Review of Palaeobotany and Palynology 229 (2016) 1–8

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Review of Palaeobotany and Palynology

journal homepage: www.elsevier.com/locate/revpalbo

The effect of preparation methods on dung fungal spores: Implications

for recognition of megafaunal populations
Eline N. van Asperen ⁎, Jason R. Kirby, Chris O. Hunt
School of Natural Sciences and Psychology, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: Spores from coprophilous fungi are emerging as an important palaeoecological indicator of the presence of large
Received 23 December 2015 herbivores, but the methods by which they are recovered may have a significant impact on their preservation
Accepted 28 February 2016 and recognition. Here, we test a number of chemical and mechanical techniques used in palynology. Spore
Available online 7 March 2016
occurrence, size and shape is affected by preparation technique and is particularly affected by acetolysis. This
has important implications for recognition of past megafaunal populations.
Keywords:
Fungal spores
© 2016 Elsevier B.V. All rights reserved.
Palynological preparation techniques
Acetolysis
Megafauna

1. Introduction (2001) cautions against including treatment with HF in the standard

procedure since its effect on fungal spores is unknown and thought
The past decades have seen an increase in the use of spores of to be deleterious. Clarke (1994) processed samples from a variety of
coprophilous fungi from sedimentary sequences as a proxy for large substrates with three different techniques:
herbivore presence and abundance (e.g. Davis, 1987; Davis and Shafer,
2006; Cugny et al., 2010; Jeffers et al., 2011; Baker et al., 2013). Although A. boiling in KOH, HF, acetolysis, mounting using tertiary butyl alcohol
they have great potential for advancing our understanding of past (TBA);
herbivore population dynamics, extinction events and the impact of B. boiling in NH4OH, sieving, swirling, mounting using TBA;
husbandry practices on the natural environment, there remains signifi- C. boiling in KOH, sieving, heavy liquid separation with ZnCl2, mounting
cant uncertainty regarding preservation and recovery of these spores using TBA.
(Feranec et al., 2011; Baker et al., 2013). The validity of this proxy
method depends heavily on a robust understanding of the relationships She found that small round to oval fungal spores behaved in a similar
between large herbivore presence and abundance, spore presence, manner to pollen. Large, buoyant forms were lost in treatment A,
variety and abundance in the natural/modern environment, spore whereas only these forms were consistently recovered in treatment B.
preservation in the soil, and spore recovery. Whilst some of these rela- Thick-walled forms were lost in treatment C. Although she did not no-
tionships are increasingly under study (e.g. Blackford and Innes, 2006; tice any deterioration of the spores, Clarke's (1994) results suggest
Raper and Bush, 2009; Dietre et al., 2012; Parker and Williams, 2012; that the preparation method chosen can affect spore representation.
Etienne et al., 2013; Gill et al., 2013), the most basic level of recovery The most common method used by pollen analysts today involves
techniques has yet to be addressed in a substantive fashion. alkali treatment followed by acetolysis. Alkali treatment is intended
In most cases, fungal spores are extracted from palaeoenvironmental for the initial disaggregation of the sample and goes back to the earliest
samples along with other relevant taxa, especially pollen, commonly days of pollen analysis, when a small piece of peat was typically boiled
using the ‘standard’ pollen preparation method (Faegri and Iversen, in a drop of water with a grain of KOH or NaOH on a microscope slide,
1989; Moore et al., 1991). This is at least in part because palaeoecologists using a candle as a heat source. Later a more standardised technique
have tended to identify and count fungal material as part of pollen-based used a known weight or volume of peat boiled in 10% KOH or NaOH
studies and because both microfossil groups are found in pollen slides. solution, followed by centrifugation, then repeated rinsing and re-
Knowledge of the effect of these chemical preparation methods on centrifuging (e.g. Godwin, 1934).
fungal spore representation in pollen slides is limited. Thus, Van Geel The acetolysis part of the standard pollen preparation method was
devised by Gunnar Erdtman, who introduced several related techniques
in the 1930s (Erdtman and Erdtman, 1933; Erdtman, 1934, 1935, 1936).
⁎ Corresponding author. This involves heating the sample in a mixture of acetic anhydride and
E-mail address: [email protected] (E.N. van Asperen). sulphuric acid (9:1), with the intention of removing the protoplasm of

http://dx.doi.org/10.1016/j.revpalbo.2016.02.004
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2 E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8

the pollen grain. Acetolysis is also known to degrade or destroy cellulose blue, and identified under a light microscope. Measurements were
(Brown, 1960, p. 144). The acetolysation process destroys all pollen taken on the fungal spores present in these preparations.
material apart from sporopollenin which forms the outer wall (exine) After an incubation period of 9–13 weeks, four 1 cm3 subsamples
of the pollen grain, which is visually inspected to enable identification were taken from the surface of each dung type (Table 1). One dung sub-
based on morphological characteristics. sample was subjected to standard pollen preparation methods (method
Alkali treatment has been implicated in the destruction of pollen A; see Table 2); a second subsample was prepared using the same meth-
grains (Brown, 1960, p. 139). Trials by Faegri and Deuse (1960) showed, od, but excluding acetolysis (methods B1 and B2); the third subsample
however, that prolonged boiling in KOH solution led to shrinkage of followed method B2 but in addition was subjected to density separation
pollen rather than destruction. Shrinkage of pollen following alkali by swirling (method C); and the fourth subsample was sieved only
treatment was also noted by Christensen (1946). It has also been argued (method D). Fungal spores present in the samples were, where possible,
that acetolysation destroys some important pollen characteristics identified and measured. The species richness, relative abundance,
including the intine structure and aperture morphology, and can also preservation state and size of spores observed in vivo were compared
degrade pollen grains with more fragile exines (Hesse and Waha, with those observed after pollen preparation. Spore counts and
1989). The use of acetolysis can lead to damage or destroy pollen of measurements are included in the Supplementary data.
Cannaceae, Juncaceae, Lauraceae and Musaceae (Erdtman, 1952, p. 9)
and causes the loss of fern perispores (Brown, 1960, p. 140), of up to
3. Results
50% of Sphagnum spores in peat samples (Wenner, 1947, p. 101) and
of most protoperidinioid dinoflagellate cysts in marine sequences
The incubated dung samples produced fruit bodies of 25 fungal taxa
(Mertens et al., 2009). In studies of the palynology of sediments
(Table 3): the Zygomycete genera Pilobolus, Mucor and Mortierella; the
known to be less than optimal for pollen preservation, including arid-
Ascomycete genera Ascobolus (4 species), Saccobolus, Cheilymenia
zone and cave sequences, acetolysis is known to degrade pollen assem-
(2 species), Sordaria, Cercophora/Podospora (10 species), Sporormiella
blages seriously (Coles, 1987; Horowitz, 1992). Furthermore, acetolysis
and Peziza; and the Basidiomycete genera Coprinus and Conocybe.
affects the size and shape of pollen grains, causing swelling of up to one
Some taxa, such as the Zygomycete genera, Sordaria, Podospora and
half (Christensen, 1946, p. 17; Faegri and Deuse, 1960, pp. 296–7). These
Coprinus, occurred on all or nearly all samples, whereas other taxa,
issues have significant implications for the recovery and identification
such as Saccobolus, Sporormiella, Peziza and Conocybe, occurred on only
of not just pollen but also a variety of microfossil groups which are likely
one or a few samples. The ubiquitous taxa also tend to be more abun-
to be similarly affected by chemical treatment.
dant when present than the less common taxa.
Mounting media have also been shown to impact pollen size and
shape. Glycerol gel was argued to cause swelling of grains (Christensen,
1946; Anderson, 1960), and although a series of experiments by Faegri 3.1. Zygomycota and Basidiomycota
and Deuse (1960) did not show the same phenomenon consistently, a
series of later papers also showed size changes in various mounting Whilst Zygomycete genera were abundantly present on the dung,
media (e.g. Reitsma, 1969; Praglowski, 1970; Large and Braggins, 1990). they were absent from most of the prepared samples. Where they
In studies of fungal spores, species identification provides crucial in- were encountered, they were present in the form of collapsed sporangia
formation, e.g. when certain species within a genus are not exclusively still containing some sporangiospores.
coprophilous, or when certain species preferentially grow on the dung Basidiomycete genera, in particular Coprinus species, were also
of particular animal species. Spore size is an important characteristic abundant on the dung. Although they are mostly absent from the
for species determination. Published spore sizes, however, refer to un- prepared samples, occasionally an abundance of spores is encountered,
processed spores, usually mounted in water (e.g. Ahmed and Cain, regardless of the preparation method employed.
1972; Lundqvist, 1972; Bell, 2005; Doveri, 2007; Guarro et al., 2012).
Spore sizes vary slightly in other mounting media, such as lactic acid,
3.2. Ascomycota
lactophenol and alcohol, or mounting agents such as Indian ink, Congo
red or Melzer's reagent that highlight certain features of particular
3.2.1. Cheilymenia and Peziza
fungal taxa (Lundqvist, 1972). So far, no studies have investigated the
When present on dung, Cheilymenia species tend to be abundant
effect of pollen preparation procedures on spore size.
(Plate I, 1a). Their spores, however, are entirely absent from samples
Building on Clarke's (1994) study, we present here an investigation
prepared with methods A and B and sparsely present in samples
into the effect of the various chemicals used in standard pollen prepara-
prepared with method C (Plate I, 1c–d). They are abundant in samples
tion procedures, as well as several mechanical preparation techniques,
which were sieved only (method D), although spores of the species
on dung fungal spore representation, preservation state and size. We
with the larger-sized spores were rare. The less common genus Peziza
then consider the potential broader implications of using traditional
was only encountered on one dung sample, which was prepared using
chemical extraction procedures in palaeoecological studies.
methods C and D (Plate I, 1b–d). A small number of Peziza spores was
found in these preparations. There are no obvious size changes in the
2. Materials and methods

Fresh dung was collected of African elephant (Loxodonta africana,

n = 2) and white rhinoceros (Ceratotherium simum, n = 2) from Table 1
Knowsley Safari Park (Prescot, UK) and wild cattle (Bos taurus, n = 4) Dung samples.
from Chillingham Wild Cattle Park (Chillingham, UK) and stored for
Sample no. Species Preparation methods
2 days in the dark at 4 °C. 100–150 g of each sample was placed on
moist paper towels in a sterilised glass dish with a glass lid and incubat- KDLA1 Loxodonta africana A & B1
KDLA2 Loxodonta africana C&D
ed under laboratory conditions (20 °C, ~12 h of daylight per day; Krug,
KDCS1 Ceratotherium simum A & B1
2004). The samples were kept moist by periodically wetting the paper KDCS2 Ceratotherium simum C&D
towel with distilled water. CDBT1 Bos taurus A & B1
During the incubation period, the dung samples were examined reg- CDBT2 Bos taurus A & B1
ularly using a stereomicroscope. Spore-producing fruit bodies growing CDBT3 Bos taurus A & B1 & B2
CDBT4 Bos taurus C&D
on the dung were isolated, mounted in alcohol and lactophenol cotton
 E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8 3

Table 2
Description of sample preparation methods.

Method Aa Method B Method C Method D

Addition of Lycopodium tracer tablets Addition of Lycopodium tracer tablets Addition of Lycopodium tracer tablets Addition of Lycopodium tracer tablets
Volumetric sampling Volumetric sampling Volumetric sampling Volumetric sampling
Heating with 10% sodium hydroxide Heating with 10% sodium hydroxide (method Heating with 10% potassium
B1) or 10% potassium hydroxide (method B2) hydroxide
Sieving out the fraction N125 μm Sieving out the fraction N125 μm Sieving out the fraction N125 μm Sieving out the fraction N125 μm
Density separation using a swirling dish
Sieving out the fraction b6 μm Sieving out the fraction b6 μm Sieving out the fraction b6 μm Sieving out the fraction b6 μm
Treatment with 10% hydrochloric acid Treatment with 10% hydrochloric acid Treatment with 10% hydrochloric
acid
Acetolysis by washing with glacial acetic acid and
heating with sulphuric acid and acetic
anhydride
Staining with safranine and mounting in silicon Staining with safranine and mounting in Staining with safranine and mounting Staining with safranine and mounting
oil using tertiary butyl alcohol silicon oil using tertiary butyl alcohol in silicon oil using tertiary butyl alcohol in silicon oil using tertiary butyl alcohol
a
Although the standard pollen preparation method generally includes treatment with HF, this was not included here due to laboratory restrictions.

spores of these two genera compared with spores mounted in alcohol lactophenol, whereas spores treated with method A are more variable
and lactophenol. than spores mounted in alcohol and lactophenol (Fig. 1).

3.2.4. Sporormiella
3.2.2. Ascobolus
A long-standing debate concerning the relationship between the
Heating with NaOH and/or acetolysis has a strong adverse effect on
genera Sporormiella and Preussia leads some authors to include
Ascobolus spores: they were absent or sparsely present in samples pre-
Sporormiella in Preussia as a later synonym (e.g. Kruys and Wedin,
pared with methods A and B1. Samples treated with KOH and HCl
2009; Guarro et al., 2012), while others regard the two genera as suffi-
(methods B2 and C) preserve a similar number of spores to samples
ciently different to retain both generic names (e.g. Lumbsch and
that were sieved only (method D). The spores found in these samples
Huhndorf, 2007; Kirk et al., 2008; Doveri, 2011). The differences
have a crumpled appearance (Plate I, 2d–g). When the sizes of the
between the two genera pertain primarily to their fruit bodies and
Ascobolus spores treated with these various methods are compared
their preferred substrate (dung for Sporormiella; plant debris, wood or
with the sizes of spores mounted directly from the dung (Plate I, 2a–c),
soil for Preussia) rather than their spores (Kruys and Wedin, 2009).
it becomes clear that the large spores (50–65 × 25–38 μm) of the species
Since pollen analysts have so far used the generic name Sporormiella
Ascobolus immersus are entirely absent from all treated samples. In com-
for 4-celled spores with germ slits, this name is used here.
parison with the spores mounted in alcohol and lactophenol, spores
Sporormiella species were not common on the dung, but where
treated with methods B1 and C appear smaller, whereas those treated
present, spores were encountered in prepared samples regardless of
with method A are larger, especially in width. The sieving-only (method
preparation technique used (Plate I, 4a–d). In samples prepared with
D) spores are most similar in size to the spores mounted in alcohol and
method A, no complete spores were found. Instead, the four-celled
lactophenol (Plate I, 2 h).
spores had broken up into their constituent cells (Plate I, 4c). Samples
prepared with the other methods did contain complete spores (Plate I,
3.2.3. Saccobolus 4d), as well as single cells. Treatment with method A in general led to
The spores of the related genus Saccobolus tend to form clusters of 4 an increase in length and width of the spores, although a wide range
or 8 spores, sticking together by the pigment in their epispore. In some of variation was documented (Fig. 1). Spores treated with method B1
species these clusters fall apart easily, while in other species the clusters are shorter but wider than spores mounted directly from the dung.
persist for longer (Bell, 2005). When species with ‘sticky’ clusters are Spores prepared with methods C and D also increased in width.
present on the dung, these clusters are commonly encountered regard-
less of the preparation method used (Plate I, 3a–e). Single spores are 3.2.5. Sordaria
rarely encountered due to their small size. Spores treated with methods Sordaria was one of the most common taxa growing on the dung
B1, C and D are smaller in size than spores mounted in alcohol and samples. Sordaria spores survive all preparation treatments tested
here, and are common to abundant in the prepared samples (Plate I,
5a–f). However, spores treated with method A are often less
well-preserved, frequently appearing wrinkled and deformed (Plate I,
Table 3
Taxa present on dung samples and in preparations. 5c–e). In addition, the pore complex tends to be more prominent,
protruding clearly from the spore, when method A is used.
Taxon Dunga Method A Method B Method C Method D
Sordaria ascospores are produced in 8-spored asci, and tend to stick
Zygomycetes +++b − − + − together in small groups as they are discharged from the fruit body
Ascobolus +++ + + ++ ++ (Ingold, 1933; Ingold and Hadland, 1959). About 39% of spores in sam-
Saccobolus ++ ++ ++ ++ ++
ples prepared with methods B1, C and D preserved these groupings,
Cheilymenia +++ − − + +++
Sordaria ++ ++ ++ ++ ++ forming groups of 2–8 spores even after preparation (Plate I, 5b). In con-
Cercophora/Podospora ++ + + + + trast, in samples prepared with method A only 9% of spores were found
Sporormiella + + + + + in groups, and no groups of more than 3 spores were found. Spores pre-
Peziza + nac na + +
pared with methods A and B1 are similar in length to spores mounted in
Basidiomycetes +++ + + + +
alcohol and lactophenol, while spores prepared with methods C and D
a
This refers to abundance when present, not to how commonly the genus is encountered are shorter (Fig. 1). However, a number of spores are clearly larger,
across the different dung samples.
b
+++ abundant; ++ common; + present but rare; − absent.
particularly in width. It is unclear whether this increased size variation
c
This genus was not present on the dung samples that were prepared using methods A is due to the chemical preparation or whether this represents another
and B. species which remained unnoticed on the dung samples.
4 E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8

3.2.6. Cercophora and Podospora withstand preparation methods using corrosive chemical methods
Although usually less abundant than Sordaria, one or more species such as acetolysis and boiling with NaOH or KOH. It is doubtful whether
of Cercophora or Podospora were present on each dung sample (Plate I, these spores could survive in soil or other substrates for extended pe-
6a–d). Their spores were present regardless of the preparation tech- riods. Furthermore, these spores are difficult to distinguish and would
nique used (Plate I, 6e–o), but often in very low numbers. As in Sordaria, not be identifiable to genus when encountered in a fossil sample.
but less frequently, spores stick together by their appendages when However, the absence of such spores in pollen preparations, whether
discharged (Ingold, 1933; Yafetto et al., 2008) and are found in small because they are not preserved in fossil samples or because they are
groups in samples prepared with methods B1, B2, C and D, but not in destroyed in the preparation procedure, could result in a lack of recog-
samples prepared with method A. Sizes are similar between the differ- nition of the presence of those dung types which are dominated by
ent preparation techniques. Only a single spore falls within the size Cheilymenia species.
range of the spores of the largest species measured when mounted in
alcohol and lactophenol, and a large number of spores fall below the 4.2.2. Genera with pigmented epispores
size range of the spores mounted directly from the dung. This could The predominantly coprophilous genus Ascobolus is readily
indicate a significant size difference in these spores when prepared recognisable by its pinkish epispores with striated, dotted or warted or-
and mounted in different fluids, although a small number of Podospora namentation (Bell, 2005; Webster and Weber, 2007; Guarro et al.,
species has such small spores, and it is therefore possible that the 2012). These epispores often disintegrate or peel off in ageing spores
small spores represent another species which remained unnoticed on (Bell, 2005). Spores in prepared samples tend to have collapsed
the dung samples. Spore size is also more variable in the treated epispores, making it difficult to ascertain the original size and ornamen-
samples, particularly in width. The preparation treatment thus seems tation of the spore. Spore size also seems to be affected by the prepara-
to have some effect on spore size and shape, although it is difficult to tion method used, with boiling with NaOH or KOH leading to shrinking,
quantify this effect from the current experiment. and acetolysis leading to swelling.
Underneath the epispore, Ascobolus spores are similar to the hyaline
4. Discussion spores of Cheilymenia and Peziza, and are therefore vulnerable when
more corrosive chemicals are used, although the presence of the epispore
4.1. Zygomycota and Basidiomycota provides limited protection. Given the tendency of the epispore to disin-
tegrate in older spores, however, these spores may not survive in soils or
Some taxa found on the dung are lost altogether or almost entirely. sediments for extended periods.
Zygomycete spores are too small to be retained in the 6 μm sieve. How- Almost all species of Saccobolus are coprophilous (Doveri, 2007;
ever, since these spores tend to have few distinguishing characteristics Guarro et al., 2012). Their spores have pinkish pigmented epispores,
that would enable identification to genus or species level, they are not which enables spore clusters to survive through all preparation treat-
regarded as having significant palaeoecological value. Although some ments. However, these clusters often fall apart with time, and it is un-
taxa, such as Pilobolus, are obligate coprophiles (Krug et al., 2004), likely that clusters persist in substrate for longer periods. Single spores
many Zygomycete taxa can grow on a wide range of substrates are often so small that they are lost through the 6 μm sieve.
(Webster and Weber, 2007). They therefore cannot be used as dung
indicators. 4.2.3. Genera with thick-walled, pigmented spores
Similarly, Basidiomycete spores are also small and mostly lost The spores of Sporormiella, Sordaria and Cercophora/Podospora are
through the 6 μm sieve. When they are sufficiently large, their pigmented more resistant to chemical degradation due to their thicker, pigmented
spore walls are tough enough to be preserved through all preparation spore walls. These three genera are generally regarded as being among
treatments. However, the spores are often difficult to distinguish and the strongest indicators of dung in palaeoecological studies (e.g. Baker
many taxa grow on a wide range of substrates (Webster and Weber, et al., 2013).
2007), limiting their usefulness as dung indicators. Most species of Sporormiella grow on dung (Doveri, 2007; Guarro
et al., 2012). The Sporormiella species present in the samples analysed
4.2. Ascomycota here were not common and developed late in the succession. The fruit
bodies, asci and 4-celled spores observed are most similar in size to
4.2.1. Genera with hyaline, thin-walled spores those of Sporormiella australis, S. lageniformis and S. tetramera, but the
Cheilymenia species characteristically grow on the dung of cattle and range of variation is relatively large, so that more than one of these spe-
other ruminants (Lundqvist, 1972; Richardson, 1972; Bell, 2005), cies may be present. In some species, the spores frequently break up
although not exclusively so. Indeed, in the experiment presented here, into their constituent cells, while in other species, the integrity of the
the genus was encountered on one sample of elephant dung as well as spore persists longer (Ahmed and Cain, 1972). The spore morphotypes
on all cattle samples (see also Ebersohn and Eicker, 1992). The spores present here seem to break up when subjected to acetolysis, whilst
of this genus, as well as those of Peziza species, are hyaline and thin- complete spores persist in larger numbers under other treatments.
walled. Peziza species are common on a wide range of substrates, al- However, such complete spores will be rare in sedimentary samples,
though they are not commonly isolated from soil (Bell, 2005; Doveri, whereas single cells are commonly encountered. It should be kept in
2007; Guarro et al., 2012). Spores of these and other genera with hyaline mind that the single cells of some Sporormiella species are so small
spores (e.g. Coprotus, Iodophanus and Thelebolus) are too fragile to that they would not be retained in the 6 μm sieve, leading to a potential

Plate I. Size and shape of spores after treatment with a range of pollen preparation methods or mounted directly from dung in alcohol and lactophenol.

1. 1a. Spores of Cheilymenia granulata mounted in alcohol and lactophenol; 1b. Spores of Peziza vesiculosa mounted in alcohol and lactophenol; 1c. Spore of Cheilymenia/Peziza
after treatment with method D; 1d. Spores of Cheilymenia after treatment with method D.
2. Spores of Ascobolus; a–c. mounted in alcohol and lactophenol; a. A. immersus; b. A. albidus; c. A. cf. michaudii; 2d. after treatment with method A; 2e–f. after treatment with
method B1; g. after treatment with method C; h. after treatment with method D.
3. Spores of Saccobolus; a. S. depauperatus mounted in alcohol and lactophenol; b. after treatment with method A; c. after treatment with method B1; d. after treatment with
method C; e. after treatment with method D.
4. Spores of Sporormiella; a–b. mounted in alcohol and lactophenol; c. after treatment with method A; d. after treatment with method D.
5. Spores of Sordaria; a. S. fimicola mounted in alcohol and lactophenol; b, f. after treatment with method B1; c–e. after treatment with method A.
6. Spores of Cercophora/Podospora; a–d. mounted in alcohol and lactophenol; a. C. mirabilis; b. P. pauciseta; c. P. fimiseda; d. P. conica; e–h. after treatment with method A; i–j.
after treatment with method B1; k–m. after treatment with method C; n–o. after treatment with method D.
E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8 5
6 E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8

Fig. 1. Histograms of length and width of spores of Saccobolus depauperatus, Sordaria fimicola and Sporormiella spp. when mounted directly from dung in alcohol and lactophenol and after
treatment with a range of pollen preparation methods.

loss of information. Most preparation methods seem to lead to swelling of the preparation method, except when acetolysis is used, which causes
of Sporormiella spores, particularly in width. deterioration of the spores.
Sordaria is almost exclusively coprophilous (Bell, 2005; Doveri, 2007), Species in the genus Podospora are mostly coprophilous, while only
although some species are frequently isolated from soil (Guarro et al., some Cercophora species are coprophilous (Bell, 2005; Doveri, 2007).
2012). Its spores are abundant and remain largely unchanged regardless The pigmented cell of the spores of these genera survives chemical
 E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8 7

preparation, whilst the hyaline cell usually collapses or is destroyed. The hind-gut fermenters such as elephants, rhinoceroses and horses
other hyaline appendages that some of these spores bear are fragile and (e.g. Alroy, 1999; Lyons et al., 2004), whose dung is richer in thick-
do not survive even sieving, and they are hard to observe without the walled, pigmented taxa. Genera with thin-walled, hyaline spores (esp.
use of Indian ink, cotton blue or Congo red (Bell, 2005). Cheilymenia) often dominate the dung of ruminants (Lundqvist, 1972;
The pigmented cells of the spores of coprophilous species of Richardson, 1972; Bell, 2005). The observed drop in dung fungal spore
Cercophora tend to be relatively small (b25 × 15 μm) compared with abundance may be influenced by the fact that the spore types which
the mostly larger pigmented cells of Podospora spores (Bell, 2005; are more characteristic of the surviving ruminants tend to be underrep-
Doveri, 2007), but there is overlap in size and shape between the two resented in pollen slides. The use of thick-walled, pigmented dung
genera (Guarro et al., 2012). However, Cercophora spores tend to remain fungal taxa as proxies for large herbivore abundance therefore needs
hyaline until after maturation and discharge (Lundqvist, 1972). Such to be revisited, especially since some large herbivore dung types are
hyaline spores are likely to be equally vulnerable to destruction in dominated by dung fungal genera that are not normally preserved in
chemical preparation as those of Cheilymenia and Peziza, although and recovered from soil samples. Absence of thick-walled, pigmented
they were sporadically present in method D samples. Furthermore, taxa cannot necessarily be taken as absence of animal dung.
Cercophora species are not commonly isolated from soils, whereas
Podospora species are (Guarro et al., 2012). It can tentatively be 5. Conclusions
assumed, therefore, that most Podospora-like spores encountered in
sedimentary samples represent coprophilous species. Different protocols for the preparation of palaeoenvironmental sam-
ples have a range of different impacts on the preservation and recovery
4.3. Taphonomic considerations of dung fungal spores. Deleterious effects vary depending on the type of
fungal spore and the chemical and mechanical treatments used. Spores
Our results have wider implications for our understanding of the of some taxa disappear from the samples entirely, either due to their
preservation potential of fungal spores in soils and sediments. It is prob- small size (Zygomycota, Basidiomycota, individual spores/cells of
able that fungal spores are not preserved uniformly. In biologically Saccobolus and Sporormiella), or due to the fragile nature of their hyaline
active soils, in particular, it is likely that there would be fairly rapid deg- and/or thin-walled cells (Cheilymenia, Peziza, unpigmented Cercophora
radation of those fungal spore types which are particularly vulnerable to upper cells, and to a lesser extent Ascobolus), which impedes both
acetolysis and other chemical treatment, since similar chemical process- their long-term preservation in substrates as well as their survival
es occur in biologically active systems. This vulnerability will likely be through preparation procedures. This is a potentially serious issue,
especially marked in genera with hyaline spores, such as Peziza, especially where such species dominate particular dung types, as is
Cheilymenia, unpigmented Cercophora upper cells, and to a lesser degree the case for Cheilymenia on cattle dung. Loss of these spores may bias
Ascobolus. If such spores do survive in favourable conditions, they reconstructions of past herbivore abundance.
will be vulnerable to chemical degradation during the preparation Pigmented, thick-walled spores are differentially impacted by differ-
procedure. ent methods. Acetolysis leads to the deterioration of the preservation
Overall, recovery is biased towards genera with thick-walled, state of certain types of spores. Sordaria spores obtain a crumpled
pigmented spores, which do not represent a comprehensive sample of appearance, making it more difficult to observe salient identification
the diversity of the dung fungal community. Furthermore, small single features. Groups of Sordaria and Podospora spores, which tend to stick
cells of Sporormiella spores may be lost when a sieving mesh with an ap- together when they are discharged from the fruit body, fall apart, and
erture size larger than the cell diameter is used. It is therefore important Sporormiella spores break up into their constituent cells when subjected
to use a sieving mesh with as small an aperture size as is practicable. to acetolysis. Where mineral-rich samples are subjected to treatment
with HF, the effect on spore preservation is likely to be even more dele-
4.4. Wider implications terious, although this remains to be shown experimentally. However,
the relative abundance of these spores appears not to be changed signif-
This study provides experimental evidence for alteration of size, icantly by the use of acetolysis, treatment with HCl, boiling in NaOH or
shape and morphological characteristics (or complete destruction) of KOH, or swirling. This parallels experience with other palynomorph
some fungal spore taxa depending on the nature of the preparatory groups including pollen, spores and dinoflagellate cysts.
treatment of samples. Other studies (e.g. Wenner, 1947; Erdtman, Spore size and shape are also affected. Spores treated by boiling in
1952; Coles, 1987; Mertens et al., 2009) have shown the impact of NaOH or KOH frequently shrink in size, or increase in width. Acetolysis
chemical pre-treatment, particularly acetolysis, on the preservation often produces swelling. Due to these size changes, measurements to
and recovery of a range of microfossil types including pollen, spores determine species on spores mounted in silicon oil or other mounting
and dinoflagellate cysts. media commonly used in pollen analysis cannot be compared directly
Use of acetolysis is confined to Quaternary pollen analysts (Brown, with measurements from the fungal literature, which are usually
1960; Wood et al., 1996), but is not used more widely in made on spores mounted directly in water. Although a large variation
palaeopalynology. Many palaeopalynologists regard the technique as a in spore size may tentatively be taken to indicate more than one species
dangerous aberration (K.J. Dorning, Pers. Comm. to COH, 1983), because is present (Johnson et al., 2015), the results of this study caution against
it is unnecessary for recognition of pollen from stratigraphic samples trying to pinpoint which species are present in the absence of morpho-
and because of the health and safety risks to the palynologist preparing logical characteristics other than size.
the sample. Efficient palynological preparation methods which avoid This study shows that the method that most closely preserves the
the use of acetolysis and most strong acids have been available for sev- diversity and relative abundance of dung fungal spores is sieving only.
eral decades (Hunt, 1985; Lentfer and Boyd, 2000; Mudie and Lalièvre, As it stands, however, this method is likely to be unsuitable for most
2013). Although it has a place in the preparation of modern type mate- other palaeoenvironmental materials (e.g. pollen) where additional con-
rial, other uses of acetolysis in palynology should be reconsidered. centration of samples is often necessary. We recommend using a method
The recovery bias towards thick-walled, pigmented genera also has that is the least corrosive as is possible given sample characteristics,
implications for their use as proxies for large herbivore abundance. starting with sieving and swirling, then boiling in KOH and treatment
The Late Quaternary extinction event is one of the key events that has with HCl, then heavy liquids, then boiling in NaOH. There appear to be
been studied using dung fungal spores as a proxy for megaherbivore no advantages in using acetolysis in the preparation of fungal spore sam-
presence (Davis, 1987; Feranec et al., 2011). The Late Quaternary extinc- ples, and fungal spore data obtained from pollen slides prepared using
tions disproportionately affected the largest species, including many acetolysis may lead to the under and over-representation of taxa.
8 E.N. van Asperen et al. / Review of Palaeobotany and Palynology 229 (2016) 1–8

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