Literature Review
Using fossilised dung fungal spores to indicate past herbivore
presence
Claire Lee Supervisor: Dr Bas van Geel
13483099 Assessor: Dr Carina Hoorn
Abstract
Fossilised fungal spores which grow on the dung of herbivores are more commonly being used by palaeoecologists during sedimentary sequence analysis to indicate the presence of herbivores in past ecosystems. This review examines the accuracy of the tool, with special interest into whether it can be used for estimating herbivore biomass and outlines how studies using the tool can be improved in the future. I present descriptions of the life cycles of individual classes of the coprophilous fungal succession seen to be lacking in current literature, and highlight the importance of spore dispersal. Current variations in
methodology across studies, and lack of analysis of these methods, hinder our abilities to compare results. Here I evaluate this variation and discuss possible solutions. Biotic and abiotic factors, such as climate, environment and livestock, also affect fungal spore
deposition. The importance of these factors is also investigated when considering these in herbivore biomass estimations. The use of SedaDNA in studies of spore of coprophilous fungi (SCF) is explored as an additional proxy and its use for species identification. Finally, I recommend studies of SCF should consider the biology of SCF life cycle in order to improve knowledge on the herbivore and SCF relationship. Further research into appropriate preparation techniques will reduce bias, while for quantifying spores, pollen and SCF concentration and influx should be used. Multiple cores should be taken in each site and multiproxy analysis should always be utilised. Lastly, investigations into quantifying the relationship between herbivore biomass and spore count is crucial for improving the accuracy of SCF herbivore biomass reconstructions.
Keywords: Coprophilous fungi . Dung . Non-pollen palynomorphs . Palaeoecology . Mycology . Herbivore biomass . Life cycle . Ecosystems .
Acknowledgements
I would like to thank Bas van Geel for his time, extensive knowledge and exceptional guidance throughout the creation of this review. Many thanks go to Carina Hoorn for support in structuring and development of the narrative. I would also like to give thanks to Giulia Devilee, Karin Nikolaus and Julia Averkamp for their continuous help and enthusiasm.
Contents:
Abstract………..2
Acknowledgements………3
1. Introduction………..5
2. Methodology………6
3. Non-Pollen Palynomorphs with special reference to spores of coprophilous fungi….……….7
3.1. The history of coprophilous fungi in Palynology.…………..………7
3.2. Fungal spore life cycle………..8
3.2.1. Mucormycetes.………9
3.2.2. Ascomycetes………..11
3.2.3. Basidiomycetes……….12
3.2.4. Fungi Imperfecti………..13
4. Preparation, Counting and Quantification……….………..14
4.1. History of NPP sample preparation……….15
4.2. History of SCF counting and quantification………15
4.2.1. Identifying genera: Sporormiella-type.………..……….16
4.2.2. Identifying genera: other spore types of coprophilous fungi……….…….17
5. External influences on coprophilous fungi abundance………19
5.1. Climatic influences………19
5.2. Livestock influences………20
5.3. Environmental influences………20
5.3.1. Lake sediments……….21
5.3.2. The transportation of spores………..22
6. New developments in research………..……….23
6.1. Environmental DNA and SCF……….………23
7. Outlook……….……….………24
7.1. Further inclusion of mycology……….24
7.2. Standardised methodology and cross site comparisons………24
7.3. Sporormiella and other SCF………25
7.4. Herbivore biomass estimations………..25
7.5. Multiproxy analysis……….26
8. Conclusion………27
References……….………..28
1. Introduction
Palynology is the study of ancient and modern pollen and non-pollen palynomorphs (NPPs) (Erdtman, 1963; Grant, 2018). Fossilised NPPs can preserve, due to their resistant organic composition (Grant, 2018), and provide information regarding the ecosystem in which they were deposited. NPP is an umbrella term for all plant microfossil material in a sample that are not pollen or fern spores, such as fungal spores, cyanobacteria, rhizopods or
invertebrate (Riding, 2004; van Geel, 2006; Cook et al., 2011; Grant, 2018). They can provide additional palaeoecological information depending on the microfossil used (van Geel, 2006). Spores of coprophilous fungi (SCFs) are NPPs which are best used along with
palaeoenvironmental proxies such as pollen and macrofossils. When synthesized it is possible to reconstruct the vegetation, climate and possibly the abundance of herbivores from cored samples. Through Lyell’s principle of uniformitarianism: the present is the key to the past (for overview see Scott, 1963), palaeoenvironmental reconstructions can provide information on current and future ecosystems. SCF play an important role but their application has been hindered because no standardised methodology for recording fungal spores exists (Hicks and Hyvärinen, 1999; Étienne and Jouffroy-Bapicot, 2014; van Asperen et al., 2016). This has impeded cross site comparisons of reconstructions (Étienne and Jouffroy-Bapicot, 2014; van Asperen et al., 2020.a) and yet the practice of SCF as indictors for herbivore biomass is increasing (Perrotti and van Asperen, 2019; van Asperen et al., 2020). It is therefore timely, to review the literature, evaluate the success of different methodologies, and summarise implications for future SCF research.
Coprophilous fungi grow primarily on the substrate of herbivore dung (Bell, 1983;
Richardson, 2001; Gauthier et al., 2010; van Asperen et al., 2020.a). When herbivores are taken out of an ecosystem, SCF will no longer be deposited (Davis and Shafer, 2006; Wood and Wilmshurst, 2013). Therefore, if SCF are found in sedimentary samples, they can indicate the presence, absence or sometimes density of herbivores at a certain time (Richardson, 2001; Johnson et al., 2015; van Asperen et al., 2020.a; Pino et al., 2020).
Despite their microscopic size, SCF have made it possible to trace back the presence of large herbivores from the Pleistocene, Holocene and Late-Quaternary. In North America,
researchers have used SCFs to create records of mass megaherbivore extinctions (Davis and Shafer, 2006; Wood and Wilmshurst, 2013; Raczka et al., 2016; Rozas-Davila et al., 2016; Birks et al., 2019; Perrotti and van Asperen, 2019; van Asperen et al., 2020.a).
In this review I provide an overview of the state of knowledge and gaps concerning SCF. Furthermore, I deliver insight into new aspects within this field of palynology. The key question I will address is: Are spores of coprophilous fungi really the accurate indicators of
herbivore presence that they are believed to be and can they be used in herbivore biomass estimations? Firstly, I discuss the development of SCF within palynology and the life cycles
of different fungal classes. I then examine preparation, counting and quantification methods. Next, I explore the popular use of the Sporormiella-type with comparisons to other SCFs. I then investigate the abiotic and biotic influences on spores, emphasising the role of transportation of spores and the effects of specific sample environments. Lastly, new research avenues and how these can be applied to the field are considered. The review ends with an explanation of how this subdiscipline can evolve and improve.
2. Methodology
The basis of this literature review started from three key articles (i) Asperen et al., (2020.a) (ii) Perrotti and van Asperen (2019) (iii) Richardson (2001). They were chosen because of their relevance to the subject and being published within the last few years by respected authors. From this point the snowball technique was used where articles or books cited within the key articles were visited to make up the reference portfolio.
For the Fungal Spore Life Cycle the best match method using Google Scholar and Scopus was chosen. The was due to lack of such details within the three key articles. Word searches within the search engines included the title, abstract and key words. These words consisted of coprophilous AND fungi AND herbivore, resulting in 64 documents. This was then
narrowed down to articles published from 2009 to present, which resulted in five
documents. The search was then changed to coprophilous AND fungi AND life AND cycle which resulted in a further five documents.
The review is a literature review but could also come under the umbrella of a state-of-the-art review. This is evident in the last section where recommendations for future studies are outlined. However, this is not done in such a rigorous way to warrant the review being categorised as systematic.
3. Non-Pollen Palynomorphs with special reference to spores of coprophilous fungi The main role of fungi in the biosphere is as decomposers and they play an important role in the nitrogen cycle (Taylor et al., 2009). Their symbiotic and vast multicellular relationship with nature has allowed fungi to thrive (Taylor et al., 2009). Some fungi grow on dung as this provides a perfect substrate (Krug et al., 2004), and directly links SCF to herbivores (Davis, 1987). SCF are the most popularly utilised NPPs because of this link.
Researchers are becoming increasingly aware of the value of NPPs within palaeoecology and archaeology (van Geel et al., 2011). Fungal spores are particularly common in fossil records, and although initially not used as widely as pollen and macrofossils, their use for
palaeoecological reconstructions should not be underestimated (Grant, 2018). The following chapters will discuss the nature of SCF, in order to highlight how they can be used as a palaeoecological tool.
3.1. The history of coprophilous fungi in palynology
Momentum has built within the field of NPPs, especially after the University of Amsterdam combined palynological studies with NPPs (van Geel, 2001; van Geel, 2006). This resulted in thousands of fungal types being identified leading to a paradigm shift which hugely
increased palaeoecological indicators (van Geel, 2006). This process was and still is interdisciplinary, gaining contributions from zoology, mycology, phycology and plant anatomy (van Geel, 2001; van Geel, 2006). Simultaneously, the process of categorisation was developed. In some cases fossil fungi are given genera names which are associated with their modern day equivalent (van Geel and Aptroot, 2006). As fossil fungal spores often cannot be identified on a family, genus or species level (Fig. 3.1), they are given a type number which sometimes follows their location of origin (van Geel et al., 2011).
Figure 3.1: The Fungal hierarchy. Scale decreasing from left to right. (Adapted after Alexopoulos et al., 1962). Coprophilous fungi have been studied for centuries (Hudson, 1968; Alexopoulos, 1962). Before they were proposed as herbivore indicators in the 1980s, they were solely considered essential biosphere decomposers (Hudson, 1968;Miranda et al., 2020) and believed to be too fragile to preserve in the fossil record (Taylor et al., 2009). This is despite Davis et al., (1975) proposal after finding Sporormiella abundance to correlate with grazing pressure in Wildcat Lake.
Today, our understanding of complex fungal successions has changed (Harper and Webster, 1964). While for much time it was postulated that fungal succession was a function of nutrition (Bell, 1983; van Asperen et al., 2020.a) this was disproven in 1964 by Harper and
Webster (Harper and Webster, 1964; Bell, 1983). It is now believed that various life cycles of fungi control the coprophilous fungal succession (Hudson, 1968).
3.2. Fungal spore life cycle
In addition to fungi class specific life cycles, some SCF also experience the cycle of being consumed and ejected by herbivores (this cycle is termed endocoprophilous) (Fig. 3.2). There are two types of SCF (i) Obligate spores which must pass through an animal’s digestion system in order to germinate (ii) Facultative spores which germinate without having to pass through a digestion system (Dix, 1995; Miranda et al., 2020). Halbwach and Bässler (2020) recently discovered that the majority of SCF are obligate, a theory which has long been postulated (van Asperen, 2020.a).
Figure 3.2: The endocoprophilous life cycle of coprophilous fungi.
As coprophilous fungi are the most commonly investigated NPPs (van Asperen et al, 2020.a), their endocoprophilous life cycle is well researched (Dix, 1995; Halbwachs and Bässler, 2020). When a herbivore eats herbage they simultaneously consume fungal spores, some of which will be coprophilous (Johnson et al., 2015; Perrotti and van Asperen, 2019). The SCF have thick melanised walls which act as protective layers, allowing them to travel through the digestive system without being harmed (Bell, 1983; Halbwachs and Bässler, 2020) and improves their likelihood of being preserved in fossil records (van Asperen et al., 2020.b). The dung and incorporated spores will then be ejected and left to germinate and grow on the dung substrate (Bell, 1983; Wood and Wilmshurst, 2013; Halbwachs and Bässler, 2020; van Asperen et al., 2020.b). At a mature stage of fructification, the fungi will eject spores away from the dung onto the surrounding herbage for the cycle to repeat itself (Bell, 1983; Perrotti and van Asperen, 2019; van Asperen et al., 2020.b). Consequently, a top-down system takes place (Baker et al., 2016) as increased production of spores reflects increased abundance of herbivores which is represented in the fossil record (Johnson et al., 2015). Dung provides a perfect substrate for fungi to grow, being high in minerals, vitamins, nitrogen, with initially relatively high temperature and moisture which reduces over time (Dix, 1995; Hudson, 1968). However, spatially the mycelium is limited to the size of the dung (Wicklow et al., 1980; Halbwachs and Bässler, 2020). Consequently, coprophilous fungi studies are likely to be related to megaherbivores rather than smaller herbivores due to the
increased size of faeces. The struggle for substrate space also affects the fungal succession. Coprophilous fungi include four classes, the three main ones being: Mucormycetes,
Ascomycetes and Basidiomycetes. Their life cycles vary, which creates different timescales for fruiting, resulting in the coprophilous fungal succession (Fig. 3.3) (Hudson, 1968). Furthermore, fruiting takes place rapidly after deposition which is likely due to microbial and invertebrate competition (Halbwachs and Bässler, 2020). Although the succession of fungi on dung is different to other fungal taxa, the coprophilous fungal succession has become known as the classic example (Fig. 3.3) (Hudson, 1968). However, mycology and the biology of spores is rarely introduced in SCF studies. Therefore, the following section aims to provide a comprehensive overview of the coprophilous fungal succession, as reproduction is an important part of spore dispersal.
3.2.1. Mucormycetes
After deposition of dung, Mucormycete fungi are the first to grow, producing between 100-100,000 spores within the first 2-3 days after deposition (Bell, 1983; Richardson, 2009). The Mucormycete or Zygomycete class are not exclusively coprophilous, however the species
Pilobolus and Pilaira only grow on dung (Bell, 1983). Their optimum growing and sporulation
temperature is 27oC with high humidity, making them ideal primary colonizers (Richardson, 2009). Their mycelium is typically aseptate (Bell, 1983) and their reproduction process is heterothallic (Krug et al., 2004). Nevertheless, they can reproduce sexually or asexually, depending on the mating type present on the substrate (Bell, 1983; Krug et al., 2004).
Figure 3.3: Coprophilous fungal class succession. Left to right: Mucromycetes image of Pilobolus crystallinus (Sharma, 2017), Ascomycetes image of Cheilymenia fimicola (Wood, n.d.), Basidiomycetes image of Panaeolus papilionaceus (Zealand, 2017).
Figure 3.1.1: Mucormycetes asexual reproduction cycle (Adapted after Bell, 1983).
Mucormycetes asexual reproduction (Fig. 3.2.1) is possible through spores produced in a sporangium (Taylor et al., 2009). When sporangiospores germinate on dung, a hyphal thallus is formed which produces sporangia or merosporangia after a few days (Krug et al., 2004). The sporangia then form sporangiaspores which are ejected once mature (Fig. 3.2.1; Richardson, 2009; Krug et al., 2004). Mucormycete fungi which are homothallic will produce zygospores simultaneously with sporangia (Krug et al., 2004). This is a classic Mucormycete characteristic (Taylor et al., 2009). Unfortunately, zygospore germination is infrequently mentioned in studies (Krug et al., 2004) and lacks research.
Figure 3.2.2: Mucormycetes sexual reproduction cycle
Heterothallic Mucormycetes species have two sexual hyphae which grow together (Krug et al., 2004). The hyphae apex is fused through the formation of progametangia once trisporic acid is produced (Krug et al., 2004). Subsequently, the progametangia becomes the
gametangia when the apex fuse, this is followed by the septum separating the apex (Krug et al., 2004). After the septum disintegrates, the nuclei in the cell are fused creating the
prozygosporangium which enlarges into zygosporangia (Krug et al., 2004). These then form hyphae or germ sporangium after germination. Germ sporangia often contain both mating types of sporagiospores (Krug et al., 2004) thus, completing the negative feedback loop (Fig. 3.2.2). The sporagiospores are then ejected through various mechanisms which are
dependent on the species and environment (Alexopoulos, 1962; Bell, 1983) but are likely dispersed through the air (Richardson, 2009).
3.2.2. Ascomycetes
Once the Mucormycetes population on the substrate has depleted, the second stage of succession takes place (Bell, 1983). Ascomycetes appear for 2-4 weeks; as their complex fruit bodies (Bell, 1983) require more time to complete their life cycle. Palaeoecological studies most commonly use ascospores which belong to the class Ascomycetes as they are usually most abundantly preserved (van Geel and Aptroot, 2006). They all have a
distinguishing characteristic called the ‘ascus’ which encompasses multiple ascospores (Alexopoulos, 1962), depicted in Fig. 3.2.3.
The life cycle pattern of Ascomycetes species can vary, however the general pattern is shown in Fig. 3.2.3. Once an ascospore germinates a mycelium forms (Alexopoulos, 1962). The nuclei in the spore will divide and the hypha will grow, branching off in some places (Alexopoulos, 1962). Fertilization is then possible if there are two opposing mating types, the male nucleus is retrieved by the female nuclei or ascogonium (Peraza Reyes and
Berteaux-Lecellier, 2013). From this the hymenium is created and ascospores are produced (Peraza Reyes and Berteaux-Lecellier, 2013), this is shown in Fig.3.2.3 and detailed below.
Figure 3.2.3: Development of Ascomycete ascospores (Adapted after Bell, 1983 and Peraza Reyes and Berteaux-Lecellier, 2013)
Ascospores form through the fusion of two nuclei after which the process of meiosis takes place where four nuclei are produced inside the ascus (Bell, 1983; Taylor et al., 2009). This division is likely to repeat, sometimes enough times to produce 64 or more ascospores in the ascus (Alexopoulos, 1962; Bell, 1983; Taylor et al., 2009). The ascospores are then ejected from the ascus once mature after mitosis, allowing the formation of mycelium post germination (Bell, 1983; Krug et al., 2004). The arrangement of the ascus varies depending on the Ascomycete genus which, if more frequently preserved, could provide a useful tool for genus identification (Bell, 1983). Many coprophilous Ascomycetes have phototropic asci, allowing them to move towards light (Bell, 1983). This characteristic guarantees that the ascospores will be ejected and discharged in the direction of herbage and not dung, so the spores can be digested (Bell, 1983). Many species are primarily transported aerially or through water (Krug et al., 2004).
3.2.3. Basidiomycetes
Basidiomycetes are a sister group to Ascomycetes (Taylor et al., 2009) and are usually the last to appear in the coprophilous fungi succession (Bell, 1983). Only few genera of this class are coprophilous (Bell, 1983; Krug et al., 2004) furthermore, their small spore size results in little representation in SCF studies. However, sometimes the parasitic order Ustiginales is preserved as their spores are generally larger (Alexopoulos, 1962).
Figure 3.2.4: Basidiomycete sexual reproduction cycle (Adapted after Taylor et al., 2009). A characteristic that makes Basidiomycete fungi so different to other classes is their production of basidiospores on the outside of the basidium (the body which produces the spores) (Alexopoulos, 1962). The formation of basidiospores is similar to that of ascospores (Alexopoulos, 1962). The basidiospores are powerfully exerted from the basidium, where they are then able to germinate and form mycelium with hyphae of uninucleate cells (Krug et al., 2004). Next, the cytoplasm from two compatible mycelia fuse to form a dicaryon which results in a binucleate cell (Alexopoulos, 1962; Krug et al., 2004; Taylor et al., 2009).
This then divides, forming a binucleate mycelium which will fruit from the hyphae (Krug et al., 2004). Fusion and meiosis of the hyphae creates the basidia, on which there are sterigmata where the basidiospores form on the outside of (Krug et al., 2004; Taylor et al., 2009). Once spores are released, if they find themselves on suitable substratum they will germinate (Krug et al., 2004) and the cycle continues (Fig. 3.2.4).
3.2.4. Fungi Imperfecti
Fungi Imperfecti, also known as Deuteromycetes, are not strictly coprophilous but do play an important role in substrate decomposition (Bell, 1983; Kiffer, 2011) and therefore fungi succession. The fungi imperfecti comprises of fungi whose sexual characteristics are
unknown but are postulated to be asexual forms of Ascomycetes or Basidiomycetes (Barnes, 1979; Kiffer, 2011). Therefore, they follow a similar life cycle, but asexual multiplication takes place through mitosis after the formation of conidia from the conidiophore (Kiffer, 2011). The structures of conidiogenesis varies across species (Kiffer, 2011).
4. Preparation, Counting and Quantification
Over the years there have been variations in the way palaeoecologists prepare and count NPP samples. The general procedure from field to microscope is as follows: As pollen and NPP samples are collected simultaneously (van Asperen et al., 2016), the field technique for Quaternary pollen analysis, as outlined by Faegri and Iversen (1989), should be conducted. Good palynological practice would be to collect both modern day reference material and fossilised material (Faegri and Iversen, 1989). Once sediment samples have been collected from the field using either a Hiller or Russian sampler, the core should not be opened unnecessarily again, to avoid contamination (Faegri and Iversen, 1989). When brought to the laboratory, preparation of samples can begin (Fig. 4.1). This is necessary to separate the pollen grains and NPPs from the sediment (Riding, 2004). Despite dispute, many studies still use the ‘standard’ pollen preparation techniques for NPPs depicted in Fig. 4.1(a) (Faegri and Iversen, 1989; van Asperen et al., 2016; Perrotti and van Asperen, 2019), while others use the University of Amsterdam (IBED-UvA) method as detailed by van Geel (2001) (Fig. 4.1(b)). After this, the samples are mounted onto slides where they can then be examined under microscopes.
Figure 4.1: Flow charts of (a) ‘Standard’ pollen preparation method as outlined by Faegri and Iversen (1989). (b) NPP preparation method as outlined by University of Amsterdam (van Geel, 2001).
4.1. History of NPP sample preparation
The morphology of a fungal spore will determine its likelihood of being preserved in the fossil record and surviving sample preparation (van Asperen et al., 2016). Spores with thicker walls will be more resistant to deterioration and larger spores are more likely to be caught in sieves (Perrotti and van Asperen, 2019). While, SCF which are round will perform similarly to pollen grains (van Asperen et al., 2016). This is a potential bias when using Faegri and Iversen (1989) as the purpose of the early sieving technique is meant for discarding clay but will inevitably also remove some smaller spores. Many Basidiomycetes spores are smaller than 10-6µm and are therefore unlikely to be retained (Perrotti and van Asperen, 2019). This causes an unbalanced source of information because Basidiomycetes fruit later in the coprophilous fungal succession. Similarly, fungal types such as Cheilymenia are abundant in cattle dung but deleterious in preparation techniques (van Asperen et al., 2016).
The UvA method requires sieving at the beginning of preparation with the intention to remove unnecessary larger vegetation remains. The recommended width of the mesh ensures that all, even very small, NPPs and pollen grains will be preserved. Another consideration is fruit bodies which are likely to break up during the preparation process (Perrotti and van Asperen , 2019) and release the previously withheld ascospores, affecting the overall spore count. However, fruit bodies are usually only preserved in macrofossil samples.
In an attempt to understand the differential effects of the many preparation techniques, van Asperen et al. (2016) tested four of them. These methods acted as a follow on from Clarke’s (1994) work and were the following:
(1) ‘Standard’ pollen preparation (Faegri and Iversen, 1989). (2) Same as Method 1 but without acetolysis.
(3) Same as Method 2 but with swirling to create density separation. (4) Only sieved.
Results from this study showed that when spores are boiled with potassium- or sodium- hydroxide (KOH, NaOH) or treated with acetolysis, their morphology will change i.e. swell or shrink. This could lead to misidentification of spores and so should be avoided in laboratory preparation techniques. Interestingly the study found Method 4 to produce the best fungal spore diversity and abundance. Yet, this is not a realistic procedure due to other proxies such as pollen requiring chemical treatment.
4.2. History of SCF counting and quantification
Unfortunately, there is yet to be an established standard practice when it comes to measuring SCF concentration in cores (van Asperen et al., 2020.a). Generally, exotic
Lycopodium spores are added to samples of a measured volume of sediment (Étienne and
Jouffroy-Bapicot, 2014) before being mounted to microscope slides. Common practice is for SCF to be identified and counted under the microscope until a predetermined number of
Lycopodium spores are counted (Stockmarr, 1971). However, this will vary depending on the
volume of the sample and number of Lycopodium tablets added (van Asperen et al.,
2020.a). Consequently, it is not possible to create a ‘one number fits all’ for SCF studies. This reduces the ease of repeatability for dung fungal spore studies, therefore not adhering to open science principles (Wilkinson et al., 2016).
Table 4.2: Quantification method, abbreviations used, units and references for each quantification method currently used in SCF studies.
Quantification Abbreviations Units References Percentage of the sum of
total pollen and total NPP % TLP + TNPP % (van Geel, 2006; Cugny et al., 2010; Wood and Wilmshurst, 2013) Percentage of total pollen
assemblage %TPA or %TP % (Perrotti and van Asperen, 2019; van Asperen et al., 2020.a) Total spore concentration Spores/cm3 (Stockmarr, 1971;
Eitenne et al., 2013; van Asperen et al., 2020.a) Pollen influx or spore
accumulation rate PI Spores/cm
2/year (Hicks and Hyvärinen, 1999; Baker et al., 2016; van Asperen et al., 2020.a)
The range of methods used to quantify SCF in samples is shown in Table 4.2. Each method has its limitations (Eitenne et al., 2013; van Asperen et al., 2020.a). For example, whilst %TPA is popular as it allows the counting of spores and pollen simultaneously. It can lead to under or overrepresentation of spores in samples as changes in pollen automatically cause changes to the spore count (Wood and Wilmhurst, 2013; van Asperen et al., 2020.a). The distinction between %TPA and %TLP+TNPP is that %TPA represents spores as a percentage within the total pollen assemblage, while %TLP+TNPP adds the percentage of spores onto the total pollen percentage. Therefore, many studies opt for %TLP+TNPP as this method counts SCF separately.
Alternatively, total pollen and spore concentration is sensitive to changes in sediment accumulation (Baker et al., 2013; van Asperen et al., 2020.a). Therefore, there is a high degree of variability across studies. Lastly, pollen influx is generally accepted as the most accurate method of quantification (Wood and Wilmshurst, 2013). Yet, a comprehensive 14C calibration and age-depth model is required, which is only sometimes available (van
Asperen et al., 2020.b). Such a model will, however, provide information on changes in sediment accumulation (Perrotti and van Asperen, 2019).
4.2.1. Identifying genera: Sporormiella-type
The Sporormiella-type has dominated palaeoecological studies after first being introduced by Davis et al. (1975) (Davis and Shafer, 2006). The spores of this genus became a popular tool due to Sporormiella species being regularly abundant in fossil samples (van Asperen et al., 2016) and displaying reliable sensitivity as an indicator of herbivore presence (Raczka et al., 2016). In many studies Sporormiella was the only SCF identified, counted and used to indicate past herbivore presence (Davis and Shafer, 2006; Raczka et al., 2016; Perrotti and van Asperen, 2019). This continual use has allowed the establishment of the practice with Étienne and Jouffroy-Bapicot (2014) stating that the “genus Sporormiella … [has]
demonstrated to be the most valuable proxy for the presence of wild and domestic herbivores”.
The best method for the Sporormiella-type is counting individual spore cells, like those in Fig. 4.2.1(a) and not complete spores (that are of rare occurrence), see Fig. 4.2.1(b) (Parker and Williams, 2012). This method is commonly applied in studies and is attributed to the fragility of the connections between spore cells (van Geel, 2006).
Figure 4.2.1: Microscope images of (a) Sporormiella separate cell of ascospore (b) Sporormiella ascospore (c) Sporormiella fruit body holding ascospore cells (provided by van Geel).
Despite successful studies using this method, it is recommended that researchers identify and count multiple SCF taxa (Perrotti and van Asperen, 2019; van Asperen et al., 2020.a), in order to reduce biases. However, further exploration into the use of other SCF is required.
4.2.2. Identifying genera: other coprophilous fungal spores
Studies which only count Sporormiella are vulnerable to inaccurate results especially when no Sporormiella spores are found, reflecting an absence of herbivores (van Asperen, 2017; Perrotti and van Asperen, 2019). Furthermore, some Sporormiella spores have a similar morphology to the non-coprophilous genus Preussia (von Arx and van der Aa, 1987; Perrotti and van Asperen, 2019; van Asperen et al., 2020.b). This could cause inconsistencies if not identified correctly. SedaDNA could be especially beneficial here, this is something detailed in section 6.
It is important for palaeoecological studies to conduct analysis of all fungal taxa found in samples (Baker et al., 2016; Perrotti and van Asperen, 2019; van Asperen et al., 2020.b) not just Sporormiella. Some SCF which have proven to be just as successful as Sporormiella have been depicted in Fig. 4.2.2. Generally, the best SCF indictors are: Cercophora-type (van Geel, 1978; van Geel et al., 2003; van Geel, 2006; Goethals and Verschuren, 2020), Podospora-type (van Geel et al., 2003; Etienne et al., 2013; Ghosh et al., 2017; van Asperen et al., 2020.b) and Sordaria-type (van Geel, 2006; van Geel et al., 2007; Ghosh et al., 2017; van Asperen et al., 2020.b). This is attributed to their ability to withstand the ‘standard’ pollen preparation procedure.
Figures 4.2.2: Timeline of when SCF were discovered and who identified them. From oldest to youngest: Sporormiella-type ascospore (provided by B. van Geel). Sordaria-type ascospore (Feeser and O’Connell, 2010). Gelasinospore-type ascospore (van Geel et al., 2011). Chaetomium-type ascospore (van Geel et al., 2003). Anthostomella fuegiana-type (van Geel, 1978). Microthyrium-type (van Geel, 1978). Coniochaeta xylariispora-type (van Geel, 1978). Pleospora-type (van Geel and Aptroot,
2006). Podospora-type ascospore (provided by B. van Geel). Cercophora-type ascospore (provided by B. van Geel). Apiosordaria-type (Gelorini et al., 2011). Coniochaeta-type (Cugny et al., 2010). Rhytidospora cf. tetraspora-type (van Geel and Aptroot). Coniochaeta ligniaria-type (van Geel and Aptroot, 2006). Arnium-type (van Geel et al., 2003). Bombardioidea-type (Bos et al., 2005). Pteridiosperma sp.-Bombardioidea-type (van Geel and Aptroot, 2006). Schizothecium conicum-Bombardioidea-type (Feeser and
5. External influences on coprophilous fungi abundance
Megaherbivore density is not the only influential factor contributing to SCF abundance in the sedimentary record (Parker and Williams, 2012). Climate, environment and herbivore type also have differential effects (Davis, 1987; Wicklow and Angel, 1980; Davis and Shafer, 2006; Parker and Williams, 2012; van Asperen, 2017; van Asperen et al., 2020.a) and must be considered when estimating herbivore biomass. Despite the knowledge we already have, our understanding of these biotic and abiotic effects on preserved SCF is still limited,
especially from a palaeoecological point of view. However, such information is crucial for improving palaeo-reconstructions and providing insight regarding the management of modern-day ecosystems (van Asperen et al., 2020.b).
Figure 5: Schematic diagram of possible external influences on spores at time of dispersal and deposition.
5.1. Climatic Influences
Climatic influences, notably temperature and moisture affect fungal growth on dung which is reflected in the amount of spores preserved. This is mainly attributed to differences in fungal species adaptability and structure (Richardson, 2001). In temperate zones more species will be found in samples from winter or spring than those from summer or autumn (Richardson, 2001; van Asperen, 2017). This is due to increased water availability which improves fruiting periods in these seasons (van Asperen et al., 2020.a), while in summer, dung is prone to drying out at a faster rate (A. J. Kuthubutheen and Webster, 1986). Alternatively, when temperatures are too low, they produce less fruit bodies (van Asperen et al., 2020.a). In general, the main climatic influence on SCF is humidity (van Asperen et al., 2020.b). Cool but moist climates are favourable for fungi community diversity (Parker and Williams, 2012; Wicklow and Angel, 1980). Furthermore, salt concentrations can hinder or aid sporulation (Perrotti and van Asperen, 2019). The initial water content of the dung
substrate will be determined by the climate and weather at the time of consumption, while salt content varies depending on deposition of animal urine (van Asperen et al., 2020.a). When temperature is too high, and moisture is low the dung will experience desiccation. This drying out of the substrate means only a few species are able to grow. However,
Sporormiella has adapted to somewhat dried out substrates (Richardson, 2001), partly, but
not fully, demonstrating reason for the popularity of the Sporormiella-type in
palaeoecological records. Larger dung is subject to less desiccation than the faeces of smaller herbivores (Richardson, 2001; Perrotti and van Asperen, 2019) giving reason to a majority of studies being related to megaherbivores. In addition, the pH of the substrate affects fungal growth. Most coprophilous species prefer a neutral pH (Krug et al., 2004). Our understanding of how preserved SCF are affected by climatic variations is incomplete and global changes in climates create more uncertainty (Baker et al., 2016). To date only one study, by van Asperen et al. (2020.b), addresses this from a palaeoecological point of view. However, as previously demonstrated, extreme wet conditions suppress fungal growth (van Asperen et al., 2020.a). Furthermore, high rainfall events could disrupt spore dispersal or facilitate transportation away from the site through runoff (van Asperen et al., 2020.b). Therefore, caution must be taken when there is an absence of SCF as this does not necessarily reflect a lack of herbivores at that time (van Asperen et al., 2020.b). Increased research into this is advisable and discussed further in section 5.3.2.
5.2. Livestock influences
Coprophilous fungi are able to grow on a wide range of faeces, however some fungal types are more likely to grow on the dung of specific herbivore species (Richardson, 2001; van Asperen, 2017; Halbwachs and Bässler, 2020). Consequently, certain herbivores will facilitate the growth of coprophilous fungi more than others, which could cause an inaccurate reconstruction of the amount of livestock present at that time (van Asperen et al., 2020.b). For example, a small collection of deer could contribute more to the fungal record despite there being a larger collection of cattle at a later time, strictly due to deer dung being more favourable for fungal growth (van Asperen et al., 2017; van Asperen et al., 2020.b). Reasons for this variation in growth has been neglected in research, however we know that diet has little influence on dung fungi community density (van Asperen, 2017). The Coprinus-type belongs to the class Basidiomycetes and does not favour a specific dung type (Richardson, 2001). Therefore, it has potential as an unbiased indicator of historic livestock abundance. However, it must be noted that Basidiomycetes are only sometimes preserved in the sedimentary record (Parker and Williams, 2012; van Asperen, 2017). Overall, our understanding of the relationship between herbivores, their surrounding landscape and how this is reflected in the fossil fungal record needs work (Baker et al., 2016).
5.3. Environmental influences
Due to the endocoprophilous nature of SCF, it is inevitable that the environment will influence the spectrum of preserved spores. Herbage consumed by herbivores is determined by surrounding ecology and biosphere, and varies in different bioclimatic regions (Fig. 5). SCF are consequently very valuable in understanding the effects of overgrazing or in some cases, human colonization (Gauthier et al., 2010). Goethals and Verschuren (2020) found that the farming style (e.g. mixed subsistence) to which livestock
are subject has an effect on the fungal spore record. Furthermore, the timing of sporulation may also be dependent on local environments and geographical evolution (Oneto et al., 2020). Therefore, it is important to compare pollen and macrofossil records when
reconstructing past megaherbivore abundance as they may provide information when SCF are lacking (van Asperen et al., 2020.a). For example, when Asteroideae, Cichorioideae,
Cirsium-type, Galium-type, Ranunculaceae, Stellaria-type and Potentilla-type pollen grains
are found together, this suggests local grazing (Mazier et al., 2006).
5.3.1. Lake sediments
SCF tend to be well preserved in cores from lake sediments (Etienne et al., 2013) and sometimes even undamaged Sporormiella asci with multiple spores are found (Parker and Williams, 2012). The excellent preservation of pollen and spores make lakes attractive study sites. However, there are biases which must be considered when reconstructing
environmental conditions based on lake deposits. Fortunately, lake area, depth and type of basin do not significantly affect SCF abundance (Parker and Williams, 2012). However, lacustrine dynamics and climatic events can vary results.
Although lake deposits can show a clearer change in SCF over time, the potential effects of the catchment area must be considered. The catchment area will be much larger for lake sediments than for peatbogs, as waterholes attract non-local herbivores (Fig. 5). Once there, visiting herbivores will eat herbage and eject dung with spores that will germinate and form fruit bodies, thus creating a condensed and potentially inaccurate record of the local
livestock (Perrotti and van Asperen, 2019; van Asperen et al., 2020.b). Furthermore, the position in a lake from which cores are taken can influence the SCF record (Etienne et al., 2013). It is for these reasons that lake sediments are more likely to produce a regional or landscape scale reconstruction rather than local conditions (Hicks and Hyvärinen, 1999; Orbay-Cerrato et al., 2017; Perrotti and van Asperen, 2019). Parker and Williams (2012) tested the variation by taking cores from the centre and margin of 24 lakes in South Dakota, Minnesota and Wisconsin. Depending on the mechanism of spore transportation some lakes have an accumulation of SCF in the centre rather than the margin, or vice versa (Etienne et al., 2013), illustrating the necessity for many more than just one core from a lake basin (Hicks and Hyvärinen, 1999).
A study by Etienne et al. (2013) assessed the relationship between SCF accumulation or concentration rate, herbivore grazing pressure and sediment accumulation. Multiple cores were taken from two lakes in the French Alps. This area was perfect for such a study as grazing pressure had been historically catalogued for the last 200 years and lake deposits contain spores which are especially influenced by transportation, livestock type and
environment. Through exploration of the lake deposits the authors could recognise the high amount of soil erosion, indicating over grazing. Furthermore, evidence of thick layers from flooding events were not analysed for coprophilous fungi as these likely contained high number of spores transported from a non-local range.
Comparison of historic data with coprophilous spore influx showed a simultaneous
reduction in sheep population between ca. 1894-1895. Overall, the pattern of coprophilous spore influx over time correlated well with historic data, thus, validating the use of SCF for reconstructing fluctuations of the population density of sheep.
5.3.2. Transportation of spores
Although the majority of fungal spores will be deposited and germinate in the local surroundings of their source (Stamets, 2005; van Geel, 2006; van Geel and Aproot, 2006; van Asperen et al., 2020.a), some may travel further (Perrotti and van Asperen, 2018; van Asperen et al., 2020.b). Spores can be transported through three means: the air,
hydrological mechanisms or animal migration (Calhim et al., 2018; van Asperen et al., 2020.a) (Fig. 5). If SCF identified in samples have been transported, it will likely have been through rivers and surface runoff (Baker et al., 2016; van Asperen et al., 2020.a) as
Basidiomycete fungi, which mainly travel through air (Krug et al., 2004), are often not preserved in core samples. Although means of transportation are usually dictated by the species morphology (Calhim et al., 2018) again, round and oval spores will act similarly to pollen (van Asperen et al., 2016).
The energy of hydrological transportation mechanisms tends to control where spores will accumulate in a lake. High energy situations such as transportation through rivers, streams or extreme surface runoff cause a concentration and settling of spores in the centre of the lake (Etienne et al., 2013). This is referred to as “degree of storminess” (Perrotti and van Asperen, 2019). However, this is also dependant on the size of the lake (Hicks and Hyvärinen, 1999). Therefore, it is important that studies take multiple cores from lake sediments, like the French Alps study by Etienne et al., (2013). Methodology such as this provides an indication of whether data is more local or regional (Orbay-Cerrato et al., 2017; Perrotti and van Asperen, 2018).
On occasions, high amounts of rainfall may inhibit SCF transportation by restricting airborne spores (Parker and Williams, 2012). A recent study found that the time of day in which sporulation takes place can have a large effect on the distance spores will travel (Oneto et al., 2020). If spores are released during the day, they could be airborne for multiple days but if sporulation takes place at night, then spores will likely only travel for a few hours (Oneto et al., 2020). This is down to differences in turbulence through the day as shown by Oneto et al. (2020) in North America. Similar research in other continents could provide
information on patterns of spore movement and additional investigations based on fossil material is necessary.
6. New developments in research
The research field of palynology is developing at a rapid pace (Perrotti and van Asperen, 2019). Due to its interdisciplinary nature, there are always new and exciting research prospects which can advance our understanding. One example is DNA research which has taken place since the 1980s but has only recently gained momentum as a palaeoecological tool (Epp et al., 2019; Kistler, 2018). Another research gap which is actively being addressed, is palaeoecological reconstructions in countries outside of Europe and North America, in an attempt to make the tools more globally applicable. In addition, periods other than the Pleistocene and Holocene should be further investigated.
6.1. Environmental DNA and SCF
SedaDNA stands for sedimentary ancient DNA (Capo et al., 2021) and involves analysing genetic information of archaeological tissues (Kistler, 2018). There are two approaches (i) metabarcoding (ii) shotgun sequencing (Edwards, 2020; Epp et al., 2019). Metabarcoding is the process of identifying single organisms through the isolation of DNA (Thomas et al., 2018). Alternatively, shotgun sequencing looks at the taxonomic community as a whole and provides DNA diversity data (Pedersen et al., 2016).
When combining DNA studies with paleoethnobotany it is possible to gain insight into past ecologies in such detail that pollen, macrofossil proxies or NPPs are unable to achieve (Ficetola et al., 2018). SedaDNA is best used alongside other proxies to produce a reconstruction of local changes in biodiversity (Capo et al., 2021). For example, a recent study used coprophilous fungi and SedaDNA to understand the evolutionary impact of an invasive rabbit species on a sub-Antarctic island (Ficetola et al., 2018). Using SedaDNA and fungal spores resulted in an in-depth reconstruction of the landscapes dynamics (Ficetola et al., 2018). In another study, ancient DNA has also been used in Scotland to accurately date exotic conifer planting (Edwards, 2020).
Another use of the DNA analysis is for taxonomic benefit. Through metabarcoding paleo environmental DNA (PalEnDNA) (Thomas et al., 2018) and phylogenetic markers it is possible to identify species assemblages (Epp et al., 2019; Stavrou et al., 2018). This allows the systematic advancement of molecular taxonomic units (MOTUs) (Edwards, 2020) and archives, like GenBank. If this were to become common practice then we can guarantee a reduction in the possibility of misidentification, an issue which is becoming increasingly evident (Stavrou et al., 2018). However, development in taxonomic resolution is required (Edwards, 2020).
Of course, this tool, like any other, has its limitations. The largest being preservation of tissue in samples. Leaching damages DNA over time and chemical build up can skew results (Kistler, 2018; Edwards, 2020). Samples which come from arid and cool environments tend to have the best preserved DNA tissue (Kistler, 2018; Edwards, 2020). However, studies from waterlogged environments have proven satisfactory too (Kistler, 2018). Epp et al. (2019) have developed a suitable standard protocol for SedaDNA sampling. Yet, our
understanding of why DNA preservation varies between samples is still under investigation (Edwards, 2020).
7. Outlook
The main purpose of this review is to synthesise current literature in order to analyse the accuracy of using SCF to indicate past herbivore presence and reconstructing herbivore biomass. In the following section I present claims regarding how future studies can develop the subdiscipline of SCF herbivore studies and substantiate these with evidence from literature previously mentioned.
7.1. Further inclusion of mycology
Current information on the succession of coprophilous fungi is something which so far has been neglected in studies (Miranda et al., 2020). SCF as indicators of herbivore populations are increasingly popular but the knowledge of many palynologists about the full fungal life cycle is insufficient. Increased consideration of this will improve our understanding of the SCF-herbivore relationship and indicate adaptations to environment and climate,
fundamentally, contributing to improving estimations of herbivore biomass.
A review by Perrotti and van Asperen (2019) aimed to analyse mycological literature and how this affects spore production yet failed to describe their life cycles. Alternatively, Richardson (2001) explored the Ascomycetes fungal communities found on specific dung types (identified on an animal species level). A similar study for Basidiomycetes fungal communities is non-existent (Halbwachs and Bässler, 2020) and would be beneficial. By including the biology of spores into palaeoecological studies we can grasp a further
understanding on why certain communities are found in specific dung types and how spores arrived in samples. Not only will this reduce irregularities in results but will also provide insight into herbivore biomass estimations.
Future studies should focus on detailing the succession of coprophilous fungi and its temporal evolution (Taylor et al., 2009) where SedaDNA could be utilised for detecting morphological changes (Kistler, 2018). Additionally, research of samples older than Quaternary would improve our understanding of the origination and development of the coprophilous fungi and herbivore relationship.
7.2. Standardised methodology and cross site comparisons
Despite large outputs of data, the progress of SCF studies has been hampered because it is often difficult to compare results, especially over different research organisations. This is due to the variations in research methodologies (van Asperen et al., 2020.a).
Firstly, there has yet to be an exhaustive study on how spore representation is affected by sample preparation (van Asperen et al., 2016). This is required before we can advance palaeoecological herbivore reconstructions as a whole. van Asperen et al. (2020.a) recommend using little or no preparation techniques whenever possible, in order to minimise damage of spores. However, this still engenders inconsistencies across the subdiscipline, consequently, cross site comparisons cannot be made. There is a need for research into a preparation method which could be applied no matter the type of
surrounding material. However, it is clear that KOH, NaOH and acetolysis should be avoided due to their influences on spore morphology (van Asperen et al., 2016).
Secondly, pollen and SCF should be counted separately when researchers are interested in the coprophilous fungi separate to vegetation. This was similarly suggested by van Asperen et al. (2020.a). Although no minimum spore count can be created, when possible
researchers should try and reach a relatively high Lycopodium spore count as this should maximise validation when data reveals an absence of herbivores (Walanus and Nalepka, 2013; van Asperen et al., 2020.a).
Finally, in future studies each SCF-type should be counted and presented separately, along with a total sum of all the SCF-types (van Asperen et al., 2020.a). When quantifying results, more than one method should be used for validation (Perrotti and van Asperen, 2019). Based on the literature reviewed, I recommend the use of pollen and SCF concentration and influx. Not only are these two methods more reliable than the percentage-method, but the age-depth model required for pollen influx provides information on sediment accumulation which can influence the total spore concentration (Perrotti and van Asperen, 2019; van Asperen et al., 2020.a). Therefore, these methods are compatible.
7.3. Sporormiella and other SCF
There are currently only four established relatively common SCF for indicating herbivore presence; Sporormiella, Podospora, Cercophora and Sordaria. Identifying and describing more fungal remains will expand this list of established SCF, fundamentally improving this palaeoecological tool (van Geel and Aptroot, 2006). The development of SedaDNA should speed up this process. Johnson et al. (2015) found by including all SCF taxa, no matter their interpretation reputation, uncertainty within their reconstructions was reduced.
A recent study in the Qinghai-Tibetan Plateau used topsoil and dung samples to assess modern fungi spore assemblages and communities (Wei et al., 2021). Results found that the
Urocytis-type correlated highly with herbivore abundance although until this study was
carried out, Urocytis was only known as a parasitic fungus (Wei et al., 2021). This not only highlights why we should be counting SCF other than Sporormiella but also expresses the need for studies to take place in more irregular environments. Palaeoecology needs to become more globally applicable as studies which take place outside of North America and Europe are at a disadvantage due to the lack of indicator species identified, as also
suggested by Montoya et al. (2010).
7.4. Herbivore biomass estimations
Currently, little is known about dung composition (Richardson, 2001) yet further research could provide insight into the relationship between SCF and the diets of herbivore species. To do this we must expand our knowledge on domesticated and wild herbivore grazing in the past (Carpenter et al., 2009; Baker et al., 2016). A study by van Asperen (2017) was aimed at comparing the fungal community found on the dung of exotic and (semi-)native herbivores. The results confirmed the validity of using megaherbivores to signal extinct herbivore presence, however this is less so when comparing with wild herbivores (van Asperen, 2017). Furthermore, Richardson (2001) recommends researchers consider the animal type studied especially when comparing dung or SCF in different geographic regions. Therefore, I believe that influences of livestock versus wild herbivores needs to be explicitly considered in future SCF and herbivore palaeoecological studies, especially if we wish to develop biomass estimations. Such field studies are highly anticipated (Johnson et al., 2015).
Lake studies benefit from multiple cores as this provides information on the method of transportation spores took but also decreases uncertainty in biomass estimations (Johnson et al., 2015). Studies similar to Etiennne et al., 2013 would be beneficial. Therefore, I recommend taking multiple cores from the same basin as good practice in SCF-herbivore studies.
Overall, our understanding of how external factors may influence the SCF which are preserved in the sedimentary recording needs improvement. Focus should be on the mechanisms of spore transportation and how/when spores are dispersed (Etienne et al., 2013). In addition, controlled modern day experiments could provide insight into the factors which influence fungal spore counts in dung (e.g. desiccation or urine concentrations) when the herbivore species and biomass is kept the same. Quantifying the relationship between herbivore biomass and fungal spore count will be a great advancement in this subdiscipline, this is in concordance with Johnson et al. (2015).
7.5. Multi-proxy analysis
SedaDNA is increasingly becoming known for its beneficial palaeoecological uses. A
comprehensive diagnosis of SedaDNA literature should be conducted so we can accelerate its practical usage. Specifically, our understanding of how different environments affect the preservation of DNA needs to be improved (Kistler, 2018).
The practice of ancient DNA analysis is likely to increase in popularity, accelerated by its reduction in cost over the years (Kistler, 2018). Not only does this open many new development possibilities, but reduces time and need for technical replicates (Edwards, 2020; Capo et al., 2021). Moreover, the use of online archives adheres more to open science principles. Overall, it enhances our ability to understand the dynamics of past ecological changes (Ficetola et al., 2018).
Although primarily ancient DNA studies have concentrated on bacteria or plants (Capo et al., 2021), there is potential for its use in fossil fungi through the fusion of techniques. In fact, it is usable on all organisms (Edwards, 2020). This will ease the very difficult task of spore species identification and provide insight into spore evolution. Overall, SedaDNA is another tool which will enhance palaeoecological studies and should be used consistently along with coprophilous fungi.
Multiproxy studies are always more concrete than single proxy studies. When using SCF, data should always be related to pollen and macrofossil proxies to provide more evidence on the timing and size of changes in previous agricultural practices (Orbay-Cerrato et al., 2017). For example, if fruit bodies are found in macrofossil analysis then this provides a indication that SCF were not transported by air (van Geel and Aptroot, 2006) and therefore evidence for samples reflecting local information. Although SedaDNA can be used for
species identification, it will likely be used as an additional proxy. This would, similarly, allow validation of records and add further information to the reconstruction.
8. Conclusion
The palaeoecological tool of using SCF to indicate past herbivore presence is sensitive to spore dispersal and transportation, lack of standardised methods and influences from climate, environment and livestock. In light of the information presented in this literature review the following conclusion is made: Yes, spores of coprophilous fungi are accurate indicators for herbivore presence, but the tool is not yet developed enough to be used in herbivore biomass estimations. In order for SCF to present accurate biomass estimations, further research must be conducted into factors which influence spore abundance and how to isolate these. Future studies should consider the use of SedaDNA and spore biology as this will likely improve our understanding of fungal communities on different herbivore species dung. However, for this to be successful our knowledge on the use of SedaDNA must be improved.
An outlook into the future of the field is presented; one which is more considerate of biological interactions which influence fungal communities. Although this subdiscipline is interdisciplinary, it is clear that biological aspects require more attention. Such attentions in future SCF studies should allow expansion of capabilities in the palaeoecological tool.
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