Manual 21423518

Manual 21423518
2.1.1 Types of introns
2.1.2 The role ofintrons in biology
2.1.3 Origin of introns
2.1.4 Positional conservation
2.2.1 Codon Bias
2.2.2 G+C content
2.2.3 Multiple overlapping substitutions
2.2.4 Transversion/transition ratio
3.1.1 Types ofmultigene families
3.1.2 Concerted evolution
3.1.3 Example of a protein encoded by a multigene family: ~-tubulin
3.2.1 Examples ofproteins encoded by single genes: largest and second largest subunits of DNA-dependent RNA polymerase II
4.2.1 Interspecific relationships
4.2.2 Intraspecific relationships
10 10 11 11 11 12 13 15 15 16 16 17 17 18 5 CONCLUSIONS
.' Since the onset of the application of molecular techniques, fungal taxonomy and phylogeny
has been dominated by the use of the ribosomal RNA genes (rrn) 18S, 28S and 5.8S, as well as the
internal transcribed spacers (ITS) separating these genes (see for example refs. 35, 119).
Depending on the particular rrn gene used, taxonomic and phylogenetic questions at all levels have
been addressed (see of example ref 247). Unfortunately, phylogenetic trees inferred using these
genes are often incongruent with fungal biology and they do not always provide sufficient
resolution ofthe taxa being studied (see for example refs. 115, 170,247,254,291,293). The major
reason for these irregularities and lack of resolution is that non-uniform evolutionary forces are
potentially acting upon the rrn genes of closely related fungi. This problem is easily solved by
including additional regions of the fungal genome in the analyses.
For this purpose, fungal
taxonomists and evolutionary biologists frequently use protein-coding genes (see for example refs.
Protein-coding genes can be applied to evolutionary questions at all taxonomic levels. They
have, for example, been used to determine the root of the tree of life and to study the ancient
eukaryotes (10, 77, 144, 149,291). Protein-coding genes can also be applied successfully to lower
(intra- and interspecies) and intermediate (intergenus or -order) taxonomic levels (18, 155, 170,
213). The use of protein-coding genes in phylogenetic studies has one major advantage over the
use of rrn genes. Whereas only specific rrn genes can be used to address questions at certain
levels, a single protein-coding gene, for example anyone of the tubulin genes, can be used to
address taxonomic questions at all levels (see for example refs. 11, 141,214,243).
Protein-coding genes are subjected to many different evolutionary forces, the effects of
which can have profound implications on the interpretation of phylogenetic data. The purpose of
this review is, partially, to discuss different forces acting on protein-coding genes and how they
influence evolutionary reconstructions.
This is done by providing background, firstly, on the
structure of protein-coding genes and secondly, on the evolutionary forces that have shaped them.
The discussion on protein-coding genes is mostly restricted to eukaryotic nuclear genes, but
organellar and prokaryotic genes are briefly considered. The remainder of this review deals with
the use of protein-coding genes in fungal taxonomy and provides some examples where they have
been used successfully.
One of the most striking features of protein-coding
is that they are generally
organized into regions of coding sequences (exons) that are interrupted by intervening non-coding
sequences (introns) (reviewed in ref 31). Since these intervening sequences confer no apparent
phenotype on the cell, and· are thought to be without function, they are subjected to fewer
evolutionary constraints than are exons (24, 65). Introns thus provide an attractive source of
sequence variation in an otherwise highly conserved gene (6). In the following sections, introns are
discussed with regard to their types, possible role in biology, origin and positional conservation. In
addition phenomena specific to exons are reviewed.
These include codon bias, G+C content,
multiple overlapping substitutions or homoplasy and transversion/transition ratios. In all cases,
special attention is given to issues pertaining to the evolutionary forces that not only gave rise to
these regions, but also those that are currently acting upon them.
2.1 Introns
2.1.1 Types of introns
Introns are divided into different classes based on their mechanisms of splicing and in which
genes they occur. Group I introns, for example, are found in the genomes of prokaryotes and
organellar genomes. They have ribozymic activity and hence encode endonucleases that assist their
splicing and trans-positioning (17, 72). Group II introns are also autocatalytic or self-splicing, but
rather than encoding endonucleases, they generally encode reverse transcriptases (17). Group II
introns are characteristic of prokaryotic and organellar genomes, where they are found in the genes
encoding proteins, transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) (194). Group ill introns
are similar to group II intron, but differ in that their self-splicing mechanisms are somewhat
defective (50, 194). Both group I and II introns are self-splicing and thus code for the 'machinery'
necessary for their spread and/or removal.
Eukaryotic nuclear genes harbor a different type of intron that is generally referred to as a
spliceosomal intron.
Removal or splicing of this type of intron involves a multi-molecular
RNA/protein complex or spliceosome that is formed through the interaction of a number of small
nuclear ribonucleoproteins (snRNPs) (88, 104, 177). The structure and splicing mechanism of
spliceosomal introns closely resembles that of group II introns, which suggests a common origin for
spliceosomal and group II introns (43, 50, 65, 137, 160,205,236).
Spliceosomal introns are subdivided into two groups based on the consensus sequences at
their splice sites (136, 200, 249, 289). The first and most common type is characterized by GT - and
AG-dinucleotides at their 5' and 3' intron boundaries (Table 1) (289). Two types of spliceosomes
can excise these GT-AG introns (Table 1). The most common type includes U2 snRNP as part of
the spliceosomal apparatus, whereas the other spliceosome include UI2 snRNP (110, l36, 249,
289). The second type of spliceosomal intron has the dinucleotides AT and AC at its 5' and 3'
boundaries (110, 200). The U2- and U12 types of spliceosomes "also excise these AT-AC introns
(Tab1e 1). These AT-AC introns are, however, very rare. U12-type AT-AC introns occur at a
frequency ofless than one in five thousand, and U2-type AT-AC introns are even rarer, since there
are only seven known examples (110, 111,200,269).
Spliceosomal introns are distinguished based on their phases. Intron phase refers to the
placement ofthe intron relative to the reading frame (58). A phase 0 intron, for example, is situated
exactly between two neighboring codons. Phase 1 and 2 introns split a codon after the first and
second bases, respectively. Long and Deutsch (173) reported that most spliceosomal introns were
ofthe phase O-type. They further showed a strong correlation between intron phase and the degree
of conservation of the splice signals in the exons surrounding an intron. In other words, the exonic
sequences flanking phase 2 introns are most variable and are not in total agreement with the
consensus sequences (Table 1). The exonic sequences surrounding a phase 0 intron usually match
the consensus exactly. According to Long and Deutsch (173), the evolutionary forces determining
the sequences surrounding a spliceosomal intron are strongly biased towards generating phase 0
introns. This is because variations in these splice signals produce phase 1 and phase 2 introns,
which may lead to intron-Ioss associated with deleterious mutations. The relatively few phase 1
and 2 introns, therefore, reflect those cases where intron-Iosses were not associated with lethal
mutations (173). This is illustrated in the
genes of a diverse group of organisms, where
more than half the introns are of the phase O-type, while the remaining introns are phase 1 and 2
(Fig. 1).
2.1.2 The role of introns in biology
Broadly speaking introns have no obvious function (24). They can be removed without any
phenotypic effect. There are, however, reports of certain introns performing regulatory functions
such as enhancing or modulating expression ofthe genes harboring them (78, 255). Several authors
have further indicated that introns might playa role in genetic recombination by creating so-called
'hot spots' for crossing-over (22,23,25,65, 96, 97). This 'loosening' of genetic linkage between
exons, forms the basis for one of the models explaining the evolution of introns. Nevertheless, if
introns do play biologically important roles, they would be subjected to evolutionary forces that are
most probably different to those acting on the exon regions.
2.1.3 Origin of introns
There are two opposing hypotheses for the origin of introns. These are known as the
'introns-early' and 'introns-late' theories (65). The 'introns-early' theory is also known as 'the exon
theory of genes' and suggests that the first exons were short (15-20 amino acid residues) and
assembled by recombination within introns. According to this theory, the short exons (encoding
functionally active polypeptides) were 'shuffled' to eventually form a gene consisting of many
introns and exons. This suggests that primitive ancestral genes would have had many introns that
separated various functionally active protein domains or exons. Divergence of prokaryotes and
eukaryotes led to the loss of these introns through genomic streamlining in the case of prokaryotes,
and retention associated with occasional loss of introns in the case of eukaryotes (65, 96, 98, 168,
Several lines ofevidence are provided for the 'introns-early' theory (24,64,96, 98, 99, 168,
211). One of these is the relative position ofintrons in genes (168, 211). As is the case in many
other genes (9, 79, 98, 185,239,281) the p-tubulin introns occur in clusters at regular intervals of
15-20 amino acid residues (Fig. 1) (168, 211). The positions of these introns also appear to be
conserved over great evolutionary distances, suggesting that modem genes evolved via intron-Ioss
from the ancestral intron-containing gene (Table 1).
The 'introns-Iate' theory suggests that prokaryotic genes most closely resemble the ancestral
state (58, 59, 65, 172, 236).
According to this model, spliceosomal introns evolved during
eukaryotic evolution. These introns were then inserted into unsplit genes after the prokaryotic­
eukaryotic 'transition' and have nothing to do with the development of genes (236). The 'introns­
late' theory is supported by the presence of so-called proto-splice sites (172). These sites serve as
recognition sequences for the insertion of introns (58). Proto-splice sites are characterized by the
sequence KAG*R (K = A or C; R
A or G and
splice junction), which closely resemble the
exonic consensus surrounding intron splice junctions (Table 1). Such proto-splice sites are present
at conserved positions in what is believed to be older intron-Iacking versions of a gene.
'introns-Iate' theory suggests that these proto-splice sites were present prior to evolutionary radiation
(58, 172). The conserved positions of the proto-splice sites, therefore, determine the positions of
introns (58, 172).
The origin of introns remains a controversial subject. Evidence for both the 'introns-Iate'
and 'introns-early' models is inconclusive. Supporters of the 'introns-early' model interpret the
presence ofproto-spij.ce sites as remnants oflost ancestral introns.
Proponents of the 'introns-Iate'
model fmd no statistil:;ally significant 'intron-splitting' of genes into functionally active domains (59,
277). They also argtIe that it is unlikely that large-scale intron-Ioss through genetic streamlining
occurred twice in evolutionary history, once in the ancestor of all eubacteria and a second time in
the archae bacterial ancestor (236). It is thus clear that neither the 'introns-Iate' nor the 'introns-early'
theories can be discredited. Recent computer assisted analyses of large numbers of conserved
protein-coding genes has provided evidence that supports both models (238,277). Some introns are
evolutionarily old and were probably involved in ancestral exon shuffling to create genes (82, 248,
281), whereas other introns were recently gained or lost (82, 172).
The opposing theories on the origin of introns complicates their use in phylogenetic
inferences. This is because evolutionary reconstructions using protein-coding genes reflect the
phylogenies of both the exons and introns.
If the introns in question originated prior to the
divergence ofthe group oforganisms included in the analyses, the intronic history would reflect the
phylogeny of the exonic regions. However, if these introns were 'acquired' during or after the
divergence of the organisms in question, the intronic and exonic phylogenies would be different.
2.1.4 Positional conservation
The nucleotide sequences of many genes display a higher degree of variability in the 5'-half,
than in the 3'-half, of the gene. This is mainly due to the fact that introns are not distributed
uniformly within genes, but appear to be more abundant in the :first or 5'-half. Examples of genes
where introns are preferentially situated in the 5'-half, are
(Fig. 1) (168), small G
proteins (59), glyceraldehyde-3-phosphate dehydrogenase (185, 228), etc. This is either because of
intron gain in the first portion ofgenes, or intron loss from the 3'- halfof genes (58, 172,236,238).
Gain and loss of introns is explained using the 'introns reinsertion-homologous
recombination' model
This model involves the spliceosome and proto-splice sites, mentioned
earlier. Based on this model the loss or gain of an intron includes a four-step process (96, 168,
236), whereby the intron is (i) spliced from the premature messenger RNA (pre-mRNA) and (ii)
reinserted into a nearby site of the pre-mRNA. This is followed by (iii) reverse transcription of the
'new' pre-mRNA into complimentary DNA (cDNA).
The final step in this model is the (iv)
reinsertion of this newly formed intron-containing cDNA into the genome via homologous
recombination with the genomic copy. The result would be intron gain, but when step two is
omitted, the result would be intron-Ioss. In other words, if the removed intron is not reinserted at a
different position in the pre-mRNA, the pre-mRNA can be reverse transcribed and recombined back
into the genome. This would generate a gene from which an intron has been deleted.
The involvement of a reverse transcription step in the 'intron reinsertion-homologous
recombination' model provides an explanation for the polarization of genes with regard to intron
position. According to this model, reverse transcription is always initiated at the 3'-end and seldom
extends fully to the 5'-end of the pre-mRNA. Homologous recombination, therefore, results in
'replacement' of the 3'-portions of the gene with a reverse transcribed intron-less cDNA copy (85,
96, 185). In some genes, the involvement of reverse transcriptional errors at the 3' end of the gene
has been used to explain why 5' intron positions are more conserved than the 3' intron positions
Intron positions in genes such as ~-tubulin, are usually conserved across great evolutionary
distances (168). Comparisons of intron positions in the
genes from a diverse group of
organisms (Fig. 1) has revealed that ascomycetous fungi are characterized by a unique intron (intron
5). The same is also true for the metazoan (intron 20) and plant lineages (intron 133) (Fig. 1). This
conservation also extends to individual lineages, since all the fungi from the pyrenomycetous order
Hypocreales have three specific introns in the first half of their
genes (introns 5, 13 and
54). All the members of the lower plants also appear to harbor a unique intron (intron 57) in their
genes. However, certain organisms have more than one
gene per individual,
each with unique intron positions. For example, the intron positions in the tube and benA
genes of Aspergillus nidulans are very different (Fig. 1).
Intron sequences are useful for answering phylogenetic questions at lower taxonomic levels.
This is because it is possible to align homologous intron sequences from closely related taxa (6, 18,
63, 83, 212, 263, 285). However, 'homologous' introns of more divergent taxa usually only share
the same position and little or no sequence homology (83, 172,263,280). For this reason the use of
intron sequences for reconstructing deeper level phylogenies are not feasible (83, 158, 191, 310).
To address this type of evolutionary question intronic regions can thus not be treated as nucleotide
bases in phylogenetic analyses, but rather as 'presence' or 'absence' characters.
2.2 Exons
Exons are the coding regions ofprotein-coding genes. Their nucleotide base composition is,
therefore, subjected to evolutionary forces that not only reflect lineage history, but also other
constraints imposed at the translational and functional levels (30, 49, 131, 132, 292). Phenomena
such as codon bias, G+C content, multiple overlapping substitutions and transversion/transition
ratios serve as indicators of these selective forces, although they may in some cases also reflect
gene and/or taxon phylogeny (1, 2, 30, 34, 55, 91, 130, 166, 171, 182, 235, 275, 298). These
phenomena are not independent of one another and changes in one results in changes in the others.
In the following sections, these interdependent factors are discussed in more detail.
2.2.1 Codon Bias
Codon bias is a phenomenon found in many protein-coding genes and is defmed as the non­
random use of synonymous codons (106). Leucine, for example, is encoded by six synonymous
codons (CUU, CUC, CUA, CUG, UUA and UUG) and the preferential use of one during translation
is referred to as codon bias. Non-biased' genes differ from biased genes, in that any of these
codons can be used during translation. Non-biased' genes are further characterized by silent or
synonymous substitutions rather than non-synonymous substitutions that would result in alteration
of the amino acid sequence (36, 45, 117, 166, 292). For example, one or two substitutions in the
three nucleotide bases specifying an amino acid will generally be synonymous or silent. This is
because the substitution results in a codon that still encodes the original amino acid.
synonymous substitutions that occur at the third nucleotide base 'are less frequent, since they will
result in alteration of an amino acid residue that can lead to loss or decrease of functionality in the
mature protein.
Codon bias is a prominent feature ofhighly expressed genes (45, 131,251). It is determined
and/or influenced by two main groups of factors. The first group of factors controls the efficiency
of translation. The second group of factors control structural aspects of the gene without regard of
translational efficiency (292). One of the factors that will influence translational efficiency is the
abundance ofa specific tRNA species (30, 49, 61, 103, 117, 130-132, 166,225,251,289). In the
yeast Saccharomyces cerevisiae, for example, the most abundant lysine tRNA species has the
anticodon CUU, which will bias the codon usage of this amino acid towards AAG. Other tRNA
species that will recognize the remaining lysine codon (AAA) are scarce and sometimes absent.
The inclusion of this codon in the genes of the fungus will thus cause a reduction in translational
efficiency. Such codons are, therefore, selected against and results in codon bias.
The second group of factors that will influence codon usage includes the requirements for
gene and RNA secondary structure (224, 253).
For example, portions of the downstream­
untranslated regions of the alcohol dehydrogenase (adh) gene of Drosophila, interacts with
nucleotides in the second exon of this gene. Synonymous substitutions (Le. those that will not
change the codon) in this exon, alter the secondary structure of the mRNA. This significantly
reduces expression ofthe adh gene (224). This type of interaction, therefore, also contributes to the
selection for specific codons.
Preferential use ofcertain codons is generally species and/or gene specific (1, 91, 159, 166),
but is sometimes associated with phylogeny (106, 131, 132). Differences in the degree of codon
bias can potentially have serious implications for determining evolutionary relationships. It is well
documented that the use ofmany codon-biased genes distorts and obscures phylogenetic histories in
many different eukaryotic and prokaryotic organisms (55, 81, 117, 125, 166, 167, 176, 182, 237,
2.2.2 G+C content
Variations in G+C content are usually located at the third bases of codons (30, 55, 166, 250,
292). This reduces the number of possible synonymous substitutions in a codon. For example, a
bias towards high G+C content will reduce the number of codons (six) specifYing a leucine residue
to three codons. The codons CUU, CVA and UUA will be selected against and thus not occur. It is
clear that G+C content and codon bias are very closely linked.
For this reason the same
evolutionary forces that affect codon bias will generally also influence G+C content (36, 55, 81,
125, 166,292).
2.2.3 Multiple overlapping substitutions
Apart from the restrictive effects of codon bias and G+C content, a multiplicity of silent or
synonymous substitutions can occur at a specific position during the evolutionary history of a gene
or species (167). For example, the third base in the codon specifying a leucine, can change from T
to C and back to T.
This is termed a 'reversal' and together with other phenomena such as
convergence and parallelism, is referred to as homoplasy (268).
Many of these overlapping
substitutions or homoplastic events will obliterate the historical information at that position. They
can thus lead to an underestimation of the degree of divergence or an overestimation of the degree
of similarity between different taxa (100, 237, 294). The use of these characters in evolutionary
inferences, therefore, results in lack of phylogenetic resolution and inconsistencies (125, 233, 237,
2.2.4 Transversion/transition ratio
A transversion is defmed as the substitution ofa pyrimidine (C or T) for a purine (A or G) or
vice versa. Transition is defined as substitution of a purine for a purine or a pyrimidine for a
pyrimidine (167). According to DeSalle et aI. (57), there is a general lack of transitional bias
between distantly related taxa, because the record of transitional events is erased by transversions.
This apparently results in an accumulation oftransversions among more divergent genomes (34, 57,
124, 275).
Transitions are, therefore, usually more abundant than transversions among closely
related organisms (34,55,57, 117, 125, 167,275).
Inference of phylogenetic relationships from protein-coding regions presupposes that
evolutionary forces underlying nucleotide variation are common to all the taxa that are examined
(167). However, many different selective forces and processes, other than those involved in the
'creation' of a lineage, are acting upon the exons and introns of protein-coding genes.
processes can distort phylogenetic information, thereby obscuring evolutionary histories and
making it impossible to reconstruct genealogical relationships. Although these forces act on genes
at all taxonomic levels, most problems are encountered at the deeper levels such as kingdom, family
and order (10, 94, 123, 125, 166, 265, 292). Among closely related taxa at the species level a
specific gene or part ofa gene is generally subjected to comparable forces (55, 117, 166,292).
Evolutionary analyses are based on the assumption that the selected gene or region of the
genome is orthologous (167). In other words, the evolutionary history for this region is similar to
that of the individuals in which it is studied. Events that will create multiple copies of genes
(duplication, hybridization and horizontal transfer) may result in non-orthology between genes.
Protein coding genes that occur in multigene families and those that occur as single genes in the
genome of an organism are further sUbjected to different evolutionary forces (217). The non­
uniform evolutionary forces acting on single and multi-copy protein-coding genes greatly
complicate their use in phylogenetic studies, since a gene phylogeny can be inaccurately interpreted
as a species phylogeny.
3.1 Multi-copy genes
3.1.1 Types of multigene families
In evolutionary biology, the duplication events that gave rise to multigene families could
have occurred very early or relatively recently. These ancient and recent duplication events are
reflected in the degree of divergence from the ancestral gene. For example, modem gene families
share a high degree of sequence homology, while ancient gene :fimrilies show very little sequence
homology. In many cases, the homology in ancient gene families will be restricted to a number of
conserved domains, which is the result of functional constraint in the mature protein (28, 133).
Examples of ancient multigene families are those that encode the different tubulin subunits of
mature microtubules (174, 178) and those encoding the subunits of eukaryotic DNA-dependent
RNA polymerases (133). Some of these ancient gene families are divided into sub:fimrilies to form
modern gene families.
Ancient multigene :fimrilies are potentially ofgreat use in evolutionary studies, especially for
the inference of deep phylogenetic relationships. For this type of study, eukaryotic protein-coding
genes are normally used. However, this approach is problematic, since suitable outgroups are not
always available (107, 144). The problem can be overcome by using the gene sequence of another
member of that ancient multigene family as an outgroup. This is, however, only possible when the
duplication event that generated the gene family predated the divergence of the taxa of interest.
This approach has been successfully employed by several research groups (107, 135, 144,254).
Modem multigene families include those encoding p-tubulins (46), chitin synthases (29),
actins (190), etc. The different members of some of these gene families are thought to be essential
at different stages of the life cycles of organisms (38, 46, 80, 128, 129, 153, 190, 246, 259, 284).
This is, however, not always the case, since disruption of genes in these :fimrilies does not always
result in lethal mutations (38, 60, 266). It is suggested that the occurrence of more than one copy of
a specific gene act as a form of multigene control of a specific trait, since another member of the
family can 'replace' a defective copy (16, 301). The"n genes also belong to the latter class of
multigene families, despite the fact that they do not encode proteins.
Members of modem multigene families can occur clustered at a specific locus on a
chromosome. For example, the genes encoding a- and l3-tubulin in some plants and metazoans are
organized as tandem repeats on a chromosome (46, 231). They can also occur at mUltiple loci on
more than one chromosome (13, 16, 46, 190, 303). Multigene families can, however, also consist
of individual genes scattered across the genome. Examples of these are the genes encoding chitin
synthases in oomycetous fungi (199) and actins in mammals (190).
3.1.2 Concerted evolution
Large-scale sequence analyses of the repeated genes constituting multigene families, have
revealed that the members of a repeat, share more similarities within a species, than between
This species-specific homogeneity is generated by a process known as 'concerted
evo lution' or 'mo lecular drive' (66, 311). There are several mechanisms through which this process
can take place e.g. unequal crossing-over, gene conversion, homologous recombination,
transposition and replication slippage. Gene conversion and unequal crossing-over are considered
the most important ofthese mechanisms.
Concerted evolution is best explained by Sanderson and Doyle (240) using a simple gene
family consisting of two members (X and Z) in each of four species (1, 2, 3, and 4) (Fig. 2A). X
and Z are paralogous genes that originated from a duplication event, prior to the radiation of species
1 - 4. The X genes in all the individuals are orthologous and trace their ancestry to a speciation
event. The same is also true for the Z genes. Phylogenetic reconstructions using either the X or the
Z genes will thus reflect the organismal evolution. However, in most cases it is impossible to
differentiate between homologous genes of paralogous and orthologous origins. In the absence of:
or prior to, homogenization (Fig. 2B), all the X genes will form a cohesive cluster, as is true for Z
genes. The lineage or species history within each of these clades, however, still reflect the 'correct'
phylogeny as depicted in the 'true genealogy' (Fig. 2A). After concerted evolution, or when it is
highly effective, all the paralogues within an individual are homogenized (Fig. 2C). In these cases,
interspecies variation by far exceeds intraspecies variation. Clearly, reconstructing evolutionary
relationships from genes that are subjected to high levels of homogenization and those where
concerted evolution is effectively absent, are relatively straightforward (52, 293).
orthologues will group together (Fig. 2B) or paralogues will be homogenized, but in both cases it
would be possible to infer the 'correct' phylogeny. However, intermediate levels of concerted
evolution introduces major complications to the inference of evolutionary relationships (183, 184,
The mechanisms through which concerted evolution take place do not exclude the potential
for 'horizontal' spread of a variant member (8, 66, 67, 120, 167). This is especially true when the
variant caries a beneficial mutation. This mutation can then be spread to all the other members of
that family, thus illustrating how a small selective advantage can become a great advantage via
concerted evolution (167). 8ince this type of mutation is not acquired through descent from a
common origin, many authors conclude that concerted evolution conceals true phylogenetic
relationships (117, 234, 240, 293). The effect of concerted evolution can be summarized most
appropriately in the words of 8chimenti (244) who states that "concerted evolution can wipe out
millions of years of divergence" or "introduce muhiple sequence changes into a member of a gene
family... in a single event".
The efficacy of concerted evolution to homogenize paralogous genes throughout in the
genome varies greatly.
In cotton (Gossypium spp.), for example, different gene families are
subjected to different levels of concerted evolution. Cronn et al. (52) showed that all the members
ofthe cotton 58 rrn family at one locus were very similar in sequence and different from the copies
at another locus. These resuhs were in contrast to those of Wendel et a1. (293) using the cotton
18S-268 rrn multigene family. They showed that all the sequenced copies of the 188-26S repeat,
whether from a single or more than one locus, had almost identical sequences. Variations in the
degree of homogenization thus occur not only among multigene families, but also among different
clusters ofthe same gene family (19,52,69, 70, 90, 114, 127, 151, 189,234, 274,293). Although
the majority of studies on the efficacy ofconcerted evolution focused on the homogenization of rrn
genes, the homogenization of protein-coding gene families is well documented (1. 47,81, 113, 114,
117, 127, 151,234,272,274).
3.1.3 Example of a protein encoded by a multigene family:
Inspection of the sequences in nucleotide databases such as GenBank: reveals that many
different protein-coding genes are currently used to address evolutionary and taxonomic issues in
diverse organisms (Table 2). The most entries for fungi are those encoding p-tubulin, translation
elongation factor la, chitin synthases, actin and glyceraldehyde-3-phosphate dehydrogenase. The
nucleotide databases for the genes encoding calmodulin, the mating type idiomorphs and histone
H3 are also relatively large.
Although the genes encoding other proteins, such as translation
elongation factor 2, H8P70 and a-tubulin are less frequently used by fungal taxonomists, they have
been successfully used in many other lower eukaryotes (Table 2). However, after the rrn genes, the
use of those encoding p-tubulin in fungi is best documented.
~- Tubulin
is one of the 50-kDa subunits of the heterodimeric protein, tubulin (46, 266).
Tubulin is the primary component of microtubules, which are the cytoskeletal filaments of
eukaryotic cells. For this reason, they are involved in determining the shape the cell and the
nucleus, as well as, as well as in cell processes such as chromosome segregation, cell division,
flagellar motility, etc. (46, 266).
From agricultural and veterinary perspectives,
is an important protein. This is
mainly due to the mode of action of benzimidazole containing fungicides and anthelmintics (54).
These drugs specifically bind to
thereby preventing the assembly of mature microtubules
and result in the inhibition of DNA synthesis (54). Single point mutations in the gene encoding
tubulin have been shown to confer resistance to benzimidazoles (7, 16, 37, 150, 220, 266, 302,
Although only one of the
loci is usually associated with benzimidazole
sensitivity or resistance, more loci can sometimes be involved (16, 266).
~- Tubulin
single genes.
is usually encoded by highly conserved multigene families or in some cases
In the best-studied higher plants, multigene families consisting of five to nine
genes have been described (108, 128, 168, 181). Similar multigene families in
animals have been reported (46, 266). Some fungi also appear to have more than one divergent
copy of the
gene. Saccharomyces cerevisiae (202), Candida albicans (257), Neurospora
crassa (220), Schizosaccharomyces pombe (121), Botrytis cinerea (304) and Fusarium species in
the Gibberellafujikuroi complex (212) all appear to have a single copy of this gene. On the other
hand, fungi such as Geotrichum candidum (101), Aspergillus nidulans (187), Colletotrichum
gleosporioides f. sp. aeschynomene (37), C. graminicola (222), Erisiphe graminis (252),
Acremonium coenophialum (278) and species in the F. solani complex (212) have at least two
different copies ofthe
gene sequence provides an excellent tool for studying phylogenetic
relationships at all taxonomic levels. This protein-coding gene has been successfully used to
determine both intra- and interspecific relationships. An example where
gene sequences
have been used at the intra-species level is in populations of the sheep gut parasite, Haemonchus
contortus (16). A well-known example where it has been used at the interspecies level is for the
molecular characterization of Fusarium species (5, 213, 216).
gene sequences have also
been used to address deep phylogeny questions. The best example is probably where this gene,
together with three other protein-coding genes, was used to demonstrate that fungi and animals are
each other's closest relatives (11).
3.2 Single copy genes
Divergence of a single copy gene and speciation are two yery close linked processes. This
is because divergence of an ancestral gene would coincide with speciation. A single copy gene
would be more 'resistant' to mutations than members of a multigene family. A lethal mutation in
one of the members of multigene family can be 'corrected' through concerted evolution.
concerted evolution fails to 'correct' the mutation, another member of the multigene family can take
on the role of the mutated gene (38, 301). A lethal mutation in a single copy gene, however, results
in death of the individual (202, 218). For this reason, non-lethal nucleotide changes in a single
copy gene will also cause changes in the individual, thus contributing to species evolution.
a single-copy gene would theoretically provide
reliable evolutionary
reconstructions than multi-copy genes (163).
Most protein-coding genes used for reconstructing phylogenetic histories, occur as
multigene families (Table 2). Some of them, however, also occur as single copy genes in lower
eukaryotes such as actin genes in certain algae, protozoans and oomycetous fungi (19, 68, 71). The
only protein-coding genes that apparently occur 'universally' as single copies, are those encoding
the largest and the second largest subunits (RPB1 and RPB2) of the DNA dependent RNA
polymerase II complex (56, 170,254). This may, however, be because of under-sampling, since the
copy numbers of these genes are seldom determined.
3.2.1 Examples of proteins encoded by single genes: largest and second largest subunits
of DNA-dependent RNA polymerase II
RNA polymerase is thought to be one ofthe earliest enzymes to have appeared (161). This
is consistent with the idea that RNA preceded DNA as genetic material.
This ancient RNA­
dependent RNA polymerase then gave rise to the modern DNA-dependent RNA and DNA
polymerases (161). The eukaryotic DNA-dependent RNA polymerases are large multi-subunit
enzyme complexes that are divided into three groups, i.e. RNA polymerase I, II and III (289).
RNA polymerase I is responsible for transcription of the 5.8S, 18S and 28S rrn genes, RNA
polymerase II transcribes nuclear protein-coding genes into mRNA and RNA polymerase III
produces tRNA and 5S ribosomal RNA (289).
The eukaryotic DNA-dependent RNA polymerases share a common ongm with the
eubacterial and archaebacterial RNA po lymerases (133). Because of this, the genes encoding their
protein-subunits closely resemble one another.
For example, the genes encoding the largest
subunits of the eukaryotic RNA polymerase I, n and In are homologous to the eubacterial
subunit (133). This homology is reflected in the nine conserved domains (I-IX) present in these
prokaryotic and eukaryotic genes.
Many researchers have indicated the potential use of the genes encoding DNA-dependent
RNA polymerase subunits in evolutionary studies (133, 161,227,254). A number of sequences for
the genes encoding the two largest subunits of RNA polyme~ase IT are available (Table 2).
However, few sequences encoding the subunits of RNA polymerase I and III are available, making
them less suitable for evolutionary analyses.
The largest subunit ofthe RNA polymerase II is encoded by the gene, RPB1, and the second
largest subunit is encoded by RPB2 (306). The GenBank nucleotide database for both these genes
is limited compared to those for other genes (Table 2), but several successes have recently been
reported on using RPBl and RPB2 gene sequences for evolutionary studies (51,56, 123, 170). Liu
et al. (170) showed that RPB2 is more useful than 18S rrn to resolve the relationships among the
different fungal orders. Croan et al. (51) showed that RPBl is useful for studying interspecific
relationships among Leishmania species. Furthermore, both these genes provide good resolution at
deeper phylogenetic levels. Denton et al. (56) reconstructed the possible phylogeny of the plant
kingdom (viridiplantae) using RPB2 gene sequence and Hirt et al. (123) placed the micro sporidia
within the fungal kingdom using RPBl gene sequence.
In recent years, protein-coding genes have increasingly been used to address phylogenetic
and taxonomic questions at all levels. These sequences have not only proven useful at deeper
(Kingdom or Division) taxonomic levels, but also at the lower (inter- and intraspecies) levels (Table
Several protein-coding genes contain sufficiently variable and conserved regions to allow
resolution at both deeper and lower taxonomic levels (Table 2). Most of the recent advances in
fungal taxonomy have, therefore, been based on sequence for protein-coding genes.
4.1 Deep level fungal taxonomy: The microsporidia-fungi relationship
Microsporidia are spore forming obligate intracellular parasites of all major animal groups
(41, 44).
Although they represent an eukaryotic lineage, the microsporidia share a surprising
number of features with prokaryotes. These include ribosomal features such as 70S rather than 80S
ribosomes and fused 5.8S and large subunit rrn genes (287). The microsporidian genomes also
correspond with those of bacteria, as they are small and rarely harbor introns (21, 83, 141). The
micro sporidia also lack eukaryotic organelles such as mitochondria (44, 141). Because of this
resemblance to prokaryotes, they were thought to represent eukaryotic lineages that evolved prior to
the acquisition ofmitochondria. They were consequently classified as Archezoa (44, 286).
The archezoan status of the micro sporidia has been supported by molecular data from the
rrn genes and those encoding the translation elongation factors, EF-la and EF-2 (139, 286). In the
microsporidian lineage, however, these genes are known to display features such as biased base
composition, unique insertions and deletions and accelerat~d rates of substitution (145).
Phylogenies based on these genes were thus not reliable (145, 226, 265), which resulted in the
erroneous placement of the microsporidia at the base of the eukaryotic tree (20, 75, 82, 122, 123,
144, 145).
Phylogenies based on a-, p-, and y-tubulin gene sequences indicated that the micro sporidia
are closely related to the fungi. Keeling et aL (145) further showed that the microsporidia evolved
from within the fungal group, sometime after the divergence of the chitrids. The idea that the
microsporidia are phylogenetically nested within the fungal kingdom is also supported by the recent
discovery of functional spliceosomal introns (20, 83). Evidence that this group of organisms once
contained mitochondria (122) also supported this finding. It thus appears that the micro sporidia
area a highly specialized fungal lineage that 'lost' many of their eukaryotic features during
adaptation to the intracellular parasitic lifestyle (20,83, 145).
The discovery of the fungal heritage of micro sporidia serves as just one example where
protein-coding sequences have been used to determine the phylogenetic position of an
evolutionarily ancient group of organisms. There are several other examples where these sequence
have been useful in reconstructing the evolutionary histories of ancient lineages (141-143). The
microsporidial example further shows that not all protein-coding genes are equally suited to address
phylogenetic questions, at all levels. In this fungal lineage, the genes encoding the translation
elongation mctors were apparently too variable, which resulted in distorted genealogies. These
genes have, however, proven useful in other fungal lineages (214, 216).
4.2 Low-level fungal taxonomy
4.2.1 Interspecific relationships
One of the best examples of a protein-coding gene being used to elucidate the relationships
among closely related species is fOund in the work of ODonnell et al. (212,213). They studied the
relationships among Fusarium species in the Gibberella fujikuroi complex using p-tubulin gene
The fungi in this complex include well-known pathogens of many important
agricultural plants (164, 165). Their classification has been hampered by the mct that they are
morphologically very similar (26, 93, 204, 209).
In the taxonomy of Fusarium species belonging to the G. fujikuroi complex, the use of
ribosomal ITS regions has proven to be problematic (212, 213). This is because they harbor non­
orthologous divergent homologues of the ITS2 region that appears to have escaped concerted
evolution (212, 213).
Apparently, these homologues were the result of an interspecific
hybridization (xenologous origin) or gene duplication (paralogous origin) event.
This event
occurred prior to the radiation of species in this complex.
The phylogenetic relationships among the Fusarium species in this complex have been
resolved using f3-tubulin gene sequences (212, 213). What was thOUght to be three to eight species
based on morphology, turned out to be more than 30 distinct Fusarium spp. (212,214,216). These
results have also been confmned using other protein-coding gene sequences, as well as
morphological characters (33, 126, 164, 209, 263). Additionally, these protein-coding sequences
have formed the basis for diagnostic techniques to identify members of this economically important
group ofplant pathogens (263).
4.2.2 Intraspecific relationships
Characterization of the intraspecific relationships among different groups of fungi is
extremely important to fungal taxonomists. Because many fungi are asexual, their populations
often constitute clones. The classification of these clones becomes increasingly important when
they are associated with the production of mycotoxins or when they are serious plant and human
pathogens (5, 42, 92, 154, 214, 270). The classification of the clonal and recombining lineages in
the aflatoxin producing fungus, Aspergillus jlavus, is one such an example. Using various protein­
coding genes, Geiser et al. (92) showed that this apparently asexual fungus is separated into groups
that correspond with their ability to produce toxin. Protein-coding gene sequences are thus valuable
tools for identifying and classifying clonal or asexual, as well as recombining fungal lineages (42,
The review of the use of protein-coding genes to study the intraspecific relationships among
populations of fungi would not be complete without reference to their value in detecting
interpopulation-recombination events. A simple method, known as 'gene-gene concordance', was
suggested to detect these recombination events (73, 74, 188, 299).
Gene-gene concordance
assesses the congruence between the phylogenetic trees constructed using several different genes.
If concordant trees are obtained from all the genes tested, it is concluded that recombination among
the individuals tested is rare. They thus represent clonal populations. Ifthe gene trees for the group
of individuals in question differ, recent recombination events among these individuals will have
occurred. Although 'gene-gene concordance' is a relatively recent introduction to fungal taxonomy
and population genetics, several authors have been able to detect sexual and asexual fungal lineages
using this approach (39,92, 154,270,271).
5 CONCLUSIONS Fungal taxonomy has entered an exciting
especially when taking into account that it is
possible to reconstruct the evolutionary history of any group of individuals by using many different
In this way, many problems associated with traditional classification (for example
morphological crypsis) have been or are in the process of being resolved using DNA sequence
information from protein-coding genes. This is especially true in cases where the rrn genes and ITS
regions display insufficient variability or where lineages are in the process of divergence.
Consequently, the available nucleotide information on many different protein-coding genes in
public domain databases is expanding continuously. Already, considerable collections of sequence
data for proteins are available for important fungal lineages such as Fusarium and Aspergillus. In
the future, these sequences will undoubtedly form the basis for DNA-based identification
techniques and classification systems.
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.' 43
Table 1. Consensus sequences for the 5'- and 3'-splice sites, as well as the putative branch sites for
the different types of spliceosomal introns (249).
Intron type
5'-Splice site I
Branch siteI
3'-splice siteI
GT-AG intron 71 6081 1__ 94 11 e\6 ___ ---- 76 91 91 99 83 --- 1176 78 79 eo eo eo so eo 82 ee87- 96_ -152-­
GT-AG intron
0000000000000 YAGIAoo
AT-AC intron
ooolATATCCTTT TTCCTTRACYCY 0000000000000 YAClooo
50 9S 90 ___ e6 95 75 es _ _ _ _ _ _ _ _ _ _ _ _ _ 95_ 96 1_ _ _
___ I_ - - - - - 96 93 70 65 95
115000 to
AT-AC intron
ARGIATAAGTooo unknown
___ I_ - e6 _ 71 ___ unknown e6
52 74 61 96 a9. 99 966B- 71
93 86
--54 - - - - - - 96 70 1
_ _ _ _ _ _ _ _ _ _ _ _ _ 96__ 154-­
_ _ _ _ _ _ _ _ _ _ _ _ _ H __
Splice junctions are indicated by vertical lines (I). Positions with no clear consensus sequences are indicated by open
circles (0), whereas R indicates either purine (A or G), Y either pyrimidine (C or T) and K either A or C. Below each
of the consensus sequences the degrees ofconservation (%) are indicated. Black dots (-) indicate 100 % conservation
in all the known sequences and horizontal lines (-) indicate the absence ofstrong conservation.
2 Dist. = distance in nucleotide bases from the putative branch site to the 3'-splice junction. nla == not available.
3 The approximate frequency at which the intron type occurs (249).
Table 2. The taxonomic level and specific problems associated with the use of selected nuclear and mitochondrial protein-coding genes in taxonomic
and phylogenetic studies.
Taxonomic levels studied
Specific problems experiencecr Species ~ Eukaryotic kingdoms (5, 11,63, 75, 92, 142145,212,213,215,216,243,245,262,278,283,290)
-Multi-copy (37, 101, 109, 112, 143,167,187,222,231,259,284)
-Lateral transfer (260)
-Distortion and lack of resolution of phylogenetic relationships due to
lineage and site-specific accelerated evolutionary rates (144, 145)
Individual ~ Eukaryotic and prokaryotic kingdoms (5,
10, 11,42,53, 135, 139, 149, 152, 157,201, 207,210,
-Multi-copy (139, 206, 233, 237)
-Distortion and lack of resolution of phylogenetic relationships due to
lineage and site-specific accelerated evolutionary rates (10, II, 123, 139,
Individuals ~ Species (140,154,191,283)
-Multi-copy (29, 192, 193, 198,199,242,301)
Elongation factor lex.
15771 Chitin synthases
272 Actin
Species ~ Eukaryotic kingdoms (4, 11, 19,68, 87, 117,
-MUlti-copy (69, 189,296, 81)
-Distortion of phylogenetic relationships because of lineage specific
nucleotide substitution rates (I, 19, 68, 11 7)
Species (2, 11, 18)
-Multi-copy (167, 300) -Distorted inter- and intrakingdom relationships because of lateral gene transfer (32,45, 84, 118, 169,258,282, 195) Calmodulin
22946 Individual
245) Mating type
Histone H3
70-kOa Heat-shock-.erotein (HSP70)
GeoBank hits1
Total 802
Eukaryotic kingdoms (11, 42, 92, 216,
-Multi-copy (162,303) Species3 (256,264,279,307)
-Sequence information from isolates with opposite mating types is not
combinable (264)
4773 Species ~ Eukaryotic kingdoms (63, 179, 262,263,272,
273,292) -MUlti-copy (79, 114, 179, 197,219,272,273,292) 482 N/u
-Multi-copy (76, 95, 196, 305, 308)
Class ~ Eukaryotic and prokaryotic kingdoms (11, 27,
77,94, 122, 157,207,245,261) -Multi-copy (261) 1140
Table 2. Continued. Protein
Specific problems experienced2 -Multi-copy (62)
Species3 (92)
Species3 (92)
Species ~ Eukaryotic kingdoms
(142, 144, 145)
-Multi-copy (143, 153, 167) -Distortion and lack of resolution of phylogenetic relationships due to lineage and site-specific accelerated evolutionary rates (144, 145) ATPase subunit 6
Species ~ Class (11, 155,207)
-Presence of hybrid genes (223) -Interspecific lateral transfer (223) 50
ATPase subunit 9
Elongation factor 2
'" O-methyltransferase
Phosphate permease
GenBank hitsr-~.. Taxonomic levels studied
Species; (92)
-Multi-copy (40, 297)
Species3 (215)
-Multi-copy (14) 93
Species ~ Eukaryotic and prokaryotic kingdoms (207,
-Multi-copy (232) 45
Species ~ Eukaryotic and prokaryotic kingdoms (116,
123, 135, 139, 157,201,207)
-Multi-copy (139) -Distortion and lack of resolution of phylogenetic relatipnships due to lineage and site-specific accelerated evolutionary rates (123) . HistoneH4
Species ~ Eukaryotic kingdoms
(142, 144, 145)
RNA polymerase II
second largest
Species ~ Eukaryotic and prokaryotic kingdoms (56,
Eukaryotic kingdoms (63, 154, 179, 272,
~ Eukaryotickingd~1lls..D2,272,
-Multi-copy (13,79,114, 186,197,219,292) -Distortion and lack of resolution of phylogenetic relationships due to lineage and site-specific accelerated evolutionary rates (I45) 280)
-Multi-copy (79, 114,148, 186, 197,219,272) Table 2. Continued.
GenBank hits 1
-'-' -
Specific problems experiencedz
Taxonomic levels studied
Species ~ Eukaryotic kingdoms (11, 48)
Nitrate reductase
Histone H2B
Species ~ Eukaryotic kingdoms (12,272)
-Multi-copy (79, 105, 114,186,197,219) Adenylate kinase
-Multi-copy (89) Eukaryotic initiation
(157) Malate
Species ~ Eukaryotic kingdoms (11, 134, 180) Glucose-6­
Species3 (92, 285)
Serine proteinase
Species3 (154)
Orotidine 5'_
Species3 (154, 230)
kinase domain of
protein kinases
RNA polymerase II
largest subunit
Species ~ Eukaryotic and prokaryotic kingdoms (15, 51,
Individuals ~ Eukaryotic and prokaryotic kingdoms (3,
86, 102, 138, 146, 155, 175, 203, 233, 241, 288)
Eukaryotic kingdoms (92, 154, 283, 309,
-Multi-copy (167) -Multi-copy (309) Eukaryotic kingdoms
Eukaryotic kingdoms (156, 245)
-Multi-copy (156) -Multi-copy (l67) -Distortion of phylogenetic relationships due to lineage specific accelerated evolutionary rates (155, 233, 298) Table 2. Continued.
, (,
GenBank bitsl
Specific problems experiencedz
Taxonomic levels studied
Eukaryotic and prokaryotic kingdoms (281)
Trichothecene 3-0acetyltransferase
Species3 (215)
ligase 8
Species3 (215) Protease I
Species3 (154) Triose phosphate
isomerase 6
Class ~ Eukaryotic kingdoms (77, 207, 208)
synthetase 6
Class ~ Eukaryotic kingdoms (77) -Multi-copy (281)
-Multi-copy(167) Aldehyde reductase
Class ~ Eukaryotic kingdoms (77,147) th
I Total number ofGenBank entries and only thOse associated with fungi (Updated November 25 2000).
2 Multi-copy refers to the presence of either non-orthologous or pseudo genes.
3 Use ofthe protein-coding gene sequence has only been tested at the level indicated by these authors. N/u
gene not used for phylogeny.
8 FIGURES .' 49
Figure 1. Intron distribution matrix for selected p-tubulin genes in eukaryotes [modified from Dibb
and Newman (58) and Liaud et al. (168)]. Intron positions and phases are indicated as defined by
Dibb and Newman (58). The intron at position 5, for example, is phase 0, which means that codon
number 5 is preceded by an intron. Phase 0 introns are indicated in blue by @. The intron at
position 10 is phase 1, which means that codon number 10 is split by an intron after the first base.
Phase 1 introns are indicated in red by
The intron at position 21 is phase 2, which means that
codon number 21 are split by an intron after the second base. Phase 2 introns are indicated in black
by (i). A vertical line (-) indicates the absence of an intron. GenBank accession numbers are
indicated in parentheses.
For the correct intron positions in Trichoderma viride (GenBank
accession number Z15054), a value of two should be added to all positions, since this strain has an
insertion of two codon residues at the beginning of the sequence. All the intron positions after
number 133 in Histoplasma capsulatum (GenBank accession number AH003038) should be
increased by one, because of a single residue insertion. All the intron positions after number 350 in
the human TUB4Q p-tubu1in gene (GenBank accession number U83668) should be decreased by
one because of a residue deletion.
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