What Defines the

“Kingdom” Fungi?
THOMAS A. RICHARDS,1,2 GUY LEONARD,1 and JEREMY G. WIDEMAN1
1
Biosciences, College of Life and Environmental Sciences, University of Exeter,
Exeter, United Kingdom; 2Integrated Microbial Biodiversity Program,
Canadian Institute for Advanced Research (CIFAR), Toronto, Canada

ABSTRACT The application of environmental DNA techniques and nonmonophyletic. These kingdoms were defined
and increased genome sequencing of microbial diversity, based on cellular organization (prokaryote or eukary-
combined with detailed study of cellular characters,
ote), multicellularity (protist or “higher” eukaryote),
has consistently led to the reexamination of our understanding
of the tree of life. This has challenged many of the definitions
and trophic level (producer [plants], consumer [ani-
of taxonomic groups, especially higher taxonomic ranks such mals], or decomposer [fungi]). Somewhat more phylo-
as eukaryotic kingdoms. The Fungi is an example of a kingdom genetically sound definitions (2) have replaced these
which, together with the features that define it and the taxa classifications, but the names, defining characters, and
that are grouped within it, has been in a continual state of flux. number of kingdoms are still not settled (e.g., 3, 4).
In this article we aim to summarize multiple lines of data When different authors classify higher units of bio-
pertinent to understanding the early evolution and definition diversity, there are few rules and many differing inter-
of the Fungi. These include ongoing cellular and genomic
pretations of what is important and what is not (see
comparisons that, we will argue, have generally undermined all
attempts to identify a synapomorphic trait that defines the Fungi. 1–3, 5–9); some authors have even abandoned the need
This article will also summarize ongoing work focusing on taxon for formal terms such as “kingdom” (10). Furthermore,
discovery, combined with phylogenomic analysis, which has mapping trait evolution onto the tree of life is difficult,
identified novel groups that lie proximate/adjacent to the fungal and even more so when these determinations are sub-
clade—wherever the boundary that defines the Fungi may be. ject to constant revision (i.e., characters mapped onto
Our hope is that, by summarizing these data in the form of a trees need to be repositioned because the tree is con-
discussion, we can illustrate the ongoing efforts to understand
stantly being redrawn and the characters are found
what drove the evolutionary diversification of fungi.
in additional taxa). The combination of these factors
means that classifications such as kingdoms are tempo-
rary, and “noisy,” especially with respect to placement
It is a certainty that the tree of life can be broken up into of taxa that branch close to the border of a kingdom
large units of biodiversity that span such great evolu-
tionary distances that one could call them kingdoms.
However, if these units are to have any meaning, they Received: 15 February 2017, Accepted: 25 February 2017,
Published: 23 June 2017
must represent radiations of diverse groups underpinned Editors: Joseph Heitman, Department of Molecular Genetics and
by unique shared derived adaptations that drove the Microbiology, Duke University Medical Center, Durham, NC 27710;
evolutionary success of each group. Without such evo- Timothy Y. James, Department of Ecology and Evolutionary Biology,
University of Michigan, Ann Arbor, MI 48109-1048
lutionary characters, these classifications are merely ab- Citation: Richards TA, Leonard G, Wideman JG. 2017. What defines
stract concepts or arbitrarily demarcated lineages. Even the “kingdom” fungi? Microbiol Spectrum 5(3):FUNK-0044-2017.
the five kingdoms (commonly called plants, animals, doi:10.1128/microbiolspec.FUNK-0044-2017.
Correspondence: Thomas A. Richards, [email protected]
fungi, protists, and prokaryotes) that gained popular
© 2017 American Society for Microbiology. All rights reserved.
acceptance from Whittaker (1) were abstractly defined

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Richards et al.

on a phylogenetic tree. Take, for example, the three thetic protist, was also reported (15, 16). Access to eu-
primary monophyletic eukaryotic kingdoms: plants karyotic and prokaryotic genomic data, and in some
(Archaeplastida [10]), animals (Metazoa), and Fungi. It cases physiological experiments, has demonstrated that
can be argued that, of the three, only the Archaeplastida the genes encoding this pathway are present through-
have a known defining evolutionary trait that under- out the eukaryotes (17, 18) and, indeed, also present in
pins their evolutionary success and diversification. This a number of prokaryotic genomes (e.g., 19). These re-
defining trait is the endosymbiotically acquired primary sults indicate that synthesis of the amino acid lysine via
plastid organelle derived from a cyanobacterium (e.g., the α-aminoadipate pathway is not a useful diagnostic
11). However, even this trait is not unique, because the character for the Fungi.
protist Paulinella (Rhizaria) also has a cyanobacterially
derived endosymbiont with some characteristics of an Ergosterol
organelle (12). For the Metazoa and Fungi, the distinc- One of the key genes for the α-aminoadipate lysine
tion is much less clear between members of these king- pathway encodes the enzyme α-aminoadipate reductase.
doms and their protist sisters, and historically, some This gene is involved in the synthesis of both lysine
authors did not separate fungi from protists (6). In the and ergosterol (18). The presence of ergosterol within
case of Metazoa, many of the cellular systems thought to the cell, and specifically the cell membrane, has also
define animal multicellularity have now been identified in been claimed to be diagnostic for fungi (e.g., 20). Some
their protist relatives (e.g., 13, 14), leading to a reeval- authors have used this biomarker for tracing fungi from
uation of the cellular systems that define membership in environmental samples (e.g., 21–23). Unfortunately, a
the animal kingdom. In the following section, we discuss large number of groups classified as Fungi, which are
this conundrum with regard to defining the boundaries placed in phylogenetic trees within the fungal radiation,
of the Fungi. We aregue that, irrespective of where tax- lack either the pathways to synthesize ergosterol or the
onomic boundaries are drawn or how we define the presence of ergosterol as a detectable sterol (24). Criti-
Fungi, understanding the changes that underpinned the cally, numerous protists that do not group on phyloge-
evolution and diversification of this group is the most netic trees with fungi also synthesize ergosterol (25).
important and interesting aspect of this research area. These include trypanosomatid flagellates and Euglena
(of the protist phylum Euglenozoa) (26–28), Chlorella
and Chlamydomonas (Archaeplastida) (29, 30), and
IN SEARCH OF A SYNAPOMORPHY Acanthamoeba (Amoebozoa) (31). The taxon distri-
FOR FUNGI bution (10) of this biochemical characteristic therefore
There have been numerous attempts to identify a ge- suggests that utilization of ergosterol is at least as old as
netic, molecular, or cellular character that defines the the major diversification of the eukaryotes, and conse-
kingdom Fungi, specifically a synapomorphy that sepa- quently ergosterol cannot be used as a synapomorphy for
rates them from other groups. To formally qualify as fungi. It is therefore important that we no longer con-
a diagnostic synapomorphy, such a character must sider ergosterol as a useful biomarker for tracing fungi in
clearly define the clade (i.e., be held by the majority of environmental samples because this is not a reliable in-
the taxa that branch within the clade), in this case fungi. dicator of fungal presence/abundance (24). Even in most
Secondary loss of a trait within the circumscribed clade of the situations in which this biomarker has been used to
is formally permitted, but patterns of loss complicate detect fungi, i.e., plant and soil environments, nonfungal
the use of the trait as a synapomorphy. Importantly, the eukaryotes that produce ergosterol are found. As such,
trait must be absent everywhere else in the tree of life. ergosterol detection should not be used as a biomarker
Historically, attempts to identify such characters have specifically to quantify fungi without further checks on
been met with limited success. Here, we summarize the community composition such as environmental DNA
several lines of evidence that have been put forward but diversity and abundance approaches, methods that es-
ultimately refuted as characters used to define the Fungi. sentially make the use of ergosterol detection as a proxy
for fungal presence/biomass redundant.
Amino Acid Biosynthesis
For over 30 years, the synthesis of the amino acid lysine The Fungal Cell Wall
via the α-aminoadipate pathway was discussed as a Another trait thought useful for diagnosing fungi was
putative diagnostic character for the Fungi, even though the carbohydrate chemical composition of the cell
the presence of the pathway in Euglena, a photosyn- wall. “Perhaps one of the most mutually satisfying by-

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 What Defines the “Kingdom” Fungi?

products of fungal cell wall research, to taxonomists such a scale and across so many major divisions of the
and biochemists alike, is the close correlation that can eukaryotes. As such, chitin as a metabolic trait and a
be established between chemical composition of the feature of cell wall composition must have predated the
cell wall and major taxonomic groupings elaborated on radiation of fungi, therefore invalidating chitin synthesis
morphological criteria,” wrote Bartnicki-Garcia in 1968 and/or the chitin cell wall as a useful character for Fungi
(32). This work showed that many major taxonomic or any groups within.
groups within the Fungi had different cell wall carbo- β-Glucans are also key components of some fun-
hydrate chemical compositions. This was cutting-edge gal cell walls (e.g., 32, 35, 36). However, a range of
research at the time and clearly demonstrated that the β-glucans are found associated with cell wall structures
cell wall diversified in composition across the major in a number of protist groups, for example, associated
fungal groups (see Table 1 in reference 32). As such, it with encystment of Acanthamoeba (Amoebozoa) (49,
was thought that these chemical characteristics were 50), the oocyst of Toxoplasma (50), and filamentous
therefore diagnostic for higher taxonomic groups within growth in the oomycetes (Stramenopiles) (39). Further-
the Fungi and not for the Fungi as a single group. Later more, the β-glucan paramylon can be produced at
work demonstrated that the chitin synthesis pathway high quantities by Euglena (Euglenozoa) (51). Taken
has been lost in several fungal groups, for example, in together, this demonstrates that β-glucan biosynthesis
Pneumocystis (33), an ascomycete fungus (34), therefore and utilization as a cell wall component predate the
showing a clear example of secondary loss of a chitin origin and diversification of fungi.
cell wall. Indeed, numerous fungal groups have different Both the β-glucan and chitin data demonstrate that
cell wall compositions, while some fungi lose their wall the term “fungal cell wall” is a misleading concept and
(e.g., many fungal zoospores) or modify its components that it is actually a group of highly heterogeneous and
dependent on the life cycle stage (see reference 35 and dynamic traits (32, 35). These traits are found in many
Fig. 3 in reference 36). taxonomic groups that do not branch with or within the
Further work has shown that several groups initially fungi; thus, it is important that we abandon this concept
classified as Fungi and thought not to have a chitinous as a diagnostic feature.
cell wall (i.e., oomycetes and hyphochytriomycetes), but
which were later shown to be members of the strameno-
piles (37), do produce chitin in their cell walls (38, 39). NO SYNAPOMORPHY FOR THE FUNGI
Indeed, the ability to lay down chitin on the cell surface The historical record of cellular and biochemical traits
is present across numerous eukaryotic groups. These used as fungal synapomorphies summarized above
include, for example, Entamoeba (of the protist phylum demonstrates that the search for a diagnostic unifying
Amoebozoa) (40, 41), Trichomonas (Excavata) (42, 43), character for the Fungi has failed. To paraphrase Tom
Diatom and Pseudofungi (stramenopiles) (38, 44–46), Bruns, there is no fungal synapomorphy; get used to it
Plasmodiophora (Rhizaria) (47), and Chlorella (Archae- (personal and public communication). And indeed, this
plastida) (48). We note that in the case of the green alga is a state of affairs eloquently outlined by others (52),
Chlorella it has been suggested that the presence of a who coined the phrase “Bruns’ law” to describe the
chitin synthesis pathway and a chitin cell wall is the situation of dissolving fungal synapomorphies. Yet the
result of horizontal gene transfer (48). Yet even with this taxa that are indisputably fungal (e.g., Dikarya) form a
caveat, the taxon distribution of the production of chitin coherent monophyletic group (e.g., 53–55) that un-
as a cell surface material suggests that this feature is doubtedly represents a highly successful group of orga-
older than the major radiations of the eukaryotes (47). nisms both in terms of evolutionary diversity (e.g. 56)
Considering the distribution of genes that encode and ecological biomass. A more important question
chitin cell wall synthesis enzymes, it is clear that many therefore is to ask what evolutionary transitions at or
of the features of chitin synthesis predate the radiation near the basal radiation of fungi tell us about the evo-
of fungi and are found across a wide diversity of eu- lution, diversification, and success of this group.
karyotes. These include both chitin synthase (divisions 1,
2, and 3), chitinase (I and II), and chitin deacetylase
enzymes (Fig. 1). These are collectively required to syn- EVOLUTION AT THE DAWN OF FUNGI AND
thesize and remodel chitin polysaccharide. It is possi- THE RISE OF FUNGAL PHENOTYPES
ble that horizontal gene transfer has led to this taxon Cavalier-Smith has set out a scenario for the origin of
distribution, but it is unlikely to have occurred on fungi from a protistan ancestor involving the loss of

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FIGURE 1 Diversity and distribution of gene families known to function in chitin cell wall
synthesis or remodeling. (A) The diversity of domain architectures (as identified using
PFAM [184]) for chitin cell wall synthesis or remodeling gene families. Note the gene fusion
between a myosin head domain motor protein and a chitin synthase which was previ-
ously suggested to be fungus-specific (92). (B) The taxonomic distribution of putative
homologues across a subset of eukaryotic taxa identified using a custom-built set of
domain-specific hidden Markov models kindly provided by Jason Stajich and Divya Sain
(185). The fungal component of the phylogenetic tree is based on Spatafora et al. (186).

phagotrophy and the coupling of absorptive trophic allowing fungal cells to develop rigid and struc-
function (also called osmotrophy) with cell wall devel- turally reinforced cellular surfaces
opment (often in the form of polarized hyphal growth) 2. Rigid reinforcement of the cell surface, which pro-
(57). Using phylogenetic reconstructions to infer char- tects the cell against high turgor pressures, allow-
acters deep within the fungal radiation, Cavalier-Smith ing cell function to flood the cytoplasm with high
also inferred that the ancestral fungus had an alter- concentrations of solutes, which (i) allows for a
nate nonflagellated trophic phase and a zoosporic phase very fast metabolic rate and (ii) allows a cell to
(spore with a flagellum) (57). Later work involving phy- assert high force to penetrate and ramify into ro-
logenetic reconstruction of the fungi with improved bust structures as part of their feeding process
taxon and gene sampling provided data consistent with 3. Evolution of rigid cells, allowing for polarized
this hypothesis (53). Therefore, loss of a flagellum repre- growth at the hyphal tip
sents one or more derived transitions within the fungal
4. A shift to external processing (digestion) of complex
radiation (53, 58). The loss of a flagellum is, for exam-
chemical compounds coupled to active transport
ple, linked to radiation of terrestrial fungi, including the
of processed metabolites into the cell (osmotrophy
diversification of the Dikarya, which encompasses the
or absorptive feeding [57])
majority of sampled fungal forms (53).
Interestingly, Cavalier-Smith’s hypothesis for the or-
Several emergent properties arise from the evolu-
igin of fungi involves changes in four linked biological/
tion of polarized, reinforced, osmotrophic functions.
cellular functions, suggesting that these characteris-
Fungi can essentially feed as they grow; i.e., they can
tic changes were synergistic. Specifically, these changes
couple the act of growth with nutrient sensing and me-
involved:
tabolite processing, allowing for improved economy.
1. Loss of phagotrophy, which removed the re- These characters have made fungi very effective feeders,
quirement for a flexible cell surface membrane, especially in environments composed of complex

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 What Defines the “Kingdom” Fungi?

biological structures where fixed carbon, phosphorus, lic goods functions drive the evolution of multicellu-
and nitrogen are packaged into recalcitrant materials larity in yeast to maximize utilization of hexose sugars
which need to be broken down for utilization. This released by the function of secreted enzymes (70). It
function is reflected by the observations that envi- has also been shown that hyphal networks allow fungi
ronments showing these characteristics, such as plant to explore wider environments, accessing additional
hosts, animal hosts, soil, sediments, and “litter,” are nutrient sources where such sources are patchy, i.e.,
often associated with high fungal biomass and diver- separated by zones where nutrients are absent. Fungal
sity (4, 53, 56, 59–62). Although this pattern of evolu- networks therefore demonstrate the properties of high
tion certainly occurred early in the radiation of the transport capacity. Importantly, these features are also
fungi, it is important to recognize that an evolutionary enhanced by larger networks, where the relative cost
transition that included these four synergistic cellular/ of such development is reduced by increase in the size of
metabolic changes occurred on multiple occasions the network formed (74). It remains to be tested if the
throughout the eukaryotic tree of life. For example, development of a hyphal network provides adaptive
similar independent transitions occurred in the lineages advantages over nonnetworked competitors in terms of
that gave rise to the Pseudofungi (i.e., oomycetes and maximizing specific osmotrophic function and public
hyphochytriomycetes) and Corallochytrium (37, 63). goods competitions (such as breakdown of recalcitrant
These features are therefore not unique to fungi, yet we material). However, as Bebber et al. (74) demonstrate,
believe that studying how these features evolved in fungi hyphal networks do maximize the economics of growth
and comparing them to other eukaryotes represents a in many environments in which fungi are both diverse
fruitful path for understanding how fungi came to be and abundant.
and why they became such a successful and highly di- Second, fungi have hugely diversified secondary me-
verse group. tabolism (e.g., 75). Many of the products of this meta-
bolic function act as antimicrobial toxins (76), and many
of these secondary metabolic properties are encoded by
OSMOTROPHY RESULTS IN PUBLIC clusters of genes (e.g., 77–81). Examples of these clus-
GOODS INTERACTIONS AND ters encode both metabolic pathway components and
SYMBIOTIC ASSOCIATIONS transporters (e.g., 82) to tightly couple expression (e.g.,
As a consequence of the fungal osmotrophic function, 83) and chromosomal linkage (and therefore coin-
nutrients are processed in the extracellular environ- heritance) of the genes encoding biosynthesis, export,
ment, a function that underpins how some fungi operate and detoxification. The role of these metabolic func-
both as parasitic and/or mutualistic symbionts of other tions is often unclear. Yet synthesis and secretion of
organisms. This function has been especially important many toxins is likely to function to exclude microbes
in how fungi form diverse and critical associations with that do not possess a cognate gene cluster encoding de-
land plants (64–68). As a result of the loss of phago- toxification phenotypes from an environment where a
trophy and diversification of osmotrophic functions, public good is present and the toxin is produced. Such
fungi must also engage in public goods interactions (e.g., traits, often described as “spiteful” in the evolutionary
64, 69–71). This is because metabolites processed by ecological literature, therefore act to police access to
secreted enzymes outside the cell are essentially ren- the public goods released by osmotrophic functions (72,
dered accessible to any other organism that occupies the 73).
same environment and can take up and make use of the Even though filamentous growth, construction of a
metabolites released (discussed in references 72, 73). As robust cell wall, loss of phagotrophy, and osmotrophic
such, communities where fungi function are also the sites feeding make up the fungal lifestyle, and underpinned
of public goods competitions. the success of fungi, it is important to note that none
Microbes evolved many strategies to maximize public of these features are diagnostic for the kingdom. This
goods competitions. In fungi, two common biological is because they are all found together elsewhere on the
features are predicted to function to maximize osmo- tree of life (e.g., 37, 63). Our current understanding of
trophic public goods functions. First, growth as multi- both fungal diversity and biomass in terrestrial envi-
cellular hyphal networks allows the colonization of ronments, however, tells us that fungi are particularly
three-dimensional space, which hypothetically acts to good at performing these functions. In the next section,
minimize loss of metabolites to competitors. Indeed, we seek to understand what underpinned the success of
comparative experiments have demonstrated that pub- this lifestyle.

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Richards et al.

WHY ARE FUNGI ECOLOGICALLY respectively (e.g., 87, 88). Other non-Dikarya fungi have
SUCCESSFUL? also been shown to have hyphal tip structures similar to
Older than Their Competitors the Spitzenkörper, specifically Allomyces, that branch
As argued by other authors (e.g., 57), the relative age of among the deepest phylogenetic branches within the
the fungi compared to other fungus-like groups, e.g., fungi (89). Yet these systems are absent in many fungi,
oomycetes (Pseudofungi), hyphochytriomycetes (Pseu- and where present, the Spitzenkörper is highly variable
dofungi), and Corallochytrium (Holozoa, Opisthokonts), in cellular structure (e.g., 87, 88). As such, it is hard to
may explain why they dominate many environments identify a cellular trait that specifically defines polarized
compared to other eukaryotic groups that grow and feed cell growth across the Fungi.
in a similar manner. Put simply, the fungi “got there first” Investigating how fungal and nonfungal microbes
and have successfully retained such niches by further couple rigid cell wall synthesis, polarized cell growth,
adaptation that underpins the competitive exclusion of and osmotrophic feeding, however, is likely to be im-
others. Molecular clock data suggest that the Dikarya, portant for understanding the evolution of characters
one of the major radiations of the terrestrial fungi, seem that underpinned the success of the Fungi. For example,
to date to ∼500 million years ago (84). In contrast, the a gene fusion between the actin cytoskeleton myosin
major radiation of the oomycetes, a group that also suc- motor head domain and a division 2 chitin synthase
cessfully couples a rigid cell wall, polarized cell growth, domain (e.g., 90, 91) was proposed to be a “unifying”
and osmotrophic feeding, seems to have occurred ∼250 character for the Fungi (92). This gene is localized to
million years ago. The largely plant-associated Perono- both vesicles and the hyphal tip and is involved in tip
sporales are estimated to date from ∼150 million years growth in Ustilago (93, 94). The myosin-chitin synthase
ago (85), some considerable time after fungal forms col- gene fusion seemed like a good candidate for a fungal
onized similar plant-associated habitats. Such hypotheses synapomorphy because the function of these protein
(57) are difficult to test, but it is likely that fungi have had domains involves cytoskeletal trafficking and cell wall
an extended time frame in which to evolve capabilities synthesis and thus is directly implicated in polarized
to maximize retention of terrestrial niches such as plant- cell growth and rigid cell wall development. However,
associated environments. it is important to note that this character is absent in
many established fungal groups (e.g., Saccharomyces
and Schizosaccharomyces [90]) (Fig. 1), though likely
Economic Coupling of Growth due to secondary loss. Furthermore, since gene fusions
and Feeding are often unstable characters undergoing both fission,
One other possibility is that fungi are just better at horizontal gene transfer, and domain rearrangement
coupling rigid cell wall synthesis, polarized cell growth, (95–97) (which has also been demonstrated in fungi
and osmotrophic feeding (i.e., feeding as they grow) than [98]), it is unclear if synapomorphies of these types
their nonfungal competitors. By “better,” we mean they can be trusted. Indeed, there is some evidence that the
can perform this function at a higher rate and/or with myosin-chitin synthase gene fusion may not be specific
greater metabolic efficiency, allowing them to outcom- to fungi and is present in other opisthokont lineages (63,
pete others. If this is the case, then it is likely due to 91) and therefore is invalid as a synapomorphy for the
adaptations in cytoskeletal functions that allow high- Fungi. However, even though it is not a unifying feature,
speed trafficking and efficient ordering of vesicles and the acquisition of the myosin-chitin synthase gene fusion
other cellular machinery which drive rapid/efficient hy- undoubtedly contributed to filamentous growth func-
phal tip growth and feeding (such machinery is discussed tions within the Fungi.
below). Indeed, some data suggest that filamentous as- Looking elsewhere in fungal growth and feeding may
comycetes can achieve very fast rates of vesicle trans- also prove fruitful for understanding what functions
portation to growing hyphal tips (86). Furthermore, underpinned the success of fungi. Cellular growth in
many fungi possess a range of unique endomembrane/ fungal filamentous structures occurs almost exclusively
cytoskeletal systems associated with the center of tip from the tip (99); this is part of the function that allows
growth. These include, for example, the cytological them to grow as they feed. This feeding process occurs
characteristic of a vesicle organizing center called the at a high rate in Pezizomycotina that specialize in fila-
Spitzenkörper and/or the apical vesical crescent, found mentous growth (86). However, these endomembrane
at the hyphal tip of many fungi, which identifies the main and cytoskeletal systems were co-opted to control bud
area of growth in filamentous Dikarya and zygomycetes, growth in Saccharomyces cerevisiae. Subsequently,

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 What Defines the “Kingdom” Fungi?

FIGURE 2 Diversity and distribution of gene families known to function in hyphal growth.
(A) A cartoon illustrating how proteins interact relating to subprocesses which govern
vesicle trafficking associated with hyphal growth. Functions are briefly discussed in the
main body of this manuscript and are marked i to viii. (B) The taxonomic distribution
of putative orthologues identified using reciprocal BLAST searches and phylogenetic
methods across a subset of eukaryotic taxa (data not shown). The fungal component of
the phylogenetic tree is based on Spatafora et al. (186).

S. cerevisiae has been developed as a model system for marker for the formation of the exocyst (Fig. 2A-vii),
studying protein function associated with polarized while Rho3 and Cdc42 mediate docking of the Golgi-
growth (99, 100) (Fig. 2). derived vesicle (Fig. 2A-viii). In filamentous fungi, ho-
Briefly, polarized growth occurs by delivery of secre- mologous sets of proteins function similarly; however,
tory vesicles from the Golgi (Fig. 2A, see step i) along an even more complex set of polarity factors are present;
cytoskeletal tracks to predetermined areas of the cell e.g., in addition to Cdc42, another RhoGTPase, Rac1,
membrane (in filamentous fungi this would be at the and an expanded set of effectors (guanine-nucleotide
hyphal tip). This process is initiated by Cdc42, which is exchange factors and GTPase-activating proteins) play
activated by its guanine-nucleotide exchange factor key roles in regulating these processes (109–111).
Cdc24 (Fig. 2A-ii), which leads to the development of a The polarisome and the exocyst, and proteins asso-
network of physically interacting proteins (99, 100) ciated with these complexes, are very important for
known as the polarisome (Fig. 2A-iii). The polarisome, establishing the temporal and spatial control of polar-
and specifically the formin Bni1, radiates actin cables ized cell growth. They also fit the criteria of key func-
(101) which play a key role in exocytosis (102). Msb3 tional adaptations that underpin the success of fungi
and Msb4 interact with Spa2, a scaffold protein of the as microbes because their function is linked to rigid
polarisome (Fig. 2A-iv), and are hypothesized to recruit cell wall development, polarized cell growth, and osmo-
Cdc42 from the cytosol at the site of tip growth (103). trophic feeding. As such, these are examples of the sys-
Post-Golgi secretory vesicles are transported along the tems we should investigate from the perspective of
actin cables using a type V myosin (Myo2) motor pro- fungal cellular function and GTPase-activating protein
tein (104, 105) (Fig. 2A-v) to dock with another mul- evolution.
tiprotein complex called the exocyst in a process that is Comparative genome analysis shows that the exo-
dependent on Sec4 and its guanine-nucleotide exchange cyst system and Sec4 orthologues are conserved across
factor Sec2 (106, 107) (Fig. 2A-vi). This process there- a diversity of eukaryotes (112, 113), suggesting a broad
fore enables the vesicle to be guided to its target site on function in directing the interaction between vesicle,
the plasma membrane (108). Cdc42, along with Rho1, is membrane, and cytoskeleton (Fig. 2B). Given current
required for localization of Sec3, which acts as a spatial genome sampling, polarisome components seem to be

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unique to fungi. However, preliminary genomic data mirroring that which occurred within or at the base of
suggest that these components could be present in un- the Fungi (63). Similarly, during host-associated stages,
published but publicly available opisthokont protists holozoan parasites Dermocystidium salmonis, Ichthy-
(data not shown), thus demonstrating further evidence ophonus hoferi, Psorospermium haeckii, and Rhino-
that many functions and gene networks associated with sporidium seeberi absorb nutrients through their cell
fungal growth are not specific to fungi. wall structures (for review, see reference 116). Members
of the Ichthyosporea (e.g., A. parasiticum and I. hoferi)
develop polar growth similar to that seen in fungal
INCREASED SAMPLING FURTHER hyphae (63, 117). Representatives of the Ichthyosporea
CHALLENGES OUR UNDERSTANDING also showed diversified sets of chitin synthase genes (63)
OF THE TREE OF LIFE presumably used as cell wall components. I. hoferi, for
So far, we have reviewed the data demonstrating that, to example, has chitin in its cell wall (118), and the Dermo-
date, no cellular or genomic characters have been iden- cystida R. seeberi appears to encode a class 2 chitin
tified that represent a synapomorphy for the Fungi. synthase (119). C. limacisporum putatively encodes a
As a consequence, it may be argued that the best defi- division II class IV/V/VII chitin synthase-myosin fusion
nition of fungi is a phylogenetic definition. Specifically, (63) previously thought to “unify” the Fungi (92). Fur-
our best approximation of defining the kingdom is de- thermore, Corallochytrium and members of the Ichthy-
claring, “Phylogenetic cluster X incorporating known osporea have a developmental cycle that involves a
taxa A, B, C, etc., represents the Fungi.” This is a chal- single-celled flagellated zoospore (e.g., Sphaerothecum
lenging state of affairs because where one draws the destructans) and a multinucleated (coenocyte) feeding
line on a phylogenetic tree to define the kingdom is structure, a developmental cycle that has similarities
also problematic. This is because the phylogenetic tree is to that of “chytrids” (i.e., fungi that form spores with
incomplete in terms of genetic data and the diversity a flagellum [zoospores] and which were at one stage
of groups sampled. Several discoveries over the past classified as Chytridiomycota but were later divided
20 years have repeatedly challenged how and where we into three phyla: Chytridiomycota, Blastocladiomycota,
mark the Fungi on a phylogenetic tree. In the next sec- and Neocallimastigomycota [63, 116, 120, 121]). Both
tion we will briefly outline these discoveries. Ichthyosporea and Corallochytrium branch on the
Holozoa side of the opisthokont tree, but current phy-
Protistan Relatives of Fungi logenetic analysis does not confirm with strong support
Multigene phylogenetic analyses have demonstrated that whether these groups are monophyletic (63), making it
the opisthokont radiation is composed of two major difficult to reconstruct the evolution of these “fungal-
evolutionary branches: (i) the Holozoa, which comprise like” characteristics in the Holozoa as either multiple
Metazoa, Choanoflagellata, Filasterea, Corallochytrium, cases of convergent evolution or retention of ancestral
and Ichthyosporea, and (ii) the Holomycota, which traits.
comprise fungi, Fonticula, and Nuclearia, with Fonticula Nuclearia are a group of pseudopodia/filipodia-
and Nuclearia forming a robustly supported clade sister forming “obligate” amoebae and were first identified as
to fungi (63, 114, 115). This branching relationship a separate protist lineage within the Opisthokonta by
suggests that Fonticula and Nuclearia should have char- small-subunit (SSU) rDNA sequencing and phylogeny
acteristics similar to fungi and to the exclusion of others (122). The related Fonticula alba is a cellular slime mold
including the Holozoa. In reality, however, what we and an amoeboid protist which can form multicellular
see is a diverse mosaic of characters, initially attributed fruiting bodies (115, 123). Both of these taxa are pha-
to fungi and now identified to be present across the gotrophic and lack a trophic phase typified by absorp-
opisthokonts, specifically in Holozoa protists. tive/osmotrophic feeding and a chitin cell wall (115); as
Holozoa comprise a wide diversity of biological forms, expected, they fail many definitions of fungi (e.g., 57).
which are not possible to discuss at length here. However, The phylogenetic placement of Fonticula/Nuclearia as a
for the purposes of this article, it is important to mention close relative of fungi (e.g., 63, 115) separates the Fungi
that representatives of the nonmetazoan Holozoa show from many holozoan protists which also have fungus-
many of the characteristics thought to typify fungi. These like characteristics (discussed above). Furthermore, this
include Corallochytrium limacisporum, a free-living pro- placement suggests that a close ancestor of fungi was a
tist which appears to have lost phagotrophy and feeds via pseudopodia/filipodia-forming phagotrophic cell with an
osmotrophic function, a pattern of convergent evolution alternative flagellated life cycle stage.

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 What Defines the “Kingdom” Fungi?

Interestingly, environmental sequencing has also sug- There are now examples of both types of these newly
gested a large diversity of phylogenetic branches asso- discovered and placed groups that collectively challenge
ciated with Fonticula in both freshwater and marine where we draw the line that defines fungi and conse-
environments. These results suggest a wealth of un- quently which characters are used for this definition.
explored biodiversity associated with this branch (124, In the following section we shall briefly introduce some
125), which could potentially further confuse evolu- of these groups and what data are available pertinent
tionary trait reconstruction at or near the origin of fungi. to our understanding of the origins and classification
of fungi. In many cases the phylogenetic placement of
New Branches Near the Origin of Fungi these groups relies on (partial) 18S SSU rDNA gene
The history of our understanding of fungal diversity has phylogenetic analysis and also includes rDNA sequences
been dominated by what can be cultured on selective that form long branches in phylogenetic trees. Phylo-
solid media (e.g., 126). Such methods have proved very genetic analysis of such datasets provides very incon-
effective at recovering some fungal groups, especially sistent and often misleading results (134–136), meaning
Dikarya, but are less effective at recovering other groups that the placement of these groups relative to each other
such as the chytrids and non-Mucorales zygomycetes. and the boundary that defines fungi is largely unresolved
Microbes that take part in complex symbiotic associa- and requires comprehensive multigene phylogenetic
tions with other organisms or are propagated in liquid analysis.
environments are often excluded by culture plate sam-
pling techniques, meaning that our understanding of Aquatic Environmental Sequencing and
biodiversity is largely incomplete. Culture plate methods the Expansion of the Chytrid Branches
also act to select for certain fungal groups that grow The use of environmental DNA methods (127) for
quickly on solid media with abundant nutrients. This sampling aquatic environments, including both fresh-
means that many fungal groups can readily be recovered water and marine environments, has revealed a diversity
from natural environments even though these microbes of novel SSU rDNA sequences that cluster among the
were not active or abundant in the initial environment deepest branches of the Fungi closest to known chytrid
sampled. Their rapid growth on culture plates means taxa (e.g., 137). The phylogenetic placement of these
they competitively exclude those microbes that were sequences often involves relatively long branches, and
actually active and dominant in the environment sam- the bootstrap support is often weak; for this reason, the
pled. Therefore, such sampling can be positively mis- phylogenetic relationships reported should be treated
leading regarding the true diversity of fungi, and indeed with caution. Nonetheless, these data suggest a diver-
other eukaryotic microbes, present in an environment. sity of undescribed and uncultured chytrid-like forms
As such, our understanding of the tree of life is partial that exist in aquatic environments (e.g., 137–141).
and biased (as discussed in references 127, 128). Strikingly, these sequences show a trend of low levels
Our understanding of how extant biodiversity maps of sequence similarity to taxonomically described fungi
onto the tree of life, specifically at the base of major compared to environmental sequences that cluster with
groups such as fungi, has been challenged by two lines the Dikarya (142), suggesting there is a wealth of un-
of research. First, environmental DNA analysis has re- explored phylogenetic diversity present in aquatic en-
vealed that uncultured groups, which are both diverse vironments and branching within or close to known
and abundant in natural environments, represent sub- chytrid groups. However, it is possible that this pattern
stantial additions to the tree of life in terms of both is exaggerated by a slower rate of rDNA variation in
branch placement and molecular diversity (e.g., 128– Dikarya as compared to the chytrids.
130). These include, for example, a group formally It has been hypothesized that this chytrid diversity is
known as soil clone group 1 (131), which has been de- an important component of the heterotrophic flagellate
tected in numerous soil environments (132) and which community in water column communities, playing a
was later classified as Archaeorhizomycetes within the key role in the microbial loop as either parasites and/
ascomycete subphylum Taphrinomycotina (133). Sec- or saprotrophs (143–145). However, the nature of the
ond, molecular sequencing combined with phylogenetic detection of these groups (environmental sequencing)
analysis of groups once thought to be distant relatives means that understanding their biology and ecology is
of fungi has shown that such groups branch within, or at difficult. However, clear examples of chytrid groups
the base of, the fungal radiation (e.g., microsporidia, (146, 147) and their affiliates (145) have been identified
discussed below). associated with diatoms, suggesting trophic interactions

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Richards et al.

with primary producers in both marine and freshwater used to argue that they were derived from an early—
environments. Their phylogenetic placement as allies to premitochondrial—phase of eukaryotic evolution (151).
the Chytridiomycota and other chytrids would, how- This hypothesis has been refuted with the demonstration
ever, suggest that they have a life cycle with alternating that microsporidia possess highly reduced mitochondria-
nonflagellated trophic and zoosporic phases and can like organelles (152) and that microsporidian nuclear-
form feeding structures reinforced with a chitin cell wall. encoded proteins are of mitochondrial ancestry (153,
However, work is needed to investigate the biology of 154). Additionally, phylogenetic analysis of nuclear-
these groups to confirm the presence of these life cycle encoded protein coding genes with appropriate correction
characteristics. for site rate variation has shown that the microsporidia
branch with fungi (e.g., 155, 156). Gene synteny analysis
Possible Fungal/Chytrid-Like Group of the sex-related locus demonstrated that fungi and
Known as Basal Clone Group 1 microsporidia share a unique syntenic pairing of the RPS9
Among the diversity of SSU rDNA sequences sampled and RPL21 encoding genes, supporting the close phylo-
from aquatic environments, one distinct group has con- genetic association of fungi and microsporidia (157). The
sistently been identified. This group of sequences was placement of microsporidia with fungi therefore demon-
initially detected in marine sediments (148) and deep-sea strates that the microsporidia are a striking example of
environments (139, 149, 150) using PCR targeting a genomic and cellular reduction when compared to their
wide diversity of eukaryotic SSU rDNA combined with fungal and protist relatives (158).
clone library selection and sequencing (see GenBank The microsporidia encompass over 1,500 described
sequence KD14-BASS [GenBank accession: EU154992] species, which are all intracellular parasites that infect
or DSGM-63 [AB275063] for exemplar representatives other eukaryotes. The defining feature of microsporidia
of this group). This clade has subsequently been detected is the development of long polar tubes coiled within
in RNA and DNA samples from a range of marine en- the parasite’s cell, which are used to infect the host cell
vironments (e.g., 141, 142), including both sediments (158) as part of their life cycle, although some micro-
and water-column samples, and is associated with both sporidia can invade host cells via host phagocyto-
large and small filtration fractions, suggesting that this sis (159). Microsporidia do not form a walled trophic
group has a complex life cycle in which it forms different form and they do not form filamentous structures simi-
cellular morphotypes or interacts with second-party cells lar to hyphal cells (57), which are characteristics argued
(141). Phylogenetic analysis of the SSU rDNA sequences to underpin the successful radiation of the fungi dis-
gives a mixed picture of the placement of this group, cussed above. The majority of microsporidia do form a
forming a relatively long and distinct branch that in chitin cell wall around their spore (57). Because micro-
some analyses is placed among the Fungi (e.g., 149), sporidia lack flagella, they do not form an alternate
while others show it as a branching sister to Ichthy- nonflagellated trophic phase and a zoosporic phase as
osporea (e.g., 139). Work is needed to assess the eco- part of their life cycle. Such characteristics are thought
logical roles and evolutionary position of this diverse to typify the ancestral fungus (discussed above) and are
and divergent group. Such analyses will likely have im- found in Rozella (discussed below). As such, indepen-
plications for understanding the evolutionary patterns dent of where the microsporidia are placed relative to
within the Opisthokonta independent of whether or not the origin of the fungi, they have secondarily lost these
this group branches within Fungi. characteristics.
The pattern of genomic and cellular evolutionary re-
Microsporidia duction that is evident in the microsporidian lineage
Microsporidia have historically been placed at numer- makes it difficult to identify characters that can be pu-
ous branching points in the tree of life, and their lack of tatively assigned as shared synapomorphies with estab-
many eukaryotic cellular structures (e.g., flagella, mito- lished fungal taxa, which in turn further complicates
chondria, and typical stacked Golgi) has made it diffi- how we define the kingdom. Specifically, it makes it very
cult to assign them to a higher taxonomic group based difficult to support either of these statements: “Micro-
on ultrastructural data (57). SSU rDNA phylogenetic sporidia belong in fungi,” or alternatively, “Microspo-
trees initially placed the microsporidia at the base of ridia belong as a sister group to fungi,” even though
the eukaryotic radiation. This result, along with the improved multigene phylogenies suggest that they branch
absence of these cellular structures and the characteris- with Rozella (55) and Rozella has historically been
tic of short prokaryotic-like SSU rDNA genes, had been classified as a fungus (e.g., 160).

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 What Defines the “Kingdom” Fungi?

Cryptomycota-Rozellomycota- from a subset of life cycle stages, although it should be

Rozellida-Rozellosporidia noted that such sampling is incomplete either for the life
A range of phylogenetic analyses suggest that Rozella cycle of an individual species or across the wider group
represents the first phylogenetic branch among fungi (165). Follow-up work has since demonstrated that
(53, 55). This conclusion is based on the premise that Rozella does indeed produce a chitin cell wall as a resting
Rozella has historically been classified as a fungus (e.g., spore as well as during the invasion process, where the
53, 121, 160), although the treatment of Rozella as a production of a cell wall is specifically linked to host
fungus is now debated (161, 162). As such, the biology penetration (55, 92), but it does not seem to use a cell
of Rozella and associated groups is very important for wall during trophic growth. This pattern of development
understanding the evolutionary transitions at, or near, has similarities with how microsporidia use their chitin
the base of fungi. Concatenated multigene phylogenetic cell wall. Consequently, Rozella, like some true fungal
analysis suggests that Rozella branches with the micro- groups (discussed above), fails the definition of fungi
sporidia, although alternative branch placement with that requires a developmental stage composed of a tro-
statistical comparisons could not reject microsporidia phic form with a chitin cell wall (57).
branching separately from Rozella as the next branch Recent exciting work has shown a number of estab-
below the fungi (55). Shared derived horizontal gene lished cultures of intracellular microbes branching
transfers (HGTs) were also identified that support the within the radiation of environmental sequences that
sisterhood of Rozella and the microsporidia (55, 163), branch with Rozella (168). This allows us to further
but again the taxon distribution of these characters investigate the phylogeny and cellular characteristics
could potentially be amended by additional genome present in this otherwise cryptic environmental group.
sequencing demonstrating a wider distribution of the One of the striking results of this work is the dem-
HGTs. onstration that subgroups within this radiation possess
Rozella has also been shown to be part of a large cellular structures similar to the microsporidia. Spe-
and diverse group of uncultured microbes, primarily cifically, Paramicrosporidium was shown to produce
identified through environmental DNA techniques (and unflagellated chitin and/or cellulose-walled spores (as
which also seem to form associations with diatoms indicated by calcofluor white staining) and grow within
[147, 164, 165]). This environmental group, along with the host as unwalled nonphagotrophic meronts (168)—
Rozella, has been given a range of different names, i.e., again like microsporidia. Nucleophaga amoebae, a
Cryptomycota, Rozellomycota, Rozellida, and Rozello- microbe initially described in 1895 (169; see 170) as
sporidia. Rozella (unlike microsporidia) has a life cycle a “chytrid” fungus, was also shown to branch within
composed of alternating nonflagellated trophic and zoo- the radiation of environmental sequences that branches
sporic phases (166). Fluorescent in situ environmental with Rozella and, also like Paramicrosporidium, pro-
DNA methods combined with antibody staining of duces unflagellated chitin and/or cellulose walled spores
microtubules demonstrated that some subgroups within (168, 170). Interestingly, Nucleophaga (see Fig. 1C
the environmental clade related to Rozella also develop and D in reference 170) and Paramicrosporidium (see
alternative nonflagellated and zoosporic phases (165). Fig. 1C–E from reference 168) also develop putative
These results are consistent with this biphasic life cycle anchoring disks and an atypical polar filament like
being present in the earliest diverging groups of the microsporidia.
Fungi. Haag et al. (171) identified a novel gut microbe of
Like microsporidia, Rozella and its uncultured rela- Daphnia named Mitosporidium daphnia. Genome se-
tives were thought to lack a cell wall during trophic quencing combined with phylogenetic analysis showed
growth within the host (160). Indeed, Rozella grows that this taxon branched at the root of the microsporidia
within its host as naked unicellular thalli (160). Fur- in a phylogeny of 53 gene families. However, rDNA
thermore, Rozella is thought to feed phagotrophically phylogenetic analysis encompassing much wider taxon
on host cellular structures because it was shown to form sampling shows that Mitosporidium branches within
pseudopod-like projections surrounding host cytoplasm the radiation of environmental sequences that branch
(167) during infection. Fluorescent in situ RNA labeling with Rozella (170). Cytological analysis shows that
combined with cell wall staining of cells captured using Mitosporidium possesses a microsporidia-like polar tube
environmental sampling allowed analysis of only frag- and lacks a flagellum for spore dispersal. Collectively,
ments of the life cycle of additional representatives of these results show, in comparison to what we know
this group but suggested that a chitin cell wall is absent about Rozella, that this group seems to be a mosaic

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Richards et al.

of fungal chytrid and microsporidian cellular features. PSEUDOPODIA FORMATION ACROSS

This mosaic pattern of features is further complicated THE DEEP BRANCHES OF THE FUNGI
by evidence that representatives of this group (e.g., FURTHER CONFUSES THE
Nucleophaga and Rozella) can form pseudopodia-like FUNGI-PROTIST DISTINCTION
cytoplasmic projections and possibly perform phago- One current suggestion is to go against the historical
trophy (167, 172), a feature that, under some defini- classification of Rozella and Nucleophaga as fungi and
tions, must be absent for a group to be classified as fungi exclude Rozella and its phylogenetic allies from the
(57). Fungi because they are thought to be phagotrophic
(161). Rozella and related groups have been shown to
Aphelids form pseudopodia-like projections (167, 170), cellular
Aphelids are amoeboflagellate parasites of microalgae features linked to phagotrophic function (172). Phago-
(173, 174) classified as protists. They form zoospores trophy is argued as necessarily absent in taxa within the
resembling those of chytrids, yet in contrast to known Fungi (161). However, it has been known for some time
chytrids, aphelids feed via phagotrophy when inside that chytrid zoospores from groups unambiguously
their host (e.g., 173). Amoebaphelidium protococcarum classified as fungi also form short pseudopodia and de-
(173), like other aphelids (175, 176), attaches to host velop amoeboid stages, e.g., Amoebochytrium, Caulo-
cells, produces a cyst cell wall, and penetrates the chytrium, Catenaria, and Paraphysoderma (see 172).
host via a germ tube, a process that is similar to how However, in these cases, pseudopodia appear to be used
Rozella and microsporidia enter the host. Once inside to crawl, and true phagocytosis, to our knowledge, has
the host, they form pseudopods and feed on host-derived never been reported.
cytoplasm via phagotrophy, unlike microsporidia but Pseudopodia (and the similar cellular structures of
possibly in a manner similar to the intracellular pseu- filopodia) are fine actin-based membrane-cytoplasmic
dopodia development observed for Rozella (167). Phy- cell surface projections used for both environmental
logenetic analysis based on three rDNA encoding genes sensing and cell motility and form structures necessary
and two protein encoding genes demonstrates strong for phagotrophy. This cellular characteristic is found
support for the monophyly of microsporidia, Rozella, throughout the eukaryotes and is thought to have
and the aphelids, which together are argued to form a evolved early in their evolution (177), long before the
sister clade to the fungi (173). These results were used, emergence of the fungal lineage. In recent work, phy-
along with similarities in germ/infection tube appa- logenomic comparisons of genes encoding actin-based
ratuses and a posterior vacuole, to suggest that these cellular protrusion proteins showed these gene families
groups form a distinct monophyletic group (named to be present in fungi (178). These include, for example,
aphelid-Rozella-microsporidia) (173) now formally clas- the Arp2/3 complex involved in actin-remodeling (179),
sified by some as Opisthosporidia (162). which in metazoans is regulated by WASP and cortactin
The data discussed in this section cover numerous function (180). The seven subunits of this complex are
groups newly and/or only partially sampled and dem- present in all major eukaryote groups and almost all
onstrate collectively that we are some distance from eukaryotes surveyed which make pseudopodia/filipodia,
achieving an accurate census of the diversity of microbial consistent with the idea that pseudopodia/filopodia
forms that branch within, or close to, the Fungi. Fur- development has a deep origin within the eukaryotes
thermore, these data also show that we still know rela- (178). In contrast, Ena/VASP, an actin-regulation pro-
tively little about the diversity of cellular and genomic tein which functions in pseudopodia/filopodia formation
features that are evident within groups that branch (181), is exclusive to Amorphea/Unikonta and appears
close to the Fungi. This state of affairs makes it very to have been secondarily lost in the Fungi (178), al-
difficult to understand the evolution of traits such as cell though genome sampling in this study only included
wall development, hyphal growth, flagellum formation, Dikarya and two chytrid genomes.
phagotrophy, and pseudopodia formation across the Recent and exciting work by Fritz-Laylin and col-
deep branches of the Fungi. However, further inves- leagues has investigated the evolution of the genes
tigation into these deep-diverging lineages of fungi/ encoding proteins with what they define as “α-motility”
protists will undoubtedly aid our understanding of the (motion driven by actin-filled pseudopods at the leading
traits present in early fungi and help to explain the gen- edge of the cell and involving fast migration and weak
eral ecological and evolutionary success of the fungal nonspecific adhesion to external surfaces [182]). Using
lineage. comparative genomics, they showed that lineages with

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 What Defines the “Kingdom” Fungi?

both WASP and SCAR/WAVE, activators of branched tune; i.e., they are very likely to be shown by further
actin assembly, make the three-dimensional pseudopods sampling to have complex evolutionary histories which
needed for α-motility. These genes encode proteins re- invalidate them as diagnostic characters. This is because
sponsible for “nucleation promoting factors” that drive the features that have been argued to define the kingdom
assembly of branched actin by the Arp2/3 complex and are either only partially present in the Fungi or, more
are distributed throughout the eukaryotic tree, suggest- importantly, are present elsewhere in the tree of life
ing an ancient origin and function (178, 182). The dis- (summarized in Fig. 3). In most cases, for features used
tribution pattern of WASP and SCAR/WAVE encoding to identify the kingdom Fungi, both situations are true.
genes was then used to identify pseudopod function It can be argued that the loss of a trait within a mono-
in the chytrid fungus Batrachochytrium dendrobatidis. phyletic group, for example, the loss of ergosterol within
Specifically, they demonstrated that zoospores, which the Fungi, does not invalidate the use of such a char-
naturally lack a cell wall, form bona fide dynamic acter as a defining synapomorphy because loss repre-
pseudopod-like protrusions, and staining of these struc- sents a secondary modification. Yet the demonstration
tures demonstrated a “shell of cortical actin surrounding that a trait is not exclusive to the group it is said to
the cell body and a dense network of filamentous actin define, and instead is present throughout the tree of
filling the pseudopod” (182). To test this observation life, is a knockout blow. This means that such charac-
further, drug treatment with an actin nucleation inhibi- ters cannot be used as a synapomorphy for the evolu-
tor significantly reduced the number of cells with iden- tionary group intended, unless a unique aspect of the
tifiable pseudopodia-like structures (182). These data trait can be identified which is specific to the group it is
confirm that species of established fungal taxonomic claimed to define. Likewise, we find the use of trait loss,
groups form pseudopodia-like structures. such as the loss of phagotrophy, as a defining synapo-
It is therefore clear that the cytoskeletal systems morphy problematic because the loss of such traits has
associated with pseudopodia, a cellular function that occurred on numerous occasions, which makes it diffi-
overlaps with phagotrophic function, were not cleanly cult to see how loss can uniquely identify a group. This
lost at the base of the Fungi and that elements of is further complicated by the observation that multiple
these cellular systems are retained within some fungal chytrid lineages build pseudopodia/filipodia. Pseudopo-
branches. As such, the hypothesis that loss of phagocy- dia/filopodia are cellular systems that are prerequisites
tosis is the defining trait for the Fungi requires further for phagotrophy, suggesting that the molecular charac-
work to understand the evolution of the cellular systems teristics that define the trait “phagotrophy” were not
that drive this function. We suspect that even with a cleanly lost prior to the diversification of taxa currently
clear set of data identifying cell/molecular traits associ- unambiguously defined as fungi.
ated with phagotrophy, and a full understanding of how This debate is further complicated by the large num-
the genes that encode this function are distributed in ber of highly diverse phylogenetic branches which are
fungi and protists, no clean separation between fungi very difficult to place with or within the Fungi (sum-
and protists will be evident for this trait. This is partly marized in Fig. 4). This confuses our understanding of
because phagotrophic function, as we currently under- character evolution. It remains a point of contention
stand it, is defined by few specific protein families (e.g., (e.g., 92, 161, 162) whether many of these groups and
183). Furthermore, if such phagotrophic genes can be the associated uncultured environmentally detected di-
found, they are likely to have also been lost in other versity should be classified as fungi (such definitions are
eukaryotic groups that have lost phagotrophy, for ex- of course dependent on what characters are used to de-
ample, plants and oomycetes, making the loss of such fine fungi).
traits an unreliable demarcation for the Fungi. We do not deny that there is a unit of biodiversity
that can be classified as fungi, but we argue that the line
between fungi and protists is, at this point, a hostage to
CONCLUSION fortune and therefore will change given further sampling
This review has discussed how the definition of the of taxa, genomes, and traits. As such, we prefer to see
Fungi, and the list of taxa that are included within this the transition from protist to fungi as gradual, with no
kingdom, have been in a continual state of flux. An clear demarcation. It therefore largely becomes a matter
additional purpose of this historical review of failed of emphasis where one draws the line which marks the
fungal synapomorphies is to demonstrate that cellular, border of the kingdom. We do not see this as a negative
molecular, and genetic characters are hostages to for- statement. Nor should this point be confused with

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Richards et al.

FIGURE 3 Cartoon illustration summarizing how features previously discussed as

defining the protist-fungal transition have been shown to have a mosaic distribution within
the Fungi and/or outside the Fungi among other eukaryotes. White connecting nodes
illustrate linked characters/traits.

philosophical arguments that can be summarized as a possible that, with the growth of comparative data sets,
disagreement between the “lumpers” and the “splitters.” a clear line can be established that separates protists and
Rather, we hope to encourage the field to focus less fungi. However, it would not surprise us, given what
on constructing categories unlikely to stand the test of we are learning about groups historically classified as
time and focus more on seeking explanations for major chytrids (e.g., 182), if many of these groups end up re-
transitions in the clade. This being said, it is perfectly sembling protists more than fungi, which would lead to

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 What Defines the “Kingdom” Fungi?

FIGURE 4 Schematic phylogenetic tree illustrating additional groups branching proxi-

mate to the origin of the fungal clade and the phylogenetic uncertainty among the deep
branches of the Fungi and associated groups. Basal clone group 1 is composed of envi-
ronmental sequences, which in some analyses is placed close to fungi (149).

much taxonomic revision if strict naming conventions Patrick Keeling, Joe Heitman, Jason Stajich, and Tom Bruns
are to be imposed. This leaves us in a confusing state of for helpful interactions or inspiring comments. We also thank
affairs because we currently lack a consensus on the Maureen O’Malley for critical comments on an earlier draft of
this manuscript. We thank Tim James and Joe Heitman for pa-
definition and composition of the kingdom Fungi such tient and wise editing of this manuscript.
that it could be argued that the definition of fungi (or
protist) does not matter at all. However, on an optimistic
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