A New Preparation Method for Testate Amoebae in Minerogenic Sediments
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A new preparation method for testate amoebae in minerogenic sediments X. Zheng1, J.B. Harper2, G. Hope3 and S.D. Mooney1 1 School of Biological, Earth & Environmental Sciences, University of New South Wales, Australia 2 School of Chemistry, University of New South Wales, Australia 3 Department of Archaeology and Natural History, Australian National University, Australia _______________________________________________________________________________________ SUMMARY Testate amoebae are one of a few moisture-sensitive proxies available to study Holocene palaeohydrology. Although the majority of research has been conducted on ombrotrophic peatlands in the Northern Hemisphere, the application of testate amoebae in minerogenic sediments, such as minerotrophic peatlands and saltmarshes, holds considerable promise but is often impeded by the low concentration of testate amoebae and by time- consuming counting. Here a new preparation protocol to concentrate testate amoebae is proposed; it removes more minerogenic particles and organic matter, but results in negligible damage to testate amoebae. Sodium pyrophosphate (Na4P2O7) is introduced to remove fine particles through deflocculation that, in contrast to the commonly used chemical digestion/deprotonation via an alkaline treatment, is more physically benign to testate amoebae. Furthermore, acetone is introduced as an organic co-solvent to increase the solubility of organic matter in the alkaline treatment. We test the new protocol against standard, water-based methods and find that the addition of sodium pyrophosphate yields the highest concentration and increases the total number of testate amoebae recovered. Statistical analyses (multivariate ANOVA and ordination) suggest that the new method retains the assemblage integrity. We conclude by recommending a protocol combining sodium pyrophosphate, acetone and a mild alkaline treatment, as this combination yields the best slide clarity, reduced counting time and results in negligible damage to testate amoebae. KEY WORDS: laboratory preparation, sodium pyrophosphate, acetone _______________________________________________________________________________________ INTRODUCTION is often the silt-sized organic matter in the minerogenic samples from peatlands that impedes The unicellular protists known as testate amoebae quantification of testate amoebae, perhaps because have been widely used as a bio-indicator to study the non-organic components can often be removed ecology and palaeoecology in peatlands (Mitchell et using sieving (or micro-sieving) (Hendon & al. 2008a). They are ubiquitous in moist environment Charman 1997) or differential settling techniques and such as soils, lakes and peatlands, and their tests the organic content in these environments is often preserve well in sediments, even dating back to the structurally more robust. The fragility of some testate Late Neoproterozoic (Porter & Knoll 2000). With amoebae means that strongly basic and strongly these features, testate amoebae have been widely oxidising conditions are avoided and hence a high used to study palaeohydrology in peatlands, organic content in the preparations for testate especially in the Holocene (Mitchell et al. 2008a) and amoebae is also clearly an issue in other settings: as it has been more than two decades since the first an example, Swindles et al. (2016) described the quantitative reconstruction of water-table depth concentration of testate amoebae in a tropical (Warner & Charman 1994). peatland as very low owing to poor preservation and Despite this capacity, the majority of research has obscuration by organic matter. been conducted in ombrotrophic peatlands, A high concentration of organic matter in a particularly in the Northern Hemisphere, and more sample prepared for the quantification of testate minerogenic sites have been used much less amoebae leads to two problems: (1) the organic frequently (Payne 2011, Charman et al. 2010, matter can aggregate (through both physical Swindles et al. 2016). As an example, Charman et al. association and chemical crosslinking), which can (2010) managed to count 50 tests in samples from obscure testate amoebae; and (2) a relatively low saltmarshes, where testate amoebae have applications concentration often requires the preparation of many relevant to sea-level change (Barnett et al. 2017). In microscope slides and extra counting time. These low our experience, and somewhat counter-intuitively, it concentrations, potentially biased results, low counts Mires and Peat, Volume 24 (2019), Article 30, 1–12, http://www.mires-and-peat.net/, ISSN 1819-754X © 2019 International Mire Conservation Group and International Peatland Society, DOI: 10.19189/MaP.2018.OMB.380 1 X. Zheng et al. NEW PREPARATION METHOD FOR TESTATE AMOEBAE and statistically unsatisfactory totals (Charman et al. Richea-Empodisma high altitude shrub bogs in 2010) have limited the application of testate amoebae Australia (Hope et al. 2009) and has an organic-rich as an independent or complementary proxy to a wider sediment that includes minerogenic material from geographical area and to other environmental settings. slope runoff and aeolian sources. Three cubic As noted, the standard methods to concentrate centimetres were sampled from a 3 cm wide section testate amoebae for quantification are designed to of the core centred on 145, 190 and 217 cm deep, and minimise damage and are based on sieving and either these were soaked in reverse osmosis (RO) water involve a mild alkaline digestion or are water-based overnight to disaggregate the sediments. A known (Hendon & Charman 1997). Although an alkaline concentration of Safranin O stained Lycopodium digestion is effective at removing unwanted organic clavatum L. spores (Stockmarr 1971) was added as matter, thereby increasing the concentration (Hendon an exotic marker to track the concentration of testate & Charman 1997) and reducing the counting time amoebae before the samples were sieved. In this, and (Barnett et al. 2013), the fragility of testate amoebae all cases mentioned below, three nested sieves (20, means that tests may be damaged and some features 215 and 250 μm) were used, with the top sieve used missing or altered (Hendon & Charman 1997). For as a cushion to reduce the strength of the flushing this reason water-based methods are often advocated water, and the material for analysis was collected (Booth et al. 2010). However, significantly between the 20 and 215 μm sieves (Zheng et al. 2019). increasing the concentration and reducing the Each sample was carefully removed from the sieve, counting time should be evaluated against the ‘cost’ homogenised and subdivided into three subsamples. of acceptable damage. Based on such considerations, Although the volume of each subsample did not Barnett et al. (2013) used a mild (less than 1 % KOH) really matter (owing to the presence of the alkaline treatment for sample solutions on a Lycopodium spores), the same volume was used. preheated hotplate, compared to 10 % KOH (in From that point on, the different subsamples were Hendon & Charman 1997), and significantly reduced subjected to different procedures, as represented the average counting time from 16 h to 8 h. diagrammatically in Figure 1. Although it has a long history in palynology as a Treatment #1 was processed using the common, deflocculant to remove fine mineral particles such as simple water-based preparation (Hendon & Charman clay and silt (Bates et al. 1978, Cwynar et al. 1979), 1997) that includes sieving only. Treatment #2 was sodium pyrophosphate is also an effective dispersant processed using two dispersant steps, separated by for fine particles of organic matter (Bremner & Lees sieving. In this research, sodium metapyrophosphate 1949, Kaur & Fanourakis 2016). In contrast to an (Na4P2O7.10H2O) was used as the dispersant: the alkaline treatment, sodium pyrophosphate is a near- subsample was placed into a 250 ml flask with neutral agent and so should not damage testate 100 ml of a 5 % aqueous sodium metapyrophosphate amoebae, but its effectiveness had not been solution, and then placed on a shaker table for 4 hours demonstrated. at 110 rpm. Treatment #3 was processed using an This research addressed several of these issues alkaline treatment adapted from Barnett et al. (2013). with the over-arching aim of improving techniques to In a 250 ml flask the subsamples were mixed with concentrate testate amoebae in more minerogenic 20 ml RO water, 25 ml acetone and 5 ml of a 10 % sediments and thereby reducing counting times. In aqueous sodium hydroxide solution (in that order) particular, the introduction of a sodium-based and then placed on a hotplate, which had been dispersant and the inclusion of an organic co-solvent preheated to 80 °C, for 5 mins. (It is important that (acetone, IUPAC name propan-2-one) to increase the the sodium hydroxide solution is not added to the effective interaction between the alkaline digestion acetone until immediately before the treatment; and organic matter was investigated. These acetone polymerises (albeit slowly) through an aldol techniques were assessed against simple water-based reaction under basic conditions.) As described above, methods and this included consideration of the acetone acts as a co-solvent to solubilise the organic damage to tests under the various protocols. matter, which may potentially have been deprotonated under the basic conditions. Although a 10 % aqueous sodium hydroxide solution was used, METHODS it is diluted in the flask and so