Diversification

About 1.4 million different species have been described as living organisms in  the world today, but this is only a fraction of the actual number of species that exist. In all groups of organisms new species are described every day, and in some habitats we are woefully ignorant of the true extent of the biological diversity that exists. A high estimate of the number of species that could exist on earth would be around 100 million species. A low estimate is 2 million. The best estimate might be around 10 million. Whichever estimate is favoured, all estimates are large numbers and they indicate the scale of the taxonomic and systematic problem faced by biologists because these organisms must be sorted into groups to aid our study of them. In dealing with diversity, classification is essential for efficient thought.

Until quite recently, living organisms were divided into two kingdoms, the animal kingdom and the plant kingdom. The animal kingdom contained mainly motile organisms which fed heterotrophically, and the plant kingdom contained mainly static organisms which fed autotrophically by photosynthesis. Fungi were regarded as plants since they have a cell wall and are immobile. With only two kingdoms however, certain organisms such as protozoa could, in effect, jump from one kingdom to the other! It was not until 1866 that this classification system was altered. Haeckel, a follower of Darwin, postulated a third kingdom: the Protista. This kingdom was to contain all microscopic organisms, including bacteria, protozoa and fungi, and for the best part of a century, biologists were content to classify living things as either a plant or animal, because in this classification fungi and lichens were classified with bacteria and algae in a group called the Thallophyta that formed part of the Kingdom Plantae. Because of this, mycology developed as a branch of botany. This is somewhat ironic since we now know that the true fungi are more closely related to animals than to plants.

With the advent of the electron microscope in the 1950s the nature of bacteria became clearer. Two domains were thus created: bacteria, with their lack of a distinct nucleus, led to their separation into the prokaryote domain; and the eukaryote domain which housed all other (so-called higher) organisms, but fungi were still classified in the Plant Kingdom (strictly speaking into the Subkingdom Cryptogamia, Division Fungi, Subdivision Eumycotina) and were separated into four classes: the Phycomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes (the latter also known as Fungi Imperfecti because they lacked a sexual cycle). These traditional groups of ‘fungi’ were identified by the morphology of their sexual organs, whether or not their hyphae had septa (cross-walls), and the ploidy (degree of repetition of the basic number of chromosomes) of nuclei in their vegetative mycelium. The slime moulds, all grouped in the Subdivision Myxomycotina, were also included in Division Fungi.

In 1969, the American biologist, R.H. Whittaker, recognised that fungi are different from other eukaryotes in many essential aspects, so he designated them to a new kingdom. Whittaker's clarification of the system attempted to place organisms in kingdoms that more nearly resembled their supposed evolutionary relationships. This five kingdom approach to classifying organisms was an important step in the attempt to form groups that contain an ancestor and all its descendants (monophyletic groups) and to create a system where similarities and relationships may be seen. It had long been accepted that evolution had occurred since the publication of The Origin of Species (Darwin, 1859); where present species had evolved from earlier species and where similar species had a recent common ancestor, different species a more distant one. Thus a natural classification should mirror descent.

Whittaker noticed, for example, the methods of nutrient intake for the three main eukaryote kingdoms (animals, plants and fungi) were completely different. Animals absorb nutrients internally, engulfing food by the action of ingestion. Plants too have a form of internal absorption, with the intake of energy from the sun by photosynthetic organelles (chloroplasts). Fungi, however, are the only eukaryote to have externalised digestion of their food prior to absorption of the digested components. Characteristically, fungi dwell in a food source releasing digestive enzymes for external digestion of that food source and absorbing the products of digestion as nutrients from the medium. This makes them the Earth's primary recyclers; but it also distinguishes them from their animal and plant relatives.

In the last 30 years, recent advancements in technology, including DNA sequencing techniques, have placed a severe strain on Whittaker's five-kingdom system (Animalia, Plantae, Fungi, Protista (eukaryotic microorganisms, and a mixed grouping of protozoa and algae) and Monera (prokaryotic microorganisms, bacteria and archaea)). Fundamentally, a distinction is made between prokaryotes (Monera) and the four eukaryotic kingdoms (plants, animals, fungi and protists). The prokaryote/eukaryote distinction recognises the ‘higher organism’ traits that eukaryotic organisms share, such as nuclei, cytoskeletons, internal membranes, and mitotic and meiotic division cycles.

The most convincing endosymbiosis theory (Margulis, 2004) accounts for the origin of the set of features that characterise the modern eukaryote through a sequence of symbiotic relationships being established between ‘prokaryotic’ partners. The mitochondria of eukaryotes evolving from aerobic ‘bacteria’ living within a host cell; chloroplasts of eukaryotes evolving from endosymbiotic ‘cyanobacteria’; eukaryotic cilia and flagella arising from endosymbiotic ‘spirochaetes’, the basal bodies from which eukaryotic cilia and flagella develop being able to create the mitotic spindle and thus contribute to the cytoskeleton.

The early eukaryotes were anaerobic, sometimes aerotolerant organisms that lacked mitochondria and peroxisomes. It is presumed that these groups diversified prior to the endosymbiotic events which gave rise to mitochondria. Today, most of these primitive eukaryotes are parasites of other eukaryotes, from microorganisms to humans, but free-living relatives of these parasites branch deep in the evolutionary tree.

These kinds of classification schemes of organisms were historically based on taxonomy according to similarity but this approach has been replaced by one firmly based on phylogenetics. The term comes from the Greek words phyle, meaning 'tribe' or 'race', and genetikos, meaning 'from birth', so it is applied to the study of evolutionary relatedness among taxa. Phylogenetic taxonomy is a natural taxonomy because the classification is based on grouping by ancestral traits. However, before the development of molecular techniques, it really was not possible to determine evolutionary relationships of present-day organisms on a sufficiently comprehensive scale to construct a meaningful tree of life.

Fossil evidence shows the fungi to be important members of terrestrial ecosystems up to 500 million years ago. Molecular phylogenetic evidence suggests that fungi are much older. To begin with, molecular phylogenetic studies tended to use single gene sequences. The most popular have been the nuclear ribosomal DNA (rDNA) locus, particularly that encoding small subunit (18S) ribosomal RNA but including the nuclear large ribosomal RNA subunit (nucLSU), mitochondrial rDNAs, and complete or near-complete mitochondrial genomes. Single-gene phylogenies may not provide sufficient information nor be truly representative to resolve a fungal phylogeny with sufficient confidence so broader studies provide better information. Sequences of protein-coding genes can be a problem because it’s difficult to design primers for PCR amplification that can be reliably applied to a wide range of taxa. Also heterozygous loci in heterokaryons can complicate interpretations. But put all the data together and a coherent story emerges that extends all the way back to the origin of eukaryotes.

The tree of life has three domains

The breakthrough was made by Carl Woese who concluded that, as all organisms possessed small subunit rRNA (SSU rRNA, so called because they form part of the small subunit of a ribosome), the small subunit rRNA gene would be a perfect candidate as the universal molecular chronometer of all life.

These SSU rRNA genes (16S rRNA in prokaryotes and mitochondria, 18S rRNA in eukaryotes) display a mosaic of conservation patterns, with rapidly evolving regions interspersed among moderately or nearly invariant regions (it has been estimated that about 56% of the nucleotide positions in 18S rRNA data sets are not free to vary and have not undergone substitutions useful in phylogenetic reconstructions). This variation in conservation permits SSU rRNA gene sequences to be used as sophisticated chronometers of evolution with the slowly evolving regions recording events that occurred many millions of years ago, and the rapidly evolving regions chronicling more recent events.

In Woese’s procedure, pairs of SSU rRNA gene sequences from different organisms were aligned, and the differences counted and considered to be some measure of evolutionary distance between the organisms. Pairwise differences between many organisms were then used to infer phylogenetic trees, maps that represent the evolutionary paths leading to the SSU rRNA gene sequences of present-day organisms. Of course, such trees rely on many assumptions, among which are assumptions about the rate of mutational change (the evolutionary clock) and that rRNA genes are free from artefacts generated by convergent evolution or lateral gene transfer (Woese, 1987; Woese et al., 1990).

Woese’s studies called into question many beliefs about evolutionary relationships between organisms and brought order to biological diversity. Most importantly, the tree of life constructed from SSU rRNA gene sequences led Woese to recognise three primary lines of evolutionary descent (first called Kingdoms, but subsequently renamed Domains, a new taxon above the level of Kingdom): the Bacteria (now called Eubacteria), the Archaea and the Eucarya (now called Eukaryota). These three domains were thought to have diverged from some universal ancestor. The first two domains contain prokaryotic microorganisms and the third domain contains all eukaryotic organisms.

There has been a good deal of speculation about the universal ancestor, which, rather than being a primitive prokaryote, might well have had a complex cell like a eukaryote, with archaea and eubacteria evolving from it by reduction and simplification (Doolittle, 2000). Modern eukaryotes use RNAs to catalyse intron splicing and stable RNA processing and suggested that these processes could be molecular fossils from the RNA-world that may have been the first step in the origin of life, before the evolution of protein catalysis. So, the universal ancestor might have possessed some extremely primitive features that are now considered to be characteristic of present-day eukaryotes. If these relics of the RNA world were present in the universal ancestor, it doesn’t mean that the ancestor was eukaryotic. Rather, the ancestor contained a mix of features that were selected and combined in different ways during evolution of the present-day archaea, eubacteria and eukaryotes.

Unfortunately, because the evolutionary clock is not constant in different lineages, the time of occurrence of evolutionary events in the tree of life cannot be extracted reliably from SSU rRNA gene sequences alone, so other sequences have to be brought into the analysis. All molecules chosen for phylogenetic studies must:

  • be universally distributed across the group chosen for study;
  • be functionally homologous;
  • change in sequence at a rate proportionate with the evolutionary distance to be measured (the broader the phylogenetic distance being measured, the slower must be the rate at which the sequence changes).

Although SSU rRNA genes satisfy these criteria, it is important not to allow phylogenetic trees based on a single gene to dominate evolutionary and systematic conclusions. Instead, account needs to be taken of phylogenetic trees based on a range of conserved molecules with a judgement being made about the weight to be given to the different lines of evidence so obtained.

Among the sequences that have been used successfully in multi-gene phylogenies are ATPase genes; the ATPase enzymes are composed of several different kinds of subunits, each related among themselves. The F-, V- and A-enzymes have catalytic and non-catalytic subunits, which are thought to have arisen during early gene duplications prior to the separation of the three domains. Because of this, the phylogenetic tree inferred from catalytic subunits can be rooted with the non-catalytic subunits. Using ATPase subunits, the root of the tree of life was placed on the eubacterial branch, making the Eukaryota and Archaea sister taxa. This conclusion was supported by studies using aminoacyl tRNA synthetase gene sequences. These genes form a series of 20 enzyme families, with each family the result of gene duplication, again argued to predate the origin of the three domains. Thus, aminoacyl tRNA synthase phylogenetic trees can be rooted in a fashion similar to that for the ATPase trees.

The ribosomal gene cluster is another regular contributor to construction of phylogenetic trees. Ribosomal genes are usually present in genomes in large numbers (100 to 200 tandem repeats) but they evolve as a single unit. The large quantities of rRNAs expressed by cells make their isolation and purification relatively easy despite the nearly ubiquitous occurrence of stable RNAses. Additionally, rDNA can be sequenced using specific oligonucleotide primers and the polymerase chain reaction (PCR). Because each genome contains many identical copies of the ribosomal genes, at least one copy for molecular analysis can usually be recovered, even from low-quality DNA preparations. Importantly, rDNA sequencing results in fewer artefacts than rRNA sequencing, and offers the opportunity of sequencing both strands of the rRNA sequences, providing a further check against sequencing errors.

Three regions of the ribosomal gene cluster in eukaryotes code for rRNA genes, which are transcribed into 5.8S, 18S and 28S RNA molecules that form part of the ribosome structure. Of the approximately 9000 nucleotides in a ribosomal repeat, the 18S gene accounts for about 1800 bp, the 5.8S gene for about 120 bp and the 28S gene for about 3200 bp. Interspersed between the rRNA genes are spacer regions. The areas that lie between the 18S and 5.8S and between the 5.8S and 28S genes are called internally transcribed spacers (ITS1 and ITS2).

The ITS1 and ITS2 regions are transcribed together as a single unit, then cleaved into separate RNA products, and finally eliminated in the process that yields rRNA. The region that separates one ribosomal gene cluster from the next is called the intergenic spacer region (IGS), which is made up of the non-transcribed spacer region (NTS) and the externally transcribed spacer region (ETS). The rRNA product from the ETS region suffers a similar fate to the internally transcribed spacers. The rRNA genes, the transcribed spacers (ITS and ETS) and the non-transcribed spacer (NTS) evolve at different rates and because of this their sequences have become widely used to discriminate between fungal taxa at levels from the kingdom to the intraspecific strains and races (Table 1).

Table 1. Characteristics of rRNA sequences used to identify and classify fungi

rRNA molecule

Structural rRNA?

Transcribed?

Level of conservation

Taxa which can be distinguished

18S rRNA (small sub-unit RNA)

Yes

Yes

Highly conserved domains interspersed with conserved domains

From domains to classes

5S rRNA

Yes

Yes

Conserved domains

Classes and orders

28S rRNA (large sub-unit RNA)

Yes

Yes

Conserved domains interspersed with variable domains

From phyla to species

Internally transcribed spacers (ITS) 1 and 2

No

Yes

Variable domains

Species and closely related genera
Intergenic spacer (IGS)
No
No
 Highly variable domains Strains and races
Table from Moore et al., 2011 (URL)

Sequences other than the ribosomal gene cluster and ATPases that have been used in phylogenetic studies of eukaryotes include: ribosomal protein factors, tubulin (alpha (a) and beta (b)), actins and cytochromes. Protein sequences, which are made up of 20 amino acids, offer several advantages over DNA sequences (made up of four nucleotides) for some phylogenetic studies because homology (similarity due to shared ancestry) is more easily distinguished from analogy (similarity in structure or function that evolved through different pathways and different ancestries, a process known as convergent evolution). Further, length changes are infrequent in protein-coding genes because insertions and deletions often lead to such large shifts in the reading frame that they are fatal, and so fail to persist in the lineage. Mitochondria and their protein coding genes are present in multiple copies in most cells and mitochondrial genes are easy to amplify, even from starting DNA of low quality. However, relatively few phylogenetic studies of fungi have used mitochondrial protein coding genes.

It has been estimated that the Domain Eubacteria and the common ancestor to the Domains Archaea and Eukaryota diverged about 4 billion years ago, with the Eukaryota arising on the Archaea lineage about 2 billion years ago (Fig. 1).

The root of the universal tree of life remains controversial, though. Evidence for ancient lateral transfers of genes and uncertainty over the ancestry of Archaea makes the distant origins of the major lineages of life uncertain. Inferring ancient relationships is full of difficulties. If the data contain too little information, random errors can swamp the truth, and random errors can be introduced by the mathematical model used to interpret the data. The method used to establish the phylogenetic trees can also introduce more systematic errors if it is too simplistic (explanations and further references in Keeling et al., 2005).

One view of the most ancient relationships of the major lineages of the domains of life
Fig. 1. One view of the most ancient relationships of the major lineages of the domains of life, redrawn from the Tree of Life Project (http://tolweb.org/Life_on_Earth/1/1997.01.01 in the Tree of Life Project, http://tolweb.org/). There are two kingdoms within the Archaea: the Euryarcheota composed of methanogens and extreme halophiles, and the Crenarcheota composed of the extreme thermophiles. Eocytes are a group of sulfur-dependent bacteria with a unique pattern of organisation of ribosomal large and small subunits. They are closely related to eukaryotes. Figure and caption from Moore et al., 2011 (URL).

Errors amplify as you attempt to reach back further in time, in what is called deep time (hundreds of millions to billions of years) where you are looking for deep divergences between major groups of organisms. Fossils are necessary to calibrate phylogenetic trees to a real timeline, but there is a very patchy fossil record and the older the fossil the greater the debate about its nature. The result of such errors and uncertainties is that the timings inferred for major events (like the divergences of major eukaryote groups) in different studies can differ by several hundred million years, or more. For example, one study claims the common ancestor of living eukaryotes existed 2.3 billion years ago. Another puts the time of eukaryote divergence at 1.26 to 0.95 billion years ago. The two studies used different methods and different phylogenetic models; their different dates could mean that the common ancestor existed for a billion years before evolving into the plant, animal and fungal lines or it could mean that the best we can say is that the divergence event occurred some time between 2.3 and 0.95 billion years ago.

Archaea and Eukaryota are sister groups with a common ancestor, so their modern representatives share many properties that differ from those found in the Eubacteria. For example, the RNA polymerase of Archaea and Eukaryota resemble each other in subunit composition and sequence far more closely than either resembles the type of polymerase found in Eubacteria. Also, the Archaea and Eukaryota use TATA-binding proteins to regulate the initiation of transcription, whilst the Eubacteria use sigma transcription factors.

Overall, the Archaea and Eukaryota are more closely related to each other than either is to the Eubacteria and it follows from this that, despite their prokaryotic nature, members of the Archaea should not be regarded as bacteria, a conclusion that some bacteriologists still find difficult to accept (similarly, some mycologists find it difficult to accept that the Oomycetes are not true fungi).

A more complete fossil record, improved molecular dating method and a better understanding of molecular evolution will be needed before the true ages of the eukaryote lines of evolution can be determined with any certainty.

The opinions presented above are based on a range of relatively recent work (for example you can read any of these for yourself by clicking on the hyperlinks: Doolittle et al., 1996; Embley & Hirt, 1998; Aravind & Subramanian, 1999; Philippe et al., 2000; Keeling et al. 2005). The studies represented in these references (together with many, many others; for a comprehensive account visit the Tree of Life Project at http://tolweb.org/Eukaryotes) build on the established schemes of evolutionary relationships developed using morphology and biochemistry. But, as we have explained above, phylogenetic trees are currently built using a wide variety of data, which are largely, but not entirely, molecular in nature. It is now accepted that molecular sequences are generally more revealing of evolutionary relationships than are classical (mainly morphological) phenotypes and this is particularly true for microorganisms.

At the moment we have to be satisfied with generalisations and the generalisations we favour are that eukaryotes and prokaryotes diverged 2 billion years ago, and plants, animals and fungi diverged 1 billion years ago.

Today microbes often live in mixed communities that are capable of rapid attachment to surfaces as a biofilm. Biofilms are an important component of our present environment, being found essentially everywhere on Earth, including in extreme environments (Sutherland, 2001). Most are beneficial or harmless but those that form on clinical equipment and many household surfaces can cause harm. Biofilm formation is so obviously beneficial to the organisms in the community that it must have arisen at an early stage in evolution. So perhaps we should think in terms of the microorganisms that existed for the first 3 billion years of life on Earth forming extensive biofilms over moist surfaces (and that would include the surfaces of bodies of shallow water). Some of those biofilms would have contained photosynthetic microbes, cyanobacterial as well as algal.

Knauth & Kennedy (2009) analysed carbon isotope ratios in ancient carbonate rocks. They concluded that the ‘greening of Earth’ started about 850 million years ago in coastal regions and resulted in an extensive spread of photosynthetic microbes (as ‘an explosion of photosynthesising communities on late Precambrian land surfaces’). They suggest that by 1000 million years ago this ‘greening’ was sufficient to increase atmospheric oxygen, alter the chemical breakdown of rocks on the Earth’s surface and increase nutrient flux and organic matter in ancient soils and sediments (Hand, 2009; for counter arguments see Arthur, 2009).

Primitive biofilms would have contained fungi, too; and an important ability of hyphal growth is that filamentous hyphae can escape from the biofilm. Even more importantly, filamentous hyphae can exploit the biofilm, digesting the adhesives, gums and other polymers that make up the biofilm matrix and parasitising the photosynthetic microbes to recruit photobionts into primitive lichen-like arrangements.

Diversity in Kingdom Fungi

There are an extremely large number of fungi that dwell in the world today. Hawksworth (1997) conducted an investigation in which he compared fungal species figures with other groups obtained from ecological regions that had been deeply researched for their biodiversity. These studies led to the conclusion that there were near 1.5 million species of fungi worldwide (Hawksworth, 2001).

Approximately 98,000 fungal species have been described to date, the majority being members of the Ascomycota (about 64,000 known species) and the Basidiomycota (about 32,000 known species), meaning there is still around 95% of the fungi population that remain undescribed. 

Organisms that have, for one reason or another, been classified as fungi have now been assigned to three different groups:

  • The monophyletic kingdom of fungi, containing a core of 'true' fungi, all seem to have a common ancestor, probably alike to the modern day choanoflagellates (a unicellular protist). The Kingdom Fungi comprises of the phyla: Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Microsporidia, Glomeromycota, Ascomycota, Basidiomycota with at least four subphyla in a (polyphyletic) group traditionally known as the Zygomycota. 

  • The Kingdom Chromista, also known as the kingdom Stramenipila, which includes Oomycota, Hyphochytriomycota and Labyrinthulomycota. All seem related to certain algae groups, but have evolved a typical fungal lifestyle. 

  • An assemblage loosely termed slime moulds, considered as protists in Kingdom Protozoa, includes the phyla Plasmodiophoromycota, Dictyosteliomycota, Acrasiomycota and Myxomycota. They grow as wall-less protoplasmic stages, often engulfing bacteria and other food particles by phagocytosis. They seemingly are not closely related to each other and their nearest relatives are not yet accurately known.

Organisms formerly classified as fungi

Several interesting points can be brought up to clarify the identity of the 'true' fungi:

  • An extremely large amount of data, including structural, biochemical, physiological and molecular, has contributed to the knowledge that three groups of organisms traditionally studied by mycologists, the Hyphochytriomycota, Labyrinthulomycota and Oomycota, are so phylogenetically remote from the kingdom fungi that they have been placed in an entirely new kingdom, the kingdom Chromista (or kingdom Stramenopiles). The kingdom, though full of diverse organisms, seems to have a biflagellate condition. Take for instance the Oomycota. This group contains organisms such as the Saprolegniales (water moulds) and Peronosporales, which contains many serious plant pathogens. They resemble fungi in being composed of hyphae and in their absorptive nutrition but differ in fundamental and biochemical features including cellulose-based walls and biochemical features resembling those of plants. It is likely they arose in the Precambrian era from the Xanthophyceae, a group of algae in which the hyphal form is common.

  • The Acrasid and Dictyostelid slime moulds (cellular slime moulds) are amoeboid organisms that phagocytose bacteria and other food particles. They are characterised by discoidal mitochondrial cristae and diverged early in the eukaryotic lineage. The Myxomycota (plasmodial slime moulds) grow as a network of protoplasm that engulfs their food. They have tubular mitochondrial cristae. Both groups differ massively to fungi including their protoplasmic somatic stage and their modes of nutrition (theirs being ingestive rather than absorptive). However, both groups form fungus-like fruiting bodies to release air-borne spores, indicating this is the reason why they were once treated as fungi.

  • Little information is known on the Plasmodiophoromycota, possibly intracellular parasites of fungi, algae and some higher plants. They can form highly persistent thick-walled resting spores, maybe a reason for the confusion with fungi. 

Success of Fungi

It should be finally noted that though many organisms, including various protists and choanoflagellate ancestors of fungi, have adapted a similar lifestyle, none have been as successful as the fungi themselves. This may be due to some sort of inherent superiority in evolutionary potential of the fungal lineage. However, it is much more likely it was due to some random chance event in which fungi exploited the niche for absorptive heterotrophs before the other various organisms could become established. They then cemented their position into all the other possible niches before the others had chance to settle, thus excluding them, it seems, for many millions upon millions of years.

Further information about the taxonomy, phylogeny and evolution of fungi can be found in the new textbook 21st Century Guidebook to Fungi by David Moore, Geoffrey D. Robson & Anthony P.J. Trinci. Published 2011 by Cambridge University Press, ISBN: 9780521186957.

URL: http://www.cambridge.org/gb/knowledge/isbn/item6026594/?site_locale=en_GB.

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Updated December 15, 2016