaploxypinus and Leiotriletes can overcrowd counts. A better test of this technique,
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though, would be further application on a wider variety of samples from different lithologies and ages. The addition of a spike of Lycopodium would also be useful to calculate the yield of
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palynomorphs per gram of sediment processed. This will allow us to find any losses related to sieving and centrifuging and help better understand statistical variability between the two
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processing techniques. Further refinements of the acid-free technique would focus on further separation of palynomorphs from phytoclasts such as through the use of peroxide treatments,
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centrifuging at different speeds, multiple density separations or sonication-assisted sieving (Riding et al., 2007; Bryant & Holloway, 2009; Caffrey & Horn, 2013; Perrotti et al., 2013)
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The limits of this technique also require further examination. Testing this on the clay-rich, organic-poor samples such as those of the Early Triassic Rewan Group may prove informative as well on carbonate-rich rocks, which might require some addition of hydrochloric acid to remove the minerals. Thermal maturity may also prove to be a significant factor and samples from the Bowen Basin would be ideal to test this parameter.
Acknowledgements The authors would like to thank the Vale UQ Coal Geoscience Program for funding this study, and AASP – The Palynological Society for providing additional funding. Many thanks also to MGPalaeo for kind assistance with sample processing. We would also like to thank Jen O‟Keefe and Jim Riding for detailed reviews that greatly improved this manuscript. Declaration of interests
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Journal Pre-proof The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Supplementary data Supplementary material 1 Supplementary material 2 References
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Bolch, C. J. S., 1997. The use of sodium polytungstate for the separation and concentration of living dinoflagellate cysts from marine sediments. Phycologia 36(6), 472-478. Brown, C.A., 2008. Palynological Techniques, In: Riding, J.B., Warny, S. (Eds.), Second Edition. American Association of Stratigraphic Palynologists Foundation, Dallas, Texas, U.S.A, pp. 137. Bryant, V. M., Holloway, R. G., 2009. Reducing charcoal abundance in archaeological pollen samples. Palynology, 33(2), 63-72. Burgess, R., Jolley, D., Hartley, A., 2020. Stratigraphic palynology of the Middle–Late Triassic successions of the Central North Sea. Petroleum Geoscience. DOI 10.1144/petgeo2019-128. Caffrey, M. A., Horn, S. P., 2013. The use of lithium heteropolytungstate in the heavy liquid separation of samples which are sparse in pollen. Palynology, 37(1), 143-150. Feist-Burkhardt, S., Götz, A. E., Ruckwied, K., Russell, J. W., 2008. Palynofacies patterns, acritarch diversity and stable isotope signatures in the Lower Muschelkalk (Middle Triassic) of N Switzerland: Evidence of third-order cyclicity. Swiss Journal of Geosciences 101(1), 115. Grimm, E.C., 2004. TGView Version 2.0.2. Illinois State Museum, Springfield. Hopkins, J. A., McCarthy, F. M. G., 2002. Post‐depositional palynomorph degradation in quaternary shelf sediments: A laboratory experiment studying the effects of progressive oxidation. Palynology 26, 167-184. Leipe, C., Kobe, F., Müller, S., 2019. Testing the performance of sodium polytungstate and lithium heteropolytungstate as non-toxic dense media for pollen extraction from lake and peat sediment samples. Quaternary International 516, 207-214. Lignum, J., Jarvis, I., Pearce, M. A., 2008. A critical assessment of standard processing methods for the preparation of palynological samples. Review of Palaeobotany and Palynology 149(3-4), 133-149. Mertens, K.N., Verhoeven, K., Verleye, T., Louwye, S., Amorim, A., Ribeiro, S., Deaf, A.S., Harding, I.C., De Schepper, S., González, C. Kodrans-Nsiah, M., 2009. Determining the absolute abundance of dinoflagellate cysts in recent marine sediments: the Lycopodium marker-grain method put to the test. Review of Palaeobotany and Palynology 157(3-4), 238252. Moss, P. T., Kershaw, A. P., 2007. A late Quaternary marine palynological record (oxygen isotope stages 1 to 7) for the humid tropics of northeastern Australia based on ODP Site 820. Palaeogeography, Palaeoclimatology, Palaeoecology 251(1), 4-22. Moss, P. T., Greenwood, D. R., Archibald, S. B., 2005. Regional and local vegetation community dynamics of the Eocene Okanagan Highlands (British Columbia Washington State) from palynology. Canadian Journal of Earth Sciences 42(2), 187-204. Moss, P. T., Smith, R. Y., Greenwood, D. R., 2016. A window into mid-latitudinal Early Eocene environmental variability: a high-resolution palynological analysis of the Falkland site, Okanagan Highlands, British Columbia, Canada. Canadian Journal of Earth Sciences 53(6), 605-613. 9
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Moss, P.T., 2013. Palynology and its application to geomorphology, In: Shroder, J.F. (Ed.), Treatise on Geomorphology. Academic Press, San Diego, pp. 315-325. Munsterman, D., Kerstholt, S., 1996. Sodium polytungstate, a new non-toxic alternative to bromoform in heavy liquid separation. Review of Palaeobotany and Palynology 91(1-4), 417422. O‟Keefe, J. M. K., Eble, C. F. 2012. A comparison of HF-based and non-HF-based palynology processing techniques in clay-rich lignites from the Claiborne Group, upper Mississippi Embayment, United States. Palynology, 36(1) 116-130. Perrotti, A. G., Siskind, T., Bryant, M. K., Bryant, V. M., 2018. Efficacy of sonicationassisted sieving on Quaternary pollen samples. Palynology, 42(4), 466-474. Phillips, L. J., Edwards, S. A., Bianchi, V., Esterle, J. S., 2017. Paleo-environmental reconstruction of Lopingian (upper Permian) sediments in the Galilee Basin, Queensland, Australia. Australian Journal of Earth Sciences 64(5), 587-609. Price, P. L., 1997. Permian to Jurassic palynostratigraphic nomenclature of the Bowen and Surat Basins. The Surat and Bowen Basins, south-east Queensland. Queensland Minerals and Energy Review Series, Queensland Department of Mines and Energy, Brisbane, 137, pp. 178. Riding, J. B., Kyffin-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), 13-44. Riding, J. B., & Kyffin‐Hughes, J. E. 2006. Further testing of a non‐acid palynological preparation procedure. Palynology 30(1), 69-87. Riding, J. B., Kyffin-Hughes, J. E., 2011. A direct comparison of three palynological preparation techniques. Review of Palaeobotany and Palynology 167(3-4), 212-221. Riding, J. B., Kyffin-Hughes, J. E., Owens, B., 2007. An effective palynological preparation procedure using hydrogen peroxide. Palynology 31(1), 19-36. Savage, N. M., 1988. The use of sodium polytungstate for conodont separations. Journal of Micropalaeontology 7(1), 39-40. Six, J., Schultz, P. A., Jastrow, J. D., Merck, R., 1999. Recycling of sodium polytungstate used in soil organic matter studies. Soil Biology & Biochemistry 31(8), 1193-1196. Traverse, A., 2007. Paleopalynology. Springer Science & Business Media, 2nd. ed., p. 813. Tyson, R.V., 1995. Sedimentary Organic Matter; Organic Facies and Palynofacies. London, Chapman & Hall, p. 615. Van der Kaars, W. A., 1991. Palynology of eastern Indonesian marine piston-cores: a Late Quaternary vegetational and climatic record for Australasia. Palaeogeography, Palaeoclimatology, Palaeoecology 85(3-4), 239-302. 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, pp. 29-50. Figure 1: Map and lithological log in stratigraphical context showing the position of Tambo 1-1A in the Galilee Basin of eastern Australia and the samples taken from the borehole. MPB – Mantuan Productus Beds, BFE – “Burngrove Formation” equivalent. Figure 2: Classification scheme for palynofacies (from Feist-Burkhardt et al., 2008). Figure 3: Relative abundance bar chart for the palynofacies data comparing the acid-free (left) and standard (right) processing techniques for each sample.
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Journal Pre-proof Figure 4: Relative abundance chart showing a comparison of the acid-free processing (red) and the standard processing (blue) for each species observed and counted. MPB – Mantuan Productus Beds, BFE – “Burngrove Formation” equivalent. Plate I: Photomicrograph comparison of the standard processing technique (left) and the acid-free technique (right). 1 – 2: TAMP23; 3 – 4: TAMP24; 5 – 6: TAMP25; 7 – 8: TAMP26; Notable features include: 1 – degraded phytoclasts; 2, 4, 6 & 7 – large opaque phytoclasts; 3, 5 & 7 – well preserved bisaccate pollen grains. Plate II: Photomicrograph comparison of the standard processing technique (left) and the acid-free technique (right). 1 – 2: TAMP27; 3 – 4: TAMP28; 5 – 6: TAMP29; 7 – 8:
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TAMP30. Notable features include: 1 – small Botryococcus; 2 – large Botryococcus; 3 & 7 –
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degraded phytoclasts; 4, 5, 6 & 8 – large, well-preserved palynomorphs and phytoclasts. Plate III: Photomicrograph comparison of the standard processing technique (left) and the
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acid-free technique (right). 1 – 2: TAMP31; 3 – 4: TAMP32; Notable features include: 1 & 3
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– Micrhystridium evansii; 2 & 4 – Micrhystridium evansii and large phytoclasts. Highlights
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An acid-free palynological processing technique is applied to Permian-aged samples for the first time. The acid-free processing technique is applied to both shallow marine and terrestrial (deltaic) sediments. Palynological and palynofacies data is compared between with the standard acid processing techniques. Palynological data between each technique is comparable with high yields of terrestrial palynomorphs and acritarchs. Quantitative differences between each technique are discussed.
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Alexander Wheeler, Patrick T. Moss, Annette E. Götz, Joan S. Esterle, Daniel Mantle PII:
S0034-6667(20)30247-5
DOI:
https://doi.org/10.1016/j.revpalbo.2020.104343
Reference:
PALBO 104343
To appear in:
Review of Palaeobotany and Palynology
Received date:
26 July 2020
Revised date:
13 October 2020
Accepted date:
27 October 2020
Please cite this article as: A. Wheeler, P.T. Moss, A.E. Götz, et al., Acid-free palynological processing: A Permian case study, Review of Palaeobotany and Palynology (2020), https://doi.org/10.1016/j.revpalbo.2020.104343
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Acid-free palynological processing: a Permian case study Alexander Wheelera,* [email protected], Patrick T. Mossa, Annette E. Götzb, , Joan S. Esterlea, Daniel Mantlec a
School of Earth and Environmental Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia
b
State Authority for Mining, Energy and Geology, Stilleweg 2, 30655 Hannover, Germany
c
MGPalaeo PTY LTD, 5 Arvida Street, Malaga, WA 6090, Australia
*
Corresponding Author.
Abstract: Acid-free palynological processing was performed on Permian material from a borehole of
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the southern Galilee Basin, eastern Australia, to test the efficacy compared to standard
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processing techniques. Both techniques yielded well-preserved assemblages of terrestrial and aquatic palynomorphs. The proportion of phytoclasts was much higher than in samples that
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underwent standard acid processing, while spores and pollen grains occur in lower abundances. A slightly higher species count was gained from samples with acid-free
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treatment but biostratigraphical index taxa were present in both sets of samples and, in
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general, the assemblages appeared to be quantitatively comparable. To improve the efficacy for future application, in particular more accurate statistical analyses with higher counts,
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absolute abundance calculations using a Lycopodium spike and refinement of the acid-free technique using hydrogen peroxide treatments, multiple density separation steps or sonication during sieving are suggested to remove phytoclasts. Further work is required to determine if
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the higher abundances of phytoplankton are influenced by the processing technique or if they better reflect a true proportion within a given sample.
Keywords: palynology; palynofacies; preparation techniques; Galilee Basin; late Permian; Australia
1. Introduction and aims The process of liberating organic material, particularly microfossils, from sedimentary successions is necessary to interpret biostratigraphy as well as environments of deposition. Many papers examining pollen and spores use the term “standard palynological processing” when referring to their processing technique. This generally concerns a series of acidtreatments (usually hydrochloric and hydrofluoric acid) that remove the mineral component and liberate the organic material within a sample. This is sometimes followed by a float-sink 1
Journal Pre-proof using heavy liquid (zinc bromide or zinc chloride, and more recently LST®) and/or sieving. However, many researchers and commercial laboratories have developed their variations on this technique that works for them and the particular type of samples they examine (some of these techniques are summarised in Wood et al., 1996; Traverse, 2007; Brown, 2008; Lignum et al., 2008). This is undertaken because sediment and rock samples containing palynomorphs vary broadly in their mineral composition, thermal maturity, grain size and organic content. The palynomorphs themselves may also be preserved in better or poorer condition in certain samples and may require acetolysis or oxidation in nitric acid for accurate examination and identification. Thus, techniques need to be tailored to the type of sample
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processed to ensure efficient extraction of the palynomorphs contained within. For example,
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Burgess et al. (2020) used sodium polytungstate (SPT; 2.2 specific gravity) to separate finer and heavier organic material to concentrate palynomorphs for examination. In similar
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fashion, Caffrey & Horn (2013) employed LST® to palynomorphs from low-yielding
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sediments.
Because of the efficacy of acid treatments, acid-free techniques are restricted to a few labs
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and are usually only applied to relatively young, i.e. Palaeogene, Neogene, Quaternary and modern samples. Acid treatments can be problematic however due to the inherent danger of
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using strong acids such as hydrofluoric acid. Safety equipment and laboratories equipped with fume hoods are required when using these acids. Stricter safety regulations applied to
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the storage, use and disposal of these have made it more difficult for palynological processing to be undertaken in many laboratories or in the field. Facing these challenges, it has become necessary to develop more effective acid-free palynological processing techniques (O‟Keefe & Eble, 2012). However, acid-free palynological techniques have only been employed on Palaeozoic-aged material on very few occasions and never before on Permian-aged samples (Riding & Kyffin-Hughes, 2006; 2011; Riding et al., 2007).
This study investigated the efficacy of a processing technique previously used only on Cenozoic-aged samples (Van der Kaars, 1991; Moss et al., 2005; Moss & Kershaw, 2007; Moss, 2013; Moss et al., 2016) to Permian-aged material. Simple biostratigraphical and palynofacies counts were used to quantify and compare yields from the standard and acidfree approaches and to see if any technique was preferential towards, or removed any component of the assemblage.
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Journal Pre-proof 2. Materials and methods 2.1. Samples Ten samples were selected from the borehole Tambo 1-1A located on the Springsure Shelf in the southern part of the Galilee Basin (Fig. 1). Samples were collected from the Mantuan Productus Beds, Black Alley Shale and „Burngrove Formation‟ equivalent. These formations were chosen to represent a progradational deltaic sequence (Phillips et al., 2017). The samples represent a consistent biostratigraphical zone but have a variety of terrestrial and aquatic (freshwater and marine) palynomorphs. In terms of thermal maturity, coeval coal measures in the Galilee Basin show low R0 values of between 0.4% to 0.8% with values of
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around 0.6% on the Springsure Shelf (Coffey et al., 2017). Approximately 3 to 5 cm thick
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half-core samples were collected preferentially selecting siltstones and mudstones. Samples
2.2.1. Standard processing (MGPalaeo)
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2.2. Palynological Processing techniques
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were then split and half were sent for standard and the other half for acid-free processing.
Samples were sent to MGPalaeo, a commercial laboratory based in Perth, Western Australia
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for standard acid processing (Wood et al., 1996). The core samples were washed to remove any drilling mud additives or modern pollen contaminants. Fifteen to 23 g of each sample
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was carefully crushed to 2–4 mm diameter pieces before being treated with 32% hydrochloric acid (HCl) to digest any carbonate minerals. The residues were then decanted and treated
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with 48% hydrofluoric acid (HF) to remove silicate minerals. When the material had been sufficiently digested (after 24 hours for these preparations), the remaining HF was decanted and the samples underwent repeated water washes with the centrifuge to reduce the concentration of any remaining acid to non-toxic levels.. A small volume (10–15 ml) of LST® (2.1 specific gravity) was added to each sample and thoroughly mixed with the residue prior to further centrifuging. This heavy liquid separation technique concentrated the organic „float‟ with the inorganic residue settling to the base of the test tube or remaining in solution. The organic float was then washed through a 10 µm polycarbonate filter to remove the unwanted fine fraction of palynodebris.
2.2.2. Acid-free processing (University of Queensland) This technique is a modification of a process used by Van der Kaars (1991) to extract palynomorphs from deep marine sediments. Samples undergoing the acid-free processing are crushed down to a fine sand to silt size fraction (<250 μm). Between 7 and 10 g of material is 3
Journal Pre-proof generally sufficient for processing. 40 ml of 10% sodium hexametaphosphate (commercially known as Calgon) is added to the samples which are heated and left to settle overnight to deflocculate the clays. Van der Kaars (1991) originally employed tetrasodium pyrophosphate as a deflocculant but sodium hexametaphosphate is an adequate replacement, the merits of which are discussed in Riding & Kyffin-Hughes (2004). Next, the material is sieved through a 250 μm sieve to remove sand and gravel particles and through an 8 μm mesh to remove clay particles. Then, the 8 to 250 μm size fraction undergoes a series of water washes using the centrifuge. 6 ml of sodium polytungstate (SPT; 1.9 specific gravity) is then added to the remaining material, which is centrifuged for 30 minutes at 2500rpm. Lastly, the organic
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material on the surface is collected, water-washed again and mounted on a slide using Eukitt,
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a resin-based mounting medium.
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2.3. Microscopy
Both sets of samples were examined on a Zeiss Photomicroscope III with an attached Leica
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MC190HD camera and photographed using the LAS® software package. Biostratigraphical counts were made up to 200 specimens. Palynofacies counts of at least 500 particles were
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done and classified according to a modification of the scheme used by Feist-Burkhardt et al. (2008) (Fig. 2). Biostratigraphical counts were made and plotted using the Tilia palynological
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software package (Grimm, 2004). Identification of key index taxa for the late Permian was
3. Results 3.1. Palynofacies
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based on the scheme of Price (1997).
All samples processed using the acid-free method apart from TAMP28 show a much higher proportion of opaque phytoclasts than the standard processing. The abundance of opaque phytoclasts in the standard samples is between 14.4% and 52.87% while the abundance in the acid-free samples is between 38.08% and 66.28%. The total relative abundance of phytoclasts (opaque and translucent) is higher in the acid-free samples. This can be seen in the counts (Fig. 3) and by visual inspection (Plates I - III). Larger opaque particles tend to clump and may obscure palynomorphs and degraded phytoclasts, refers to clumps of fragmented phytoclast material. These are particularly abundant in the standard samples TAMP23, TAMP28 and TAMP30. High abundances of degraded phytoclasts in these samples made identification and counting of palynomorphs challenging and time consuming, potentially obscuring some key taxa. Spore and pollen grain abundances are also relatively low in the 4
Journal Pre-proof acid-free samples but yields and preservation are generally good enough for sporomorph counts. The standard samples have pollen grain and spore yields that tend to vary but are highest in samples TAMP26 and TAMP27. TAMP23 produced very low yields of palynomorphs using both processing techniques. While some pollen grains and spores are observed in the standard sample, they are too damaged or obscured by degraded phytoclasts to count and thus both samples were regarded as barren. Freshwater and brackish algal abundances tend to be relatively low in all samples apart from a small peak in Botryococcus in the acid-free sample TAMP27. The standard sample also features some Botryococcus, but
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these specimens are not as large or as abundant.
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3.2. Palynology
The acid-free processing yielded 85 different taxa in all 10 samples while the standard
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processing yielded only 77 species (Fig. 4). The species counts also show some differences between the processing techniques. All of the major expected pollen and spore taxa appear in
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both sample sets. The common spore taxon Leiotriletes directus appears to occur at a higher relative abundance in the standard samples, particularly in the lower part of the studied
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interval. Striate bisaccate pollen grains (Protohaploxypinus sp., Alisporites sp.) appear to occur at slightly higher abundances in the acid-free samples but this can vary between
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different taxa. Protohaploxypinus limpidus is the most commonly observed bisaccate pollen taxon and tends to have very similar abundances for each processing technique in most
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samples. Dulhuntyispora parvithola, a key index taxon for the late Permian in Australia (Price, 1997), appears in eight samples in the acid-free samples compared to six in the standard samples. It appears at a particularly high abundance in the acid-free sample TAMP28 (17%). Another key index taxon for the late Permian in Australia is Microreticulatisporites bitriangularis. It appears in four of the acid-free samples compared to just one of the standard samples. The abundance and diversity of algae appears to be different for each type of processing. The acid-free samples appear to yield a more diverse assemblage of algae and comparatively higher abundances of acritarchs in samples TAMP31 and TAMP32 as well as higher abundances of algae, particularly Botryococcus in TAMP27 (38%). However, the Micrhystridium evansii acritarch acme is well represented in both techniques. M. evansii makes up 76% and 83% in the acid-free samples TAMP31 and TAMP32 respectively and 68.5% and 75.5% in the standard samples. In samples TAMP28 and TAMP29, the abundance of Micrhystridium sp. is higher in the standard samples (3.5% and 4.5% respectively) compared to the acid-free samples (2% and 0.5% respectively). 5
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4. Discussion 4.1. Is the acid-free process effective? The acid-free process effectively yields a diverse range of well-preserved palynomorphs though there are some caveats. While there are apparent differences in species diversity between the two processing techniques, these extra species that appear in the acid-free processing all occur in very rare abundances and thus do not suggest a significant difference in species diversity. However, even without measuring concentrations using a marker species such as Lycopodium (Mertens et al., 2009), it is clear that palynomorph yields are generally
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higher when employing the standard processing. This is partly due to the numerous large
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phytoclast fragments overcrowding and covering palynomorphs but also potentially because palynomorphs may struggle to work through the sediments during centrifuging and density
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separation. This is most apparent in samples which contain a high abundance of clay even after significant sieving. The clays overlie the sand and silt particles during centrifuging
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forming a cap that may potentially block palynomorphs from rising to the top of the sodium polytungstate. This problem can usually be alleviated with multiple treatments of sodium
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polytungstate to increase yields. But even with this issue, yields were suitable in the processed samples to easily count to 200 palynomorphs in every sample apart from TAMP23,
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which was barren of palynomorphs in both instances.
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4.2. How does the acid-free process compare to the standard process? Even small differences in processing techniques can have strong effects on the palynomorph yield and composition of the assemblage. Lignum et al. (2008) demonstrated that just changing the type of mesh used for sieving can greatly influence the dinoflagellate cyst assemblage. In this work, each technique uses meshes of slightly different sizes (8 and 10 µm), but more importantly of different construction (nylon vs polycarbonate). Sieving is also done at different stages of the process for different purposes. Sieving is done in the acid-free technique before density separation to remove clay particles, while it is used post-density separation in the standard technique to remove fine organic material. Having said that, the palynological data presented herein show comparable results even though some differences in the composition of the assemblages could potentially be attributed to the different processing techniques. Any differences noted are also not surprising based on the relatively low number of palynomorphs counted, and the biostratigraphically important late Permian taxa for eastern Australia are represented in both assemblages. Sieving of the deflocculated clay particles 6
Journal Pre-proof potentially results in lower palynomorph yields than the standard processing technique (Riding & Kyffin-Hughes, 2011) and must be done carefully to remove as many fine clay particles as possible that can obscure the palynomorphs. Sodium polytungstate (SPT) has previously been used instead of the more toxic zinc bromide to density separate a variety of living and fossilised microorganisms (Savage, 1988; Munsterman & Kerstholt, 1996; Bolsch, 1997). While the initial costs of purchasing sodium polytungstate can be high, it can also be recycled and reused multiple times (Six et al., 1999). Tests on modern lake and peat samples using more statistically rigorous counts show different but relatively comparable pollen yields when comparing HF and sodium polytungstate (Leipe et al., 2019). Moss et al. (2005)
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achieved pollen yields from the mid Eocene Republic Formation in Washington, USA using
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the acid-free techniques, which had been previously described as barren using the standard acid technique. The high abundance of degraded phytoclasts in several samples processed
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using the standard technique and the comparatively low abundance in their acid-free counterparts suggests that these are an artefact of the processing technique. They are easy to
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recognise and differentiate from amorphous organic matter (AOM) and true plant detritus (Tyson, 1995); however, their presence can be problematic as they can affect palynofacies
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counts and obscure palynomorphs. This is potentially the reason for the large difference in the abundance of the relatively fragile Brazilea scissa in TAMP30, whereas Leiotriletes
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directus is robust and easily identifiable.
methods?
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4.3. How does this acid-free technique compare with other non-standard processing
Riding & Kyffin-Hughes (2004; 2006) and Riding et al. (2007) developed and refined a technique employing sodium hexametaphosphate and treatments of hydrogen peroxide to initially deflocculate the clays and then oxidise the phytoclast material to concentrate palynomorphs. This technique was tested on samples of a variety of ages (Ordovician, Carboniferous, Jurassic and Palaeogene) with variable results. Hydrogen peroxide is a powerful oxidising agent and can also damage palynomorph assemblages (Hopkins & McCarthy, 2002). It has also been noted to selectively damage dinoflagellate cysts (Hopkins & McCarthy, 2002). Algal bodies such as Botryococcus have relatively low preservation potential and are easily broken up during sieving and centrifuging (Tyson, 1995; FeistBurkhardt et al., 2008). While the acid-free technique appears to favour preservation of Botryococcus, further work is required to determine if this is due to the processing technique, and if it accurately reflects the true assemblage. 7
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5. Conclusions and outlook The standard processing technique using HF and HCl remains an effective way to liberate palynomorphs from most types of pre-Cenozoic rock and sediments. However, this study shows that the acid-free treatment is also effective at liberating palynomorphs from Permianaged samples. The advantages of this acid-free palynological processing technique lie in its safety relative to HF treatments and the simplicity of the technique, which offers wide applicability. However, further testing and refinement of this technique would be useful to improve its efficacy and to better understand how it compares to the standard technique. For
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more accurate statistical analysis higher counts of up to 1000 specimens would be more
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suitable particularly in the case of Permian samples where common taxa such as Protohaploxypinus and Leiotriletes can overcrowd counts. A better test of this technique,
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though, would be further application on a wider variety of samples from different lithologies and ages. The addition of a spike of Lycopodium would also be useful to calculate the yield of
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palynomorphs per gram of sediment processed. This will allow us to find any losses related to sieving and centrifuging and help better understand statistical variability between the two
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processing techniques. Further refinements of the acid-free technique would focus on further separation of palynomorphs from phytoclasts such as through the use of peroxide treatments,
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centrifuging at different speeds, multiple density separations or sonication-assisted sieving (Riding et al., 2007; Bryant & Holloway, 2009; Caffrey & Horn, 2013; Perrotti et al., 2013)
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The limits of this technique also require further examination. Testing this on the clay-rich, organic-poor samples such as those of the Early Triassic Rewan Group may prove informative as well on carbonate-rich rocks, which might require some addition of hydrochloric acid to remove the minerals. Thermal maturity may also prove to be a significant factor and samples from the Bowen Basin would be ideal to test this parameter.
Acknowledgements The authors would like to thank the Vale UQ Coal Geoscience Program for funding this study, and AASP – The Palynological Society for providing additional funding. Many thanks also to MGPalaeo for kind assistance with sample processing. We would also like to thank Jen O‟Keefe and Jim Riding for detailed reviews that greatly improved this manuscript. Declaration of interests
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Journal Pre-proof The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Supplementary data Supplementary material 1 Supplementary material 2 References
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Bolch, C. J. S., 1997. The use of sodium polytungstate for the separation and concentration of living dinoflagellate cysts from marine sediments. Phycologia 36(6), 472-478. Brown, C.A., 2008. Palynological Techniques, In: Riding, J.B., Warny, S. (Eds.), Second Edition. American Association of Stratigraphic Palynologists Foundation, Dallas, Texas, U.S.A, pp. 137. Bryant, V. M., Holloway, R. G., 2009. Reducing charcoal abundance in archaeological pollen samples. Palynology, 33(2), 63-72. Burgess, R., Jolley, D., Hartley, A., 2020. Stratigraphic palynology of the Middle–Late Triassic successions of the Central North Sea. Petroleum Geoscience. DOI 10.1144/petgeo2019-128. Caffrey, M. A., Horn, S. P., 2013. The use of lithium heteropolytungstate in the heavy liquid separation of samples which are sparse in pollen. Palynology, 37(1), 143-150. Feist-Burkhardt, S., Götz, A. E., Ruckwied, K., Russell, J. W., 2008. Palynofacies patterns, acritarch diversity and stable isotope signatures in the Lower Muschelkalk (Middle Triassic) of N Switzerland: Evidence of third-order cyclicity. Swiss Journal of Geosciences 101(1), 115. Grimm, E.C., 2004. TGView Version 2.0.2. Illinois State Museum, Springfield. Hopkins, J. A., McCarthy, F. M. G., 2002. Post‐depositional palynomorph degradation in quaternary shelf sediments: A laboratory experiment studying the effects of progressive oxidation. Palynology 26, 167-184. Leipe, C., Kobe, F., Müller, S., 2019. Testing the performance of sodium polytungstate and lithium heteropolytungstate as non-toxic dense media for pollen extraction from lake and peat sediment samples. Quaternary International 516, 207-214. Lignum, J., Jarvis, I., Pearce, M. A., 2008. A critical assessment of standard processing methods for the preparation of palynological samples. Review of Palaeobotany and Palynology 149(3-4), 133-149. Mertens, K.N., Verhoeven, K., Verleye, T., Louwye, S., Amorim, A., Ribeiro, S., Deaf, A.S., Harding, I.C., De Schepper, S., González, C. Kodrans-Nsiah, M., 2009. Determining the absolute abundance of dinoflagellate cysts in recent marine sediments: the Lycopodium marker-grain method put to the test. Review of Palaeobotany and Palynology 157(3-4), 238252. Moss, P. T., Kershaw, A. P., 2007. A late Quaternary marine palynological record (oxygen isotope stages 1 to 7) for the humid tropics of northeastern Australia based on ODP Site 820. Palaeogeography, Palaeoclimatology, Palaeoecology 251(1), 4-22. Moss, P. T., Greenwood, D. R., Archibald, S. B., 2005. Regional and local vegetation community dynamics of the Eocene Okanagan Highlands (British Columbia Washington State) from palynology. Canadian Journal of Earth Sciences 42(2), 187-204. Moss, P. T., Smith, R. Y., Greenwood, D. R., 2016. A window into mid-latitudinal Early Eocene environmental variability: a high-resolution palynological analysis of the Falkland site, Okanagan Highlands, British Columbia, Canada. Canadian Journal of Earth Sciences 53(6), 605-613. 9
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Moss, P.T., 2013. Palynology and its application to geomorphology, In: Shroder, J.F. (Ed.), Treatise on Geomorphology. Academic Press, San Diego, pp. 315-325. Munsterman, D., Kerstholt, S., 1996. Sodium polytungstate, a new non-toxic alternative to bromoform in heavy liquid separation. Review of Palaeobotany and Palynology 91(1-4), 417422. O‟Keefe, J. M. K., Eble, C. F. 2012. A comparison of HF-based and non-HF-based palynology processing techniques in clay-rich lignites from the Claiborne Group, upper Mississippi Embayment, United States. Palynology, 36(1) 116-130. Perrotti, A. G., Siskind, T., Bryant, M. K., Bryant, V. M., 2018. Efficacy of sonicationassisted sieving on Quaternary pollen samples. Palynology, 42(4), 466-474. Phillips, L. J., Edwards, S. A., Bianchi, V., Esterle, J. S., 2017. Paleo-environmental reconstruction of Lopingian (upper Permian) sediments in the Galilee Basin, Queensland, Australia. Australian Journal of Earth Sciences 64(5), 587-609. Price, P. L., 1997. Permian to Jurassic palynostratigraphic nomenclature of the Bowen and Surat Basins. The Surat and Bowen Basins, south-east Queensland. Queensland Minerals and Energy Review Series, Queensland Department of Mines and Energy, Brisbane, 137, pp. 178. Riding, J. B., Kyffin-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), 13-44. Riding, J. B., & Kyffin‐Hughes, J. E. 2006. Further testing of a non‐acid palynological preparation procedure. Palynology 30(1), 69-87. Riding, J. B., Kyffin-Hughes, J. E., 2011. A direct comparison of three palynological preparation techniques. Review of Palaeobotany and Palynology 167(3-4), 212-221. Riding, J. B., Kyffin-Hughes, J. E., Owens, B., 2007. An effective palynological preparation procedure using hydrogen peroxide. Palynology 31(1), 19-36. Savage, N. M., 1988. The use of sodium polytungstate for conodont separations. Journal of Micropalaeontology 7(1), 39-40. Six, J., Schultz, P. A., Jastrow, J. D., Merck, R., 1999. Recycling of sodium polytungstate used in soil organic matter studies. Soil Biology & Biochemistry 31(8), 1193-1196. Traverse, A., 2007. Paleopalynology. Springer Science & Business Media, 2nd. ed., p. 813. Tyson, R.V., 1995. Sedimentary Organic Matter; Organic Facies and Palynofacies. London, Chapman & Hall, p. 615. Van der Kaars, W. A., 1991. Palynology of eastern Indonesian marine piston-cores: a Late Quaternary vegetational and climatic record for Australasia. Palaeogeography, Palaeoclimatology, Palaeoecology 85(3-4), 239-302. 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, pp. 29-50. Figure 1: Map and lithological log in stratigraphical context showing the position of Tambo 1-1A in the Galilee Basin of eastern Australia and the samples taken from the borehole. MPB – Mantuan Productus Beds, BFE – “Burngrove Formation” equivalent. Figure 2: Classification scheme for palynofacies (from Feist-Burkhardt et al., 2008). Figure 3: Relative abundance bar chart for the palynofacies data comparing the acid-free (left) and standard (right) processing techniques for each sample.
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Journal Pre-proof Figure 4: Relative abundance chart showing a comparison of the acid-free processing (red) and the standard processing (blue) for each species observed and counted. MPB – Mantuan Productus Beds, BFE – “Burngrove Formation” equivalent. Plate I: Photomicrograph comparison of the standard processing technique (left) and the acid-free technique (right). 1 – 2: TAMP23; 3 – 4: TAMP24; 5 – 6: TAMP25; 7 – 8: TAMP26; Notable features include: 1 – degraded phytoclasts; 2, 4, 6 & 7 – large opaque phytoclasts; 3, 5 & 7 – well preserved bisaccate pollen grains. Plate II: Photomicrograph comparison of the standard processing technique (left) and the acid-free technique (right). 1 – 2: TAMP27; 3 – 4: TAMP28; 5 – 6: TAMP29; 7 – 8:
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TAMP30. Notable features include: 1 – small Botryococcus; 2 – large Botryococcus; 3 & 7 –
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degraded phytoclasts; 4, 5, 6 & 8 – large, well-preserved palynomorphs and phytoclasts. Plate III: Photomicrograph comparison of the standard processing technique (left) and the
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acid-free technique (right). 1 – 2: TAMP31; 3 – 4: TAMP32; Notable features include: 1 & 3
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– Micrhystridium evansii; 2 & 4 – Micrhystridium evansii and large phytoclasts. Highlights
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An acid-free palynological processing technique is applied to Permian-aged samples for the first time. The acid-free processing technique is applied to both shallow marine and terrestrial (deltaic) sediments. Palynological and palynofacies data is compared between with the standard acid processing techniques. Palynological data between each technique is comparable with high yields of terrestrial palynomorphs and acritarchs. Quantitative differences between each technique are discussed.
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