Preparation and analysis of testate amoebae in peatland paleoenvironmental studies

Abstract

The use of testate amoebae in studies of peatland hydrology and palaeohydrology has been increasing, and considerable efforts have been made over the past decade to standardise techniques as much as possible. In this article we briefly describe the common procedures used to collect, prepare and analyse testate amoebae in peat-based studies of palaeohydrology and palaeoclimatology. Although specific methods are dependent on the questions and objectives of individual studies, the methods presented here should serve as a good starting point for peatland palaeoenvironmental applications.

Full text

Mires and Peat, Volume 7 (2010/11), Article 02, 1–7, http://www.mires-and-peat.net/, ISSN 1819-754X
© 2010 International Mire Conservation Group and International Peat Society
1
Preparation and analysis of testate amoebae
in peatland palaeoenvironmental studies
R.K. Booth
1
, M. Lamentowicz
2
and D.J. Charman
3
1
Earth and Environmental Science Department, Lehigh University, Bethlehem, PA, USA
2
Department of Biogeography and Palaeoecology, Adam Mickiewicz University, Poznan, Poland
3
Department of Geography, University of Exeter, UK
_______________________________________________________________________________________
SUMMARY
The use of testate amoebae in studies of peatland hydrology and palaeohydrology has been increasing, and
considerable efforts have been made over the past decade to standardise techniques as much as possible. In
this article we briefly describe the common procedures used to collect, prepare and analyse testate amoebae
in peat-based studies of palaeohydrology and palaeoclimatology. Although specific methods are dependent
on the questions and objectives of individual studies, the methods presented here should serve as a good
starting point for peatland palaeoenvironmental applications.
KEY WORDS: palaeoclimate, palaeohydrology, transfer function, water table depth.
_______________________________________________________________________________________
1. INTRODUCTION
Testate amoebae are routinely used as indicators of
past changes in peatland hydrology (Charman 2001,
Mitchell et al. 2008). These single-celled organisms
respond quickly to environmental change, produce
decay-resistant and taxonomically distinctive shells
(Figure 1), and are generally well preserved and
abundant in Holocene peat deposits. In oligotrophic
peatlands, testate amoeba community composition is
primarily controlled by the moisture content of the
surface peat, allowing the development of transfer
functions to infer changes in past water table depths.
Over the past couple of decades, transfer functions
have been developed and validated in many regions
of the world (e.g. Charman & Warner 1992,
Charman 1997, Charman & Warner 1997,
Woodland et al. 1998, Bobrov et al. 1999, Mitchell
et al. 1999, Lamentowicz & Mitchell 2005,
Schnitchen et al. 2006, Payne et al. 2006, 2008,
Charman et al. 2007, Payne & Mitchell 2007, Booth
2008, Lamentowicz et al. 2008a,c, Swindles et al.
2009, Markel et al. 2010). Cross-validation of these
transfer functions reveals that water table depths can
typically be reconstructed with a mean error of
about 6–8 cm (Figure 2). Additional validation for
the use of testate amoebae in studies of past climate
variability has come from comparative studies of
testate amoeba-inferred water table depths and
instrumental records of climate for the past few
centuries (Charman et al. 2004, 2009, Schoning et
al. 2005, Charman 2007, Booth 2010, Lamentowicz
et al. 2010), comparison of Holocene water table
depth reconstructions from within the same region
(e.g. Hendon et al. 2001, Booth et al. 2006,
Charman et al. 2006), multi-proxy studies from
within the same sediment cores (e.g. Nichols et al.
2006, Lamentowicz et al. 2008b) and comparison
with long-term observed water table measurements
(Charman et al. 2004).
A range of techniques has been used to collect,
isolate, identify and analyse testate amoebae from
modern and fossil peats, but recent research has
mostly followed the protocols outlined by Hendon
& Charman (1997) and Charman et al. (2000).
However, increasing numbers of researchers are
now utilising testate amoebae in palaeo-
environmental studies, leading to some
modifications of these protocols. In this paper, we
briefly describe common field and laboratory
procedures that are used to reconstruct peatland
palaeohydrology and palaeoclimate using testate
amoebae. These general procedures can be modified
to address the specific questions and objectives of
individual studies.
2. MODERN SAMPLING METHODS
For modern calibration studies, communities of
testate amoebae are collected from surface peat.
Sampling should be directed toward capturing the
range of environmental variability within a peatland
(e.g. microtopography, vegetation). At sites where

R.K. Booth et al. PREPARATION AND ANALYSIS OF TESTATE AMOEBAE
Mires and Peat, Volume 7 (2010/11), Article 02, 1–7, http://www.mires-and-peat.net/, ISSN 1819-754X
© 2010 International Mire Conservation Group and International Peat Society
2
Figure 1. Photomicrographs of some testate amoeba taxa commonly encountered in peatland studies:
a) Archerella (Amphitrema) flavum, b) Amphitrema wrightianum, c) Arcella discoides type,
d) Assulina muscorum, e) Bullinularia indica, f) Centropyxis aculeata type, g) Centropyxis ecornis type,
h) Centropyxis platystoma type, i) Cyclopyxis arcelloides type, j) Difflugia pulex type,
k) Heleopera sylvatica, l) Hyalosphenia elegans, m) Hyalosphenia papilio, n) Hyalosphenia subflava,
o) Nebela carinata, p) Trigonopyxis arcula.
Figure 2. Cross-validation (leave-one-out) of a transfer function developed from North American
peatlands, including sites in mid-continental and eastern North America (Booth 2008), the Rocky
Mountains (Booth & Zygmunt 2005) and Alaska (Markel et al. 2010), using a simple weighted average
model.

R.K. Booth et al. PREPARATION AND ANALYSIS OF TESTATE AMOEBAE
Mires and Peat, Volume 7 (2010/11), Article 02, 1–7, http://www.mires-and-peat.net/, ISSN 1819-754X
© 2010 International Mire Conservation Group and International Peat Society
3
Sphagnum moss dominates, about 10cm
3
of the
upper photosynthetic part of the moss is usually
collected for analysis. This typically represents the
uppermost ca. 5cm of the moss, although more or
less may be collected depending on the density of
the Sphagnum. The upper 1–2 cm (i.e. capitulum) is
often removed prior to analysis because vertical
variation in testate amoebae occurs along the stem,
and samples collected from the lower portion of the
stem exhibit higher taxonomic diversity (Mitchell &
Gilbert 2004) and are generally thought to be more
similar to the death assemblage that is incorporated
into the peat record. At sites lacking Sphagnum,
samples are generally taken from brown moss
carpets and vascular plant remains.
In association with each modern testate amoeba
sample, water table depth is measured, often in
conjunction with other environmental variables (e.g.
pH, conductivity, N, P, Ca, Mg, DOC). Water table
depth measurements that reflect the average
experienced during the growing season are best for
comparison with testate amoeba communities, and
these can be obtained through repeat measurements
(e.g. Woodland et al. 1998) or other integrative
estimates such as polyvinyl chloride (PVC) tape
discolouration (Belyea 1999, Booth et al. 2005).
However, PVC tape discolouration has had mixed
success in recent studies (Payne et al. 2006,
Schnitchen et al. 2006, Booth 2008, Markel et al.
2010), and when integrative estimates of water table
depth are not possible, instantaneous measurements
(i.e. measured on the day of sampling) are still
useful, so long as extremely dry conditions are
avoided (Charman et al. 2000, Booth 2008).
3. FOSSIL SAMPLING METHODS
Subsamples of 1–2 cm
3
are collected from along a
peat core, each typically spanning 0.5–1 cm of peat.
Given the rapid response time of testate amoebae to
environmental change and the likely increasing
sensitivity of peatland hydrology to autogenic
change over longer timescales (Charman et al.
2006), analysis of contiguous or nearly contiguous
subsamples is recommended for studies of past
hydroclimatic variability.
4. ISOLATION OF TESTS FROM PEAT
Testate amoebae are usually isolated from modern
and fossil peat using a sieving procedure without
any chemical reagents (Hendon & Charman 1997,
Charman et al. 2000). A modified version of this
procedure is presented here.
1. Each peat sample is placed in beaker (100–250
ml) with distilled water (~50–100 ml) and a
clean stirring rod. One or two tablets of
Lycopodium spores can be added as an exotic
marker to permit the calculation of test
concentrations (tests cm
-3
) and accumulation or
influx rates (tests cm
-2
year
-1
). The number of
tablets is dependent on the peat volume used,
with one tablet per cm
3
of peat typically
adequate.
2. Samples are boiled in distilled water for
approximately ten minutes, stirring occasionally
to disaggregate peat and disperse the
Lycopodium spores. Alternatively, some analysts
recommend soaking the samples overnight in
distilled water. The boiling step may be omitted
if living and dead individuals are to be
differentiated in modern samples, as boiling may
remove some cells from the tests. However, for
transfer function development, both living and
dead tests are usually tallied together because the
objective is to characterise the assemblage that
becomes incorporated into the fossil record.
3. Distilled water is added to cool off the samples,
and the material is typically washed through
300 µm and 15 µm sieves. The 300 µm sieve
removes coarse particulate matter from the
samples, and the 15 µm sieve filters some of the
smaller particulates and tends to make analysis
easier and more efficient. A source of vibration
can be held against the 15 µm sieve to speed the
fine-sieving process (a dremel tool works quite
well). Some analysts recommend using a 10 µm
sieve (Beyens & Meisterfeld 2001) or no
microsieve at all (Payne 2009), to avoid the loss
of particularly small taxa (Wall et al. 2009).
However, most palaeoclimate work requires the
examination of numerous samples (often
continuous analysis along sediment cores), and
microsieving makes analysis more efficient. The
choice of whether or not to use fine sieving
depends on the objectives of the study, but for
quantitative environmental reconstructions the
calibration and fossil data should ideally be
obtained using the same procedure (Payne 2009).
The material retained in the 15 µm sieve is
washed into 50 ml centrifuge tubes and
centrifuged at 3,000 rpm for five minutes.
4. Water is decanted and the residues may be
stained with Safranine to help highlight the tests
during analysis, although this depends on the
preference of the analyst. In modern samples,
empty and living tests can be distinguished by

R.K. Booth et al. PREPARATION AND ANALYSIS OF TESTATE AMOEBAE
Mires and Peat, Volume 7 (2010/11), Article 02, 1–7, http://www.mires-and-peat.net/, ISSN 1819-754X
© 2010 International Mire Conservation Group and International Peat Society
4
staining with Rose Bengal, which tends to
preferentially stain cytoplasm.
5. After staining, an additional water wash followed
by centrifuging is typically performed to remove
residual stain.
6. Residues are then transferred into stoppered vials
where they can be stored in water, glycerol, or
some other storage medium. Water is
recommended if scanning electron microscope
work may be undertaken. Slides can be made
using water, glycerol, or another mounting
medium. Although glycerol performs adequately
for routine analysis, the optical properties of
water are better.
5. TAXONOMY AND QUANTIFICATION
The large amount of morphological variability
amongst the testate amoebae has given rise to a
diverse and confusing literature. For peatland
research, some good resources for test identification
include: Leidy (1879), Penard (1902), Cash &
Hopkinson (1905, 1909), Cash et al. (1915),
Deflandre (1936), Grospietsch (1958), Corbet
(1973), Ogden & Hedley (1980), Meisterfeld
(2001a,b) and Charman et al. (2000). There are also
numerous monographs and descriptions of
individual taxa in the literature, many of which are
referred to by the sources listed here. For routine
peatland palaeohydrological applications, a
recommended starting point for test identification is
Charman et al. (2000), as this taxonomic approach
has been followed for the majority of modern
peatland palaeohydrological studies. Useful
modifications to the identification scheme of
Charman et al. (2000) are presented elsewhere
(Booth 2002, 2008). Most common taxa are
relatively easily identified, but there are a number of
taxon groups that can be difficult to separate from
one another and these may be grouped or split at
different levels. Because the purpose of many
studies is to apply transfer functions, which are
often derived by different analysts, taxonomic
harmonisation of datasets is important. In general, it
is recommended that a high-level taxonomy such as
that used by Charman et al. (2000) is applied. Taxa
may be split to lower levels in counting but may
need to be grouped to ensure reliable application of
the transfer function. There is evidence that
relatively minor differences in morphology such as
size variants (e.g. Bobrov et al. 1999) and number
of pores (Booth & Meyers 2010) can add power to
environmental inferences, so it may be worth
recording these attributes during counting.
As in other types of microfossil analysis, slides
are usually scanned and the abundance of the
different testate amoeba taxa is tallied. Slides are
typically scanned at 400x magnification, although
1000x is useful for the identification of some taxa.
Traditionally, a total of 150 tests has been tallied,
but for transfer function applications a total count of
100 individuals is likely to be sufficient for most
samples (Payne & Mitchell 2009). Higher counts are
needed to identify all taxa and to estimate the
relative abundance of rare taxa accurately (Wall et
al. 2009). Relative abundances of taxa are typically
expressed as percentages of the total counted,
although concentration and influx can also be
calculated.
6. TRANSFER FUNCTION DEVELOPMENT
AND APPLICATION
Transfer functions are widely used in palaeoecology
and palaeoclimate research to provide quantitative
reconstructions of environmental or climatic
variables based on a modern training set
(assemblage data and associated environmental
variables) and a fossil assemblage dataset. Inferred
values of the variable of interest are calculated by
applying a regression model developed from the
modern dataset to the fossil data (Birks 1998).
Standard software is now available for developing,
testing and applying a range of models (Juggins
2007). A typical procedure would be to test a range
of potential models against a modern dataset. There
are many possible models including those based on
Gaussian responses of taxa to the environmental
variable (mostly forms of ‘weighted averaging’),
those based on linear responses, and those based on
matching total assemblages (‘modern analogue
techniques’). Most studies suggest that weighted
averaging produces easily understood, accurate and
precise estimates of water table position from testate
amoeba assemblages. Once the best-performing
model has been identified, it can be applied to the
fossil data to produce inferred water table values.
Sample-specific error estimates for reconstructed
water tables are estimated by using a bootstrapping
procedure to assist detection of significant changes.
7. ACKNOWLEDGEMENTS
This article was improved by the thoughtful
comments of Dmitri Mauquoy and Edward
Mitchell.

R.K. Booth et al. PREPARATION AND ANALYSIS OF TESTATE AMOEBAE
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© 2010 International Mire Conservation Group and International Peat Society
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Submitted 30 Apr 2010, revision 13 May 2010
Editor: Olivia Bragg
_______________________________________________________________________________________
Author for correspondence:
Dr Robert (Bob) Booth, Earth and Environmental Science Department, Lehigh University, Bethlehem, PA,
USA. Tel: +1 610 758-6580; Fax: +1 610 758-3677; E-mail: rkb205@lehigh.edu