Biodivers Conserv (****) **:**** ****

DOI **.****/s10531-007-9221-3

O R I G I NA L P AP E R

Testate amoebae analysis in ecological

and paleoecological studies of wetlands: past,

present and future

Edward A. D. Mitchell Daniel J. Charman

Barry G. Warner

Received: 21 August 2007 / Accepted: 21 August 2007 / Published online: 30 October 2007

Springer Science+Business Media B.V. 2007

Abstract Testate amoebae are an abundant and diverse polyphyletic group of shelled

protozoa living in aquatic to moist habitats ranging from estuaries to lakes, rivers, wet-

lands, soils, litter, and moss habitats. Owing to the preservation of shells in sediments,

testate amoebae are useful proxy indicators complementary to long-established indicators

such as pollen and spores or macrofossils. Their primary use to date has been for inferring

past moisture conditions and climate in ombrotrophic peatlands and, to a lesser extent, to

infer pH in peatlands and the trophic or nutrient status of lakes. Recent research on these

organisms suggests other possible uses in paleoecology and ecology such as sea-level

reconstruction in estuarine environments, as indicators of soil or air pollution, and monitor-

ing recovery of peatland. We review the past and present use of testate amoebae, the chal-

lenges in current research, and provide some ideas on future research directions.

Keywords Testate amoebae Protozoa Peatland Sphagnum Paleoecology

Bioindicators Community ecology Transfer functions Peatland management

Restoration ecology

E. A. D. Mitchell Swiss Federal Research Institute WSL, Wetlands Research Group, Station 2, Lausanne CH-1015,

Switzerland

e-mail: abpmr2@r.postjobfree.com

E. A. D. Mitchell

EPFL, Laboratoire des Syst mes cologiques (ECOS), Station 2, Lausanne CH-1015, Switzerland

D. J. Charman

School of Geography, University of Plymouth, Plymouth, Devon PL4 8AA, UK

B. G. Warner

Department of Earth and Environmental Sciences, University of Waterloo, Waterloo,

ON, Canada, N2L 3G1

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Introduction

Testate amoebae are unicellular protists, which are ubiquitous in environments such as

lakes, rivers, mosses, and soils, but also occur in estuarine environments (Meisterfeld

2002a, b). They are small (mostly 20 200 m roughly the size of pollen grains), abun-

dant (e.g., 103 104 individuals g 1 dry weight peat), and diverse (about 2000 species

described so far, and usually between 10 and 30 species in any given sample). Testate

amoebae build a test (shell) either from proteinaceous, calcareous, or siliceous material.

Some of them form agglutinated tests by gluing together organic or mineral particles from

their surrounding environment. These tests cover a relatively broad range of sizes (over one

order of magnitude) and a high diversity of morphologies (Fig. 1), which allows identiWca-

tion to species level. These tests are usually well preserved in peat and lake sediments

(Warner 1990).

Testate amoebae have a long fossil history, with fossils dated from the Cretaceous

(Boeuf and Gilbert 1997; Foissner and Schiller 2001; Patterson and Kumar 2002; Schmidt

et al. 2001; Sch nborn et al. 1999), the Carboniferous (Loeblich and Tappan 1964), and

perhaps even Late Neoproterozoic (Porter and Knoll 2000). Despite this long fossil history,

most paleontological studies of testate amoebae focus on the Quaternary and especially on

the Holocene where they are used as paleobioindicators in lakes and peatlands (Charman

2001; Medioli et al. 1990; Tolonen 1986).

Fig. 1 Light- and scanning electron micrographs of some testate amoebae found in peatlands, illustrating the

range of morphological variability; (a) Trigonopyxis arcula, (b) Hyalosphenia subXava, (c) Bullinularia in-

dica, (d) Nebela tincta, (e) Nebela militaris, (f) Assulina muscorum, (g) Assulina seminulum, (h) Arcella arto-

crea, (i) H. elegans, (j) Physochila griseola, (k) H. papilio, (l) Centropyxis aculeata, (m) Archerella Xavum,

(n) Placocista spinosa var. hyalina, (o) DiZugia bacillifera, (p) N. carinata, (q) Amphitrema wrightianum.

Scale bars indicate approximately 50 m except for A. muscorum: 20 m. SEM were conducted at the Uni-

versity of Alaska Anchorage by K. Kishaba, J. Kudenov and E. Mitchell

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In this review, we cover selected aspects of the phylogeny, taxonomy, biogeography,

biology and ecology of testate amoebae. We then discuss the application of this knowl-

edge to paleoecological research and present some new Welds of research and application.

This review also constitutes a guide to an extensive selection of the literature on testate

amoebae.

Testate amoeba taxonomy and biogeography: facts and open questions

Higher taxonomic levels: phylogeny

Testate amoebae are a polyphyletic assemblage of at least two major, unrelated taxonomic

groups of mostly heterotrophic unicellular eukaryotes (Meisterfeld 2002a, b; Wylezich

et al. 2002). Testate amoebae have been divided based on pseudopod morphology. The

Wrst group has lobose pseudopodia (order Arcellinida). The second group with Wlose pseu-

dopodia is represented mainly by order Euglyphida. Some testate amoebae possessing

anastomosing networks of reticulopodia, and traditionally classiWed in the phylum Gran-

uloreticulosea (Bovee 1985), have recently been placed among testate amoebae with Wlose

pseudopodia (Meisterfeld 2002b). The phylogenetic position of Arcellinida and Euglyph-

ida, among Amoebozoa and Cercozoa, respectively, has been established based on ribo-

somal DNA sequences (Cavalier-Smith and Chao 1997; Meisterfeld 2002a, b; Nikolaev

et al. 2005; Wylezich et al. 2002). However, DNA sequences are available only for a

small number of taxa and therefore the higher-level taxonomy of both orders still rests

entirely on morphological characters. It is urgent to complete the sequence data for all

families and genera, in order to establish a solid phylogeny based on molecular and mor-

phological characters.

Perhaps because of the uncertainty about their phylogeny, many diVerent names have

been used to describe these organisms: thecamoebians, rhizopods, testate amoebae, testa-

ceans, and arcellaceans. Some of these names implicitly suggest a taxonomic aYliation.

For example, the term arcellaceans implies relationships to the order Arcellinida or the

genus Arcella, which is incomplete and unrepresentative of all testate amoebae. Rhizopods

on the other hand are not restricted to testate amoebae and may include naked amoebae.

We suggest that the terms testaceans or testate amoebae should be preferred as they are

unambiguous.

Lower taxonomic levels: genera and species

The taxonomy of testate amoebae at lower taxonomic levels is largely based on shell mor-

phology and is also unresolved (Lee et al. 2000; Patterson 1996). Although uncertainties at

the higher taxonomic levels have little impact on the usefulness of testate amoebae in eco-

logical and paleoecological research, the establishment of solid phylogenetic relationships

is necessary for determining the taxonomic signiWcance of some important morphological

features that are used to identify the species.

Despite the fact that the common species can usually be identiWed with conWdence, there

is an urgent need for taxonomic revision and a synthesis of the existing data. There are no

recent updated complete monographs or even species lists and many of the approximately

2000 described species are probably never securely identiWed by most ecologists for lack of

appropriate identiWcation criteria, the diYculty in accessing the original descriptions, or

simply because no up to date synthesis exists in which the identiWcation characteristics of

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all species are clearly described. The identiWcation of species is usually based on mono-

graphs, which often date back to decades or even over a century and which, despite the fact

that none of them includes all the described species, are still the most useful resources

(Cash and Wailes 1915; Chardez 1969, 1991; Corbet 1973; DeXandre 1928, 1929, 1936;

Grospietsch 1958, 1964; Harnisch 1958; Ogden 1983; Ogden and Hedley 1980; Penard

1902; Sch nborn 1965b). A few more recent studies provided clariWcations on some

selected taxa (Foissner and Korganova 1995, 2000; L ftenegger et al. 1988; Sch nborn

et al. 1983). A valuable guide for paleoecologists was recently published by the Quaternary

Research Association (Charman et al. 2000). However, much remains to be done to make

the identiWcation of testate amoebae more straightforward for ecologists and paleoecolo-

gists, and through this, the comparison among studies more reliable. The amount of work

involved in such a task is huge and will require a signiWcant investment.

Moreover, the intra-speciWc variability of test morphology in most taxa has not yet been

satisfactorily assessed. It is quite likely that many species have been described based on the

extremes of a continuum of morphotypes of the same variable species (Bobrov and Mazei

2004). Recent studies show that (1) abiotic and biotic environmental factors such as food

source, temperature, and insecticides aVect the shell morphology (Chardez 1989; Sch nborn

1992a; Wanner 1999; Wanner et al. 1994; Wanner and Meisterfeld 1994), and (2) testate

amoebae are characterized by a high degree of morphological variability both among and

within populations (Bobrov and Mazei 2004), and (3) even under controlled conditions, the

morphological variability of the shell increases with time (Sch nborn 1992a). Such variabil-

ity is apparently at least partly genetically determined and allows the species to adapt to the

spatial or temporal heterogeneity of their environment (Bobrov and Mazei 2004).

Phenotypic plasticity may however not necessarily mean that testate amoebae are less

useful as a tool for ecologists and paleoecologists. Indeed if the relationships between

environmental conditions and shell morphology were well understood and could be

reliably predicted, then this information could be used to infer environmental conditions

on the basis not only of community composition but also of shell morphological charac-

teristics. Instead of having discrete categories (species) along a gradient, ecologists and

paleoecologists would have a continuum. Inference (forecasting) models could then be

built from the analysis of such continua, with linear or polynomial regression. We there-

fore call for more detailed morphometric analyses of (1) living taxa under known environ-

mental conditions, and (2) cultures grown under controlled conditions. This approach will

enable a predictive model of morphological characteristics as a function of environmental

conditions to be created. Such a model would have direct applications for paleoenviron-

mental reconstructions.

The fact that testate amoebae have been found to be useful despite these imperfections

suggests that they could become an even better tool for ecologists if the taxonomy was

improved. The present state of testate amoebae taxonomy and the above-mentioned Wnd-

ings call for (1) a conservative approach to the description of new species and (2) a test of

the morphology-based taxonomy using molecular data (e.g., phylogenetic trees derived

from ribosomal RNA sequences) with the long-term goal of revising the taxonomy of

testate amoebae based on the combination of molecular and morphological data. We

suggest a combined approach focusing on the morphology, molecular taxonomy, and

ecology of problematic groups of species such as the genera DiZugia, Centropyxis,

Phryganella, Trinema, and Euglypha or groups of species within otherwise easy genera

such as the Nebela tincta species complex.

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Fig. 2 Apodera vas (Certes), a testate amoeba found in South and Central America and Sub-Sahara Africa

but lacking from North America and Eurasia. This individual is from a Sphagnum moss collected in Tanzania.

SEM conducted at the University of Alaska Anchorage by K. Kishaba, J. Kudenov and E. Mitchell

The debate over cosmopolitanism

The question of the cosmopolitanism of microorganisms is of great importance to the glo-

bal use of testate amoebae as bioindicators and paleobioindicators in peatland. There is cur-

rently a lively debate as to whether free-living protists are ubiquitous (Finlay 2002; Finlay

and Clarke 1999; Finlay et al. 2001; Finlay et al. 1999; Finlay and Fenchel 1999) or

whether some, and perhaps indeed most, of them have limited geographical distributions

(Foissner 1997a, 1998, 1999). Testate amoebae represent an interesting paradox as they

provide evidence for both views. Indeed, the available data suggests that they include both

species small enough to be transported passively over long distances and species large

enough for this to be impossible (Wilkinson 1994; Wilkinson 2001). An often-cited exam-

ple of the latter is Apodera (Nebela) vas (Fig. 2), a species that has so far been found

mostly in the Southern Hemisphere, with some Northern Hemosphere records in Hawaii,

Venezuela, and Central America, but no records from Eurasia or North America (DeXandre

1936; Van Oye 1944). Given the large size of this species (130 210 m), its distinct mor-

phological characteristics, and the larger number of studies on testate amoebae of the

Northern Hemisphere as compared to the Southern Hemisphere, it is very unlikely that it

would have been overlooked.

Excessive splitting of taxa may also give the impression of a higher rate of endemism

than actually exists. Given the variability of shell morphology discussed above, we believe

that many of the described species and even more of the subspecies and varieties should not

be used as evidence for limited distribution ranges as many of them were not conWrmed or

observed by anyone else than the person who Wrst described them. On the other hand, the

existing literature on testate amoebae biogeography is mostly based on the observation of

morphotypes. It remains possible that a given morphological species may in fact hide sev-

eral genetically distinct species (i.e. cryptic species) that may diVer with respect to geo-

graphical distributions and/or ecological requirements. In a diVerent context, cryptic

species of both benthic and planktonic foraminifera have recently been discovered, some of

which have cosmopolitan distribution while others have not (Darling et al. 2004; Darling

et al. 2000; Hayward et al. 2004).

In addition to size, there might also be a diVerence in the cosmopolitanism of species

depending on their habitat: wetland species and species living in the soil litter and mosses

are more likely to be transported over long distances than species living deeper in the soil.

Clearly the debate on cosmopolitan distribution versus local endemism is unlikely to be

resolved unless the size range and habitat are speciWed. Even so, the possible existence of

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cryptic species calls for caution and further studies of global and local distribution patterns,

ideally with an approach combining morphological, ecological, and molecular methods

such as ribosomal RNA sequencing (Mitchell and Meisterfeld 2005).

Biology

A review of the present knowledge of the biology of testate amoebae is beyond the scope of

this paper. Much remains unknown about their life, for example the relative importance of

asexual and sexual reproduction (Mignot and Raikov 1992), and their physiology. In the

following paragraphs we will focus only on two aspects that are most relevant to the ecolo-

gist: their shell characteristics and their general role in the ecosystem.

Source of material for test construction

Testate amoebae need to Wnd the required material to build their test and this requirement

may be one of the constraints that determine the micro-distribution of species (Meisterfeld

1977; Sch nborn 1963). An example of niche separation between two closely related taxa

can be seen in the vertical micro-distribution of Amphitrema wrightianum and A. Xavum.

Both species may both be found in wet and oligotrophic conditions such as pools in the

centre of raised bogs, with A. Xavum usually with its optima in slightly drier conditions

than A. wrightianum. A. Xavum produces a shell that is entirely composed of proteinaceous

material whereas A. wrightianum uses xenosomes (usually minute organic debris) and

cements them together to create a test. This diVerence in shell construction may prevent A.

wrightianum from fully colonizing the uppermost part of Sphagnum mosses (the capitu-

lum) because there may not be enough free organic or mineral material for shell construc-

tion. A further example of this kind is the occurrence of a number of taxa of the genus

DiZugia (e.g., D. bacillifera, D. elegans, D. leydyi) in very wet conditions on oligotrophic

peatlands. The tests of this genus are also xenosomic and it is likely that the silica particles

predominantly used in the tests are more abundant in shallow pools. These constraints may

also explain some of the patterns of species distribution along micro-environmental gradi-

ents, which are usually interpreted as representing responses to variables such as pH or

moisture that are more easily measured than the availability of xenosomes for shell

construction.

Functional role of testate amoebae

Testate amoebae are usually considered to be predators of bacteria and fungi (Hausmann

et al. 2003). As such they play an important role in the cycling of nutrients in soil, although

clearly Xagellates, naked amoebae and/or ciliates are probably more important, at least in

mineral soils (Clarholm 1981, 1985, 2002; Clarhom and Rosswall 1980; Co teaux and

Pussard 1983; Sch nborn 1992b). However, despite the fact that there are few data on the

functional role of testate amoebae in peatlands, the observation that they are by far the

dominant group of protozoa in Sphagnum suggests that in these ecosystems they play a

signiWcant role (Gilbert et al. 1998a; Gilbert et al. b). In support of this idea, peatland

testate amoebae have also been found to use a wide variety of food sources including other

protists, fungi, organic matter and micro-metazoa such as rotifera (Gilbert et al. 2000).

Despite this general knowledge, it is striking that we still do not have a good idea about the

exact trophic role of most soil protozoa, even of the dominant species.

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Environmental inXuences on testate amoebae include many potential direct and indirect

eVects. Environmental conditions, such as rising atmospheric CO2 concentrations, global

warming, increased nitrogen deposition, drainage, or changes in rainfall patterns may aVect

soil protozoa directly and, perhaps more importantly, also indirectly through their eVect on

the vegetation and prey organisms (Gilbert et al. 1998a, b; Lussenhop et al. 1998; Mitchell

2004; Mitchell et al. 2003; Treonis and Lussenhop 1997).

Ecology and paleoecology

Community structure and responses to ecological gradients

The responses of testate amoebae to the major ecological gradients in peatlands, such as the

fen-bog transition and the humidity gradient, have long been established (Harnisch 1925,

1927; Heal 1961, 1962, 1964; Sch nborn 1962). Later work has provided a more quantita-

tive understanding of these relationships and tested them over a larger geographical range.

The earliest ecological classiWcation of peatland testate amoebae is the eight-class wetness

scale of Jung (1936). More recently, multivariate classiWcations of communities (Meister-

feld 1978, 1979; Mitchell et al. 1999; Tolonen et al. 1994) and quantitative relationships

between community structure and speciWc environmental variables have been explored

using univariate and multivariate statistics (Bobrov et al. 1999; Booth 2001; Charman

1997; Charman and Warner 1992, 1997; Lamentowicz and Mitchell 2005; Mitchell et al.

2000b; Mitchell et al. 1999). These studies generally show that testate amoebae respond

primarily to some measure of moisture (usually either the moisture content of the sample or

the water table depth). Seasonal changes in soil moisture content appear to inXuence shifts

in abundance and community composition (Heal 1964; Quinn 2003; Warner et al. 2007).

SigniWcant relationships have also been found with pH and macro-nutrients when a signiW-

cant range of variability has been sampled (Tolonen et al. 1992). However, in general, the

relationship with pH is secondary to that of hydrology (Booth 2001).

It is also important to emphasise that these empirical relationships do not necessarily

reXect the causes of species responses and are not yet based on a thorough physiological

understanding of testate amoebae. Indirect eVects such as materials for test construction

(see above) may be important, but processes related more directly to hydrology can be sug-

gested. For example, higher soil moisture may allow species with larger tests to success-

fully reproduce because of the thicker water Wlms on plant surfaces (e.g. Assulina

seminulum vs A. muscorum; Nebela tincta var. major vs. N. militaris). In addition to size,

shape is important: Thin tests may be better suited to life in the thin water Wlms under drier

conditions (e.g., Xattened Arcella species such as A. arenaria versus rounder ones such as

A. gibbosa). The spines on the surface of some taxa may be an adaptation to limit move-

ment in very wet conditions and where similar spined and glabrous taxa occur, the former

mostly occupies wetter niches (e.g., Euglypha ciliata and E. ciliata var. glabra, Placocista

spinosa and P. spinosa var. hyalina) (Bobrov et al. 1999). Further work on the ecological

and physiological mechanisms underlying the recorded gradients is required to fully under-

stand why testate amoebae communities vary in response to the key variables of hydrology

and water chemistry.

Comparisons among diVerent areas of the world reveal that many taxa have very similar

water table depth optima but that others (e.g., Centropyxis aculeata, Hyalosphenia papilio,

H. elegans, Cyclopyxis arcelloides, Nebela tincta, H. subXava) vary in their relative posi-

tion on the hydrological gradient (Fig. 3). Such comparative results can however be

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Fig. 3 Water table depth optima of testate amoebae in Sphagnum peatlands: a summary of studies from

Europe, North America and New Zealand, modiWed from Booth (2001), with kind permission from the Jour-

nal Wetlands

strongly biased by (1) the range of hydrological conditions sampled, (2) the way the water

table was measured (e.g., single point measurement or long-term average), and (3) the tax-

onomic resolution (see above). The only way to truly ascertain that testate amoebae indeed

have similar responses to ecological gradients in diVerent regions is to carry out a multi-site

study with a standard protocol, from sampling strategy to numerical analyses. This has not

been done to date. This question also relates to two other fundamental questions: (1) the

supposed cosmopolitanism of testate amoebae (see above), and (2) the possible response of

testate amoebae to climate (see below). If testate amoebae are not truly cosmopolitan, or if

they respond to temperature in addition to responding to moisture and pH, then it may be

possible that the ecological preferences of a given morphological species is not identical in

diVerent geographical areas. If true this could also mean that the ecological preferences of

species could have change over time, perhaps even within the Holocene.

Within each community or microhabitat type there are more subtle diVerences in faunas

such as changes in the relative abundance of species. For example, the micro-distribution of

testate amoebae along Sphagnum stems suggests that they are highly sensitive to vertical

micro-environmental gradients (e.g., light, moisture) (Bonnet 1958; Booth 2002; Chacharo-

nis 1954, 1956; Heal 1962, 1964; Meisterfeld 1977; Mitchell and Gilbert 2004; Sch nborn

1963). Furthermore, even in an apparently homogeneous surface such as an almost Xat,

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1.0

Axis 2

-1.0

-1.0 Axis 1 1.0

Fig. 4 Sample plot of canonical correspondence analysis (CCA) of testate amoebae data in an open peatland

area. The study site is a small schwingmoor peatland complex dominated by Sphagnum in southern Ontar-

io, Canada. The site is dominated by Chamaedaphne calyculata and Vaccinium oxycoccus with hummocks

of S. fuscum and S. capillifolium, and lawns of S. magellanicum, and hollows of S. cuspidatum. Samples of

Sphagnum were collected for testate amoebae analysis. Species data were log-transformed, and rare species

were down-weighted. Empty circle: spring; Wlled circle: summer; triangle: fall (October). Warner et al.

(2007)

monospeciWc Sphagnum lawn, complex patterns of community structure can be observed.

Using geostatistical tools, such patterns may be explained by the heterogeneity of the envi-

ronment such as the micro-topography (e.g. maximum vertical diVerence of 6 cm within a

40 60 cm area) and associated small diVerences in moisture conditions and water chemis-

try (Mitchell et al. 2000a).

Questions remain surrounding the degree to which seasonal changes and microsite spa-

tial patterns in faunal abundances and composition are controlled by biological inXuences

on the animals themselves or by environmental factors or both. In a recent study, Warner

et al. (2007) collected Sphagnum from various microhabitats an open peatland for seasonal

comparisons of testate amoebae faunas. A moderate shift in testate amoebae community

composition is observed in spring compared to summer and fall faunas (Fig. 4). These

changes may be due to moisture Xuctuations, or to diVerences in seasonal dynamics among

testate amoebae species.

Much of the work to date has focussed on testate amoebae in Sphagnum-dominated

peatlands, primarily in the northern hemisphere. Very little is known about testate amoe-

bae faunas in other types of peatlands, especially in tropical and subtropical latitudes.

For example, testate amoebae have been found in peat cores from herbaceous popales

peatlands in tropical central Mexico (Primeau 2004). Similarly, testate amoebae have

been found in the isolated high-altitude bofedales Juncaceae-dominated peatlands in

the central Andes of South America (Earle 2000; Warner unpublished). Much remains to

be learned about the ecology and paleoecology of testate amoebae in the lesser-known

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peatlands in other geographic regions of the world. Perhaps some light could be shed on

the evolutionary and biogeographic questions discussed above and help assess the

reliability of testate amoebae-derived transfer functions over broad geographical areas

and using that as a substitute for time, the long-term stability of these models.

Transfer functions using testate amoebae in peatland paleoecology

The development of large ecological data sets relating testate amoebae to major ecological

gradients has led to the development of transfer functions used in paleoecology. Transfer

functions describe the relationship between species and an environmental variable of inter-

est (e.g., depth to water table) statistically, and then apply this relationship to fossil assem-

blages to provide estimates of changes in the environmental parameter through time.

Several characteristics make testate amoebae especially useful in paleoecological studies:

(1) the tests of many taxa of testate amoebae are well preserved in a variety of lacustrine

and terrestrial organic sediments, (2) they are abundant and diverse (Warner 1987), (3) they

respond quickly to environmental changes (Buttler et al. 1996), (4) they provide informa-

tion about the exact coring location (as opposed to e.g. pollen and spores), and (5) they may

not respond to exactly the same factors as other indicators such as vascular plants (Mitchell

et al. 2000b) and thus provide an independent line of evidence for multi-proxy paleoeco-

logical reconstructions (Schreve and Thomas 2001). The growing number of paleoecologi-

cal studies that have used testate amoebae now attest to the value of the transfer function

approach to palaeoenvironmental reconstruction (Beyens and Chardez 1987; Charman

2001; Charman et al. 2001; Charman and Hendon 2000; Charman et al. 1999; Charman

et al. 1998; Mitchell et al. 1999; Warner and Charman 1994; Warner and Chmielewski

1992; Woodland et al. 1998). Testate amoebae are also central to an EU-funded research

project (ACCROTELM), which aims to provide data on mid-late Holocene climate

changes by analysing testate amoebae in peat sediments.

The robustness of any transfer function approach depends partly on the availability of

good quality modern data sets but also on an assumption that the species-environment

relationship has been stable through time. It is impossible to verify that the ecology of the

species has remained constant through time, which is one of the basic assumptions of

paleoecology. But it is possible to assess if species-environment relationships are similar

across broad geographical areas. The rationale for this approach is that if testate amoebae

were able to adapt to local conditions relatively rapidly, it would then be possible to detect

diVerences in their ecological optima among study sites. As noted above, when this is done,

the ecological preferences of most species generally compare quite well even between

widely spaced sample regions (Booth 2001; Lamentowicz and Mitchell 2005), and this

provides a good basis for the application of transfer functions over at least recent geological

time periods. But there are also exceptions and these call for further studies.

Another possible limitation of testate amoeba analysis is the lack of modern analogues

to some past communities. One such example has recently been stressed for a community

strongly dominated by Hyalosphenia subXava and DiZugia pulex (Caseldine and Gearey

2005). It remains to be established is such lack of modern analogues are real or if they

perhaps reXect an incomplete sampling for the development of transfer functions. It is

indeed possible that such communities represent very degraded situations that could be

avoided, consciously or not, when collecting surface samples. For example, Hyalosphenia

subXava was found to be associated with forested peatlands in Ontario (Charman and

Warner 1992), while it was characteristic for drained peatlands in Finland (Tolonen et al.

1994). The case of DiZugia pulex is more complex as this species has not been found to

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dominate any surface sample to this date. Perhaps we should aim speciWcally for unusual

habitats within peatlands to Wnd communities such as the above-mentioned one?

In ombrotrophic peatlands, the moisture content of the Sphagnum mosses and the depth

to the water table are directly linked to climate, and more speciWcally to the balance

between evaporation and precipitation (direct responses to temperature are treated further).

The response of testate amoebae to moisture in these ecosystems therefore makes them

good indicators of paleoclimate and this is how reconstructed water table depths are now

often being interpreted (Charman et al. 2004; Chiverrell 2001; Hendon and Charman 2004;

Hendon et al. 2001; Langdon et al. 2003; Mauquoy and Barber 1999, 2002; McGlone and

Wilmshurst 1999; Wilmshurst et al. 2002). In these cases, modelling the relationship

between testate amoebae and hydrology is only one part of the process. The reconstructions

that can be obtained using this approach have been tested against other hydrological prox-

ies such as plant macrofossils and peat humiWcation (Blundell and Barber 2005; Booth and

Jackson 2003; Booth et al. 2004; Charman et al. 2001; Charman et al. 1999; Lavoie and

Richard 2000; Mauquoy and Barber 2002; McGlone and Wilmshurst 1999; McMullen

et al. 2004; Mitchell et al. 2001; Wilmshurst et al. 2002). Blundell and Barber (2005) con-

cluded that testate amoebae provided more consistently reliable reconstructions that the

other techniques, but that a multi-proxy approach is nevertheless preferable. The accuracy

of reconstructions that can be obtained can also be demonstrated by comparisons with

instrumental records of water table change and climate variability over the relatively recent

past (Charman et al. 2004). This study showed that even the relatively low amplitude

changes in water table that have occurred over the past 50 200 years can be reconstructed

with some conWdence using a transfer function approach. The root mean squared error of

prediction for the transfer function used in this study (Woodland et al. 1998) was only

3.5 cm.

The degree of preservation of subfossil testate amoebae in peat or other sediments can

be quite variable and may depend on both the condition under which the sediment was pro-

duced and its subsequent history. A major gap exists in our understanding of taphonomic

processes associated with transformation of living plant to peat, and hence transformation

of living testate amoebae faunas to dead faunal assemblages. If we consider that Sphagnum

plants and litter generally lose integrity and mass rapidly immediately upon senescence and

early stages of decomposition compared to later stages, and how Sphagnum litter travels

through the oxic layer before entering the deeper anoxic layers, how might such processes

and length of time shape dead testate amoebae communities? Is it possible that dead fossil

assemblages are mixed assemblages that originated from diVerent microhabitats? In order

to provide answers to such questions, more attention should be devoted testate amoeba

taphonomy; it could indeed become a discrete Weld of study in itself.

Future applications of testate amoebae

The short generation time and wide distribution (albeit with reservations in mind regarding

cosmopolitanism) of testate amoebae makes them good indicators for the monitoring of

environmental change. Their usefulness as bioindicators is not restricted to long-term

hydrological development of Sphagnum peatlands. Although a large proportion of the work

on peatland ecology has focused on the reconstruction of former hydrological conditions,

other applications have also been developed. In particular, pH changes can be reconstructed

eVectively (Mitchell et al. 2001). Testate amoebae can also be used to assess the ecological

impact of some speciWc events such as the deposition of volcanic ash on peatlands (Dwyer

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2126 Biodivers Conserv (2008) 17:2115 2137

and Mitchell 1997). A range of new applications has thus been emerging over the past

10 years and others are being reinvigorated by renewed interest. A number of examples are

given here. Such new developments are very promising and may still represent only a frac-

tion of the full potential of testate amoebae in ecological and paleoecological research.

Limnology and palaeolimnology

Studies on the ecology of lake and river testate amoebae have shown that they respond to

the trophic status, pH, and pollution (Beyens et al. 1986; Burbidge and Schr der-Adams

1998; Dalby et al. 2000; Kumar and Patterson 2000; Moraczewski 1962; Ruzicka 1982;

Sch nborn 1965a, 1966, 1973; Sch nborn et al. 1965). Testate amoebae communities may

also respond to large scale changes in land use such as deforestation and subsequent

watershed management such as fertilizer and pesticide use (Patterson et al. 2002; Scott and

Medioli 1983). It has also been suggested that some taxa respond directly to climate and

mean temperature change (McCarthy et al. 1995). As in peatlands, the preservation of tests

in many lake sediments means that subfossil testate amoebae communities can be used to

trace changes in these environmental conditions over time (Ellison 1995; Ruzicka 1982;

Sch nborn 1973). It is important that interpretation of lacustrine assemblages take into

account the diverse range of inXuences on testate amoebae in these environments. For

example changes to more acidic indicators in a lake may reXect soil development in the

catchment and transport of soil dwelling tests in run oV water rather than a change in lake

water conditions (Ellison 1995).

Sea-level change

Testate amoebae have been found in estuarine environments, where their range overlaps

with that of foraminifera, a large group of protists much better known in the marine realm

(Charman et al. 1998; Gehrels et al. 2001; Medioli et al. 1990; Scott et al. 2001). As with

other habitats there are still diverse opinions on the taxonomy of this particular group of

testate amoebae but it is clear that in marginal coastal settings there is a strong zonation of

testate amoebae assemblages. This has been shown in a number of locations in the UK

(Charman et al. 2002), and North America, where the testate data are normally considered

along with foraminifera (e.g., Scott et al. 2001). Earlier studies tended only to examine

larger taxa found in the same size range as foraminifera (>63 m) but more recent studies

have demonstrated that a much greater abundance and diversity of taxa can be found when

a smaller size range is included (Charman et al. 1998).

In a similar approach to that used for peatland water tables (see above), multivariate sta-

tistical methods can also be applied to the sea-level data to provide estimates of individual

taxon optima and their ability to predict mean sea-level elevation (Fig. 5). By comparison

with other bio-indicators of sea level, testate amoebae are particularly abundant and show

strongest zonation in the upper parts of saltmarshes. In these situations, they have the

potential to produce much more precise estimates of sea-level elevation than groups such as

foraminifera and diatoms. Combining groups together may provide an optimum approach

to reconstruction of former sea levels from saltmarsh sediments (Gehrels et al. 2001). Use

can also be made of testate amoebae in understanding the rates of transition from saline

lagoon to freshwater lake environments in isolation basins where sea level has fallen. As

with saltmarsh sediments, extending the range of size fractions analysed yields a much

richer assemblage of tests (Lloyd 2000; Roe et al. 2002).

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Fig. 5 Optimum position in the tidal frame of testate amoebae in British saltmarshes. The circles show the

optimum and the bars the tolerance of each taxon. Data are plotted as the % of time (square root) that a loca-

tion is Xooded by the tide over an annual cycle. i.e., lower values are higher up the saltmarsh and subject to

less Xooding. See Gehrels et al. (2001) for details. Reproduced with permission of the Quaternary Research

Association

While all the modern studies conWrm that elevation in relation to mean sea level is by far

the most important determinant of assemblage composition in coastal environments, it is

clear that other factors may also play a signiWcant role. Elevation is a proxy for period of

tidal inundation, which creates diVerences in hydrological and salinity conditions across a

saltmarsh. However, salinity may vary independently from elevation (for example in rela-

tion to micromorphology of a saltmarsh) and other factors such as particle size and organic

content of the substrate probably also play a role (Charman et al. 2002).

To date, most of the work on coastal testate amoebae has focused on contemporary ecol-

ogy. The ultimate aim of much of the work is to apply it in palaeo-reconstructions. Initial

assessments of the abundance of fossil tests in a variety of coastal sediments suggests preser-

vation of tests may be quite variable and that capitalising on this potential application will

need careful site selection (Roe et al. 2002). Certainly conditions in coastal settings are more

physically dynamic and geochemically variable than those found in acid peatlands and lakes,

and this probably explains why Wnding well-preserved tests is still a challenging prospect.

Paleoclimatology

Testate amoebae are now being used as indicators of paleoclimate in ombrotrophic peat-

lands only indirectly, through their response to hydrological changes. Their usefulness as a

paleoecological tool would be much greater if one could show that they also respond

directly to temperature changes. Such a response is suggested by patterns of testate amoe-

bae diversity in relation to latitude. Indeed, testate amoeba diversity decreases with increas-

ing latitude: 300 taxa in the Arctic (Beyens and Chardez 1995) from a total of 2000,

and a decline in nebelid (Nebela species and closely related taxa) species richness were

observed towards high southern latitudes (Smith 1992, 1996; Wilkinson 1994). But are

these trends real or are they rather due to lower available and therefore sampled habitat

diversity?

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Fig. 6 Testate amoeba diagram

showing the vertical distribution,

as relative proportions, of testate

amoebae in a peat monolith from

Le Cachot, a cutover bog in the

Swiss Jura Mountains. Zone des-

ignations are: L, living Sphagnum

mosses; S, secondary peat; D&O,

detrital and old cutover horizon;

DC, highly decomposed catotelm

peat; CA, catotelm peat. See But-

tler et al. (1996) for details.

Reproduced with permission of

the New Phytologist

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The main diYculty to address the possible inXuence of temperature on testate amoebae

is to take into account the confounding eVects of factors such as water table, pH, and host

plants. Indeed, plant communities and soil types are generally strongly correlated to latitu-

dinal and altitudinal gradient (Odum 1971). It is not surprising that micro-organisms living

in soil or mosses also respond strongly to this gradient if they are analysed in the diVerent

habitats (soil type or moss species) found along a climatic gradient, as has usually been the

case (Loranger et al. 2001; Todorov 1998).

One way to avoid confounding factors is to limit the study to a very speciWc habitat such

as one moss species. To date the only study in which a standard substrate was used (a single

moss species, Hylocomium splendens, sampled in similar situations along three altitudinal

gradients) has failed to demonstrate a signiWcant overall reduction in diversity with increas-

ing altitude (Mitchell et al. 2004). Such a result suggests that testate amoebae may not

respond very clearly to temperature, at least not in terms of overall diversity. Further stud-

ies are needed, perhaps in diVerent regions or habitats. To properly address this issue

requires (1) a well-deWned sampling protocol to explore the natural patterns, and (2) an

experimental approach to test the eVect of temperature alone on the amoebae.

Peatland regeneration and management monitoring

Much of this review concerns applications of testate amoebae analysis to palaeoenviron-

mental reconstructions and interpretation of past conditions. Some of these cover the recent

past (i.e., less than 300 years) (Buttler et al. 1996; Hendon and Charman 2004; Patterson

et al. 2002; Reinhardt et al. 2001). However, there is a growing recognition that testate

amoebae can also be used in shorter-term biomonitoring. For example, testate amoebae are

starting to be used in the monitoring of peatland regeneration (Davis and Wilkinson 2004;

Jauhiainen 2002; Laggoun-D farge et al. 2004), and peatland management (McMullen

et al. 2004; Vickery and Charman 2004) (Fig. 6).

In wetlands, the small scale variability of testate amoebae assemblages may make represen-

tative repeat sampling diYcult (Mitchell et al. 2000a) and careful sampling strategies are

needed to reliably identify statistically signiWcant diVerences over time or between manage-

ment treatments. Experiments need to include adequate replication to account for spatial vari-

ability. Vickery and Charman (2004) compared testate amoebae populations in experimental

plots following diVerent forestry harvest and hydrological management on a Scottish raised

bog. The overall health of testate amoebae communities was assessed by the percentage of liv-

ing tests present. Improved conditions were created by leaving some tree remains on the bog

rather than clearing away all remnants of harvested trees, and this probably reXects a mulching

eVect, which keeps the moisture content of surface moss higher than in open conditions.

The response of testate amoebae to soil moisture rather than water table depth suggests

that major vegetation change on the mire surface may aVect palaeohydrological reconstruc-

tions independently of climate change. Such changes are only likely to arise from major

human disturbance or severe Wre, which would generally be visible in the pollen or plant

macrofossil record.

Soil and air pollution monitoring

Given the sensitivity of testate amoebae to water chemistry and other micro-environmental

gradients, it is not very surprising that several studies have illustrated how they respond to

soil pollution (Kandeler et al. 1992; Wanner and Dunger 2001, 2002), and to atmospheric

pollution (Balik 1991; Nguyen-Viet et al. 2004). These studies showed that the diversity

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and in some cases the density of testate amoebae was lower in polluted situations. As was

observed in lake studies, some species seem to tolerate higher pollution levels than others

and may even be found only in the polluted sites (Nguyen-Viet et al. 2007). The exact

reason why some species are more tolerant than others remains to be determined. Very lit-

tle is known about the exact mechanisms by which water chemistry inXuences testate

amoebae.

This response of testate amoebae to pollution could make them very valuable as integra-

tive monitors of pollution. Indeed one major challenge of pollution monitoring is the spatial

and temporal variability of the pollution. To accurately describe the pollution, and hence to

take appropriate actions to limit its impact on the health of Humans and the environment, it

is necessary to have continuous or very regular measurements. This approach, combined

with the observation of living organisms, constitutes the optimal approach. This may be an

option in developed countries, but is not in many poor regions. In such cases, a biomonitor-

ing approach, either alone or combined with a lower resolution sampling for chemical anal-

yses, could constitute a viable low-cost alternative.

Other possible Welds of study or applications

The reasons that testate amoebae oVer so much potential for biomonitoring applications are

similar to those listed for palaeoenvironmental applications; the speed of response to change is of

the order of days to weeks, and the diversity of the fauna is high. A unique characteristic of tes-

tate amoebae (as compared with ciliates or nematodes for example) is the preservation of the

tests of dead individuals, at least over short periods of time (months to years) in soils. This

means that living and dead assemblages could be used to provide information on very short-term

or long-term conditions respectively. Some comparative studies suggest that testate amoebae

respond more dramatically than ciliates to the conversion of a soil to agriculture, by a reduction

in species richness of up to 50% as compared with neighbouring natural soils (Foissner 1997b).

We believe that we are only seeing a fraction of the potential applications of testate

amoeba analysis as an ecological tool. As examples of some unusual application (albeit far

from the general topic of this review), testate amoebae have been shown to be reliable indi-

cators of the truZe-bearing potential of soils in the South of France (Bonnet 1977, 1979)

and were also used in a murder case: by analysing the testate amoebae found in the mud on

the shoes of a suspect, it could be proven that he had been present at the murder scene

(Lambert and Chardez 1978)!

Concluding remarks

Testate amoebae are clearly an interesting group of bioindicator organisms in peatland

ecology and paleoecology. Like any method, this one currently suVers from some limita-

tions. Some of these are certainly inherent to the organisms themselves but others are due

to current limitations in the knowledge we have on their taxonomy, ecology and biogeogra-

phy. The current interest and research eVort in these organisms will no-doubt increase their

reliability and also extent the range of possible applications in the future.

Acknowledgements Edward Mitchell is supported by Swiss NSF project n 205***-******/1 and was

supported by EU project RECIPE, partly funded by the European Commission (n EVK2-2002-00269) and

partly, for the Swiss partners, by the State Secretariat for Education and Research, Switzerland. SEM illus-

trations were produced at the SEM lab of the University of Alaska Anchorage under the direction of Dr. Jerry

Kudenov. Jan Pawlowski of the University of Geneva provided critical comments on parts of the manuscript.

The critical comments of two anonymous reviewers on the manuscript are gratefully acknowledged.

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