Journal of Heredity 2009:100(5):618–623
doi:10.1093/jhered/esp056
Advance Access publication July 17, 2009
Published by Oxford University Press 2009.
Intron-Dominated Genomes of Early
Ancestors of Eukaryotes
EUGENE V. KOONIN
From the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health,
Bethesda, MD 20894.
Address correspondence to Eugene V. Koonin at the address above, e-mail: [email protected].
Abstract
Evolutionary reconstructions using maximum likelihood methods point to unexpectedly high densities of introns in proteincoding genes of ancestral eukaryotic forms including the last common ancestor of all extant eukaryotes. Combined with the
evidence of the origin of spliceosomal introns from invading Group II self-splicing introns, these results suggest that early
ancestral eukaryotic genomes consisted of up to 80% sequences derived from Group II introns, a much greater contribution
of introns than that seen in any extant genome. An organism with such an unusual genome architecture could survive only
under conditions of a severe population bottleneck.
Key words: effective population size, endosymbiosis, group II self-splicing introns, origin of eukaryotes, spliceosomal introns
Spliceosomal introns that interrupt the protein-coding genes
and the spliceosome, a highly complex RNA–protein
machine that mediates intron excision and exon splicing is
among the hallmarks of eukaryotes (Doolittle 1978; Gilbert
1978; Mattick 1994; Deutsch and Long 1999). All eukaryotes
with sequenced genomes, including unicellular organisms
with compact genomes, previously suspected to be intronless, have been shown to possess at least a few introns
(Nixon et al. 2002; Simpson et al. 2002; Vanacova et al.
2005) and a (nearly) complete spliceosome (Collins and
Penny 2005). The intron density in eukaryotes ranges from
a few introns per genome to more than 8 per gene (Logsdon
1998; Mourier and Jeffares 2003; Jeffares et al. 2006). The
intron content of a genome is generally thought to be
determined by the efficiency of purifying selection that itself
depends on the effective population size of the corresponding organism (Lynch 2007). The population-genetic theory
has it that, because of their large effective population size
leading to highly efficient purifying selection, most of the
unicellular eukaryotes cannot sustain more than a small
number of short introns (Lynch 2002, 2006; Lynch and
Richardson 2002; Lynch and Conery 2003). In contrast,
proliferation of introns is possible in genomes of multicellular eukaryotes (plants and animals) that have small
populations subject to relatively inefficient purifying
selection pressure but high levels of random drift.
For the first 25 years after the discovery of splicing, the
study of intron evolution revolved, primarily, around the
more or less speculative debate between the introns-early
and introns-late concepts. The introns-early hypothesis
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(more recently recast in the form of ‘‘introns-first’’) posits
that very first protein-coding genes already contained
introns that contributed to the emergence of proteins via
recombination between RNA molecules that encoded short
peptides (Doolittle 1978; Gilbert 1978; Gilbert and Glynias
1993; Gilbert et al. 1997; Jeffares et al. 2006). The intronslate concept counters that the primordial genes were
intronless, and prokaryotic genes have retained that
primitive state, whereas eukaryotic genes were invaded by
introns only after (or during) the onset of the eukaryotic
lineage (Stoltzfus et al. 1994; Logsdon et al. 1995; Logsdon
1998). Given the absence of the spliceosome and spliceosomal introns in prokaryotes, the failure of key predictions of the introns-early hypothesis, such as those on
differences in intron phase distributions among ancient and
more recent introns (Rogozin et al. 2003) and conservation
of intron positions between ancient paralogs (Cho and
Doolittle 1997; Sverdlov et al. 2007), and the uncertainty
surrounding other types of evidence, such as intron–domain
correspondence (Roy and Gilbert 2006), the original
introns-early concept does not seem to be a viable
contender anymore (Koonin 2006).
However, as far as evolution of eukaryotes themselves is
concerned, introns do seem to be a very early acquisition,
perhaps, contemporary with the origin of the eukaryotic cell,
as indicated by the presence of at least a few introns and,
most importantly, a (nearly) full-fledged spliceosome in all
eukaryotes with sequenced genomes (Collins and Penny
2005; Koonin 2006; Roy 2006; Roy and Gilbert 2006). An
intriguing question is, what were the dynamics of intron
Koonin Intron-Dominated Genomes
content over the course of eukaryotic evolution? Given that
the last eukaryotic common ancestor (LECA) was, in all
likelihood, unicellular, and most of the extant unicellular
eukaryotes have intron-poor genes, it might seem logical to
infer that LECA had a low intron density, and subsequently,
some of the eukaryotic lineages have accrued many more
introns. However, the latest comparative genomic analyses
increasingly suggest that this scenario is unlikely to be correct.
Reconstruction of Intron Gain–Loss Events
during the Evolution of Eukaryotes
Numerous intron positions in orthologous genes are
conserved at great evolutionary depths: for instance,
orthologous genes from human and Arabidopsis share up
to 25% of intron positions (Fedorov et al. 2002; Rogozin
et al. 2003). Sequencing of multiple genomes of diverse
eukaryotes created the possibility for reconstruction of
intron content in ancestral forms for which purpose maximum parsimony and maximum likelihood (ML) methods
have been applied (Rogozin et al. 2005). The maximum
parsimony approaches are straightforward but are intrinsically overreliant on the conservation of the analyzed
characters (intron positions, in this case) and might severely
underestimate the intron content in ancestral genomes.
More sophisticated ML methods that model the rates of
intron gain and loss along branches of a phylogenetic tree
have the potential to yield higher and, conceivably, more
realistic estimates. The results of such reconstructions
strongly suggest that protein-coding genes of ancient
eukaryotic ancestors including LECA already possessed
intron density comparable to that found in modern,
moderately intron-rich genomes (Csuros 2005; Nguyen
et al. 2005; Roy and Gilbert 2005a, 2005b; Carmel et al.
2007). The estimate for LECA, although characterized by
a relatively high uncertainty due to methodological reasons,
conservatively indicates the presence of at least 2 introns per
kilobase of protein-coding DNA (Figure 1). Moreover,
a recent reconstruction of intron content in the ancestors of
alveolates and chromalveolates that employed an advanced
ML technique yielded an unexpected result: It was inferred
that the ancestors of these intron-poor unicellular eukaryotes had high intron densities, comparable to those in
vertebrates, and also that LECA contained at least 3 introns
per kilobase of coding DNA (Csuros et al. 2008). Thus, the
ancestors of at least 3 of the 5 eukaryotic supergroups
(Keeling et al. 2005), Chromalveolata, Plantae, and Unikonts
(animals, fungi, and amoebozoa), most likely, had intronrich genomes (the genomic data for the remaining 2
supergroups are currently insufficient for specific inferences). Accordingly, the history of eukaryotic genes appears to
have been, to a large extent, a story of extensive loss of
ancestral introns, perhaps, punctuated with a few episodes
of major gain (Roy 2006; Carmel et al. 2007). These
conclusions, which suggest that the above estimate of at
least 2 introns per kilobase of protein-coding DNA in
LECA is highly conservative, have interesting implications
Figure 1. Intron densities in selected extant eukaryotic
genomes and inferred intron densities in ancestral genomes. The
data are from Carmel et al. (2007). Intron density is the number
of introns per kilobase of protein-coding DNA. Color code: blue,
extant eukaryotic genomes; gray, ancestral forms; and red, LECA.
For the ancestral forms, the low bound of intron density is
indicated. Species names: Anoga, Anopheles gambiae; Arath,
Arabidopsis thaliana; Aspfu, Aspergillus fumigates; Caeel, Caenorhabditis
elegans; Cioin, Ciona intestinalis; Cryne, Cryptococcus neoformis; Danre,
Danio rerio; Dicdi, Dictyostelium discoideum; Drome, Drosophila
melanogaster; Homsa, Homo sapiens; Neucr, Neurospora crassa; Plafa,
Plasmodium falciparum; Schpo, Schizosaccharomyces pombae; Strpu,
Strongylocentrotus purpuratum; and Thepa, Theileria parva.
for the genome architecture and population dynamics of
early eukaryotic ancestors.
The First Eukaryotes: Did They Have
Intron-Dominated Genomes?
Although the ultimate origin of eukaryotic spliceosomal
introns is not known with certainty, the current dominant
hypothesis has it that they are derivatives of prokaryotic Group
II self-splicing introns that gave rise both to introns and to the
active RNA moieties of the spliceosome (Lambowitz and
Zimmerly 2004; Robart and Zimmerly 2005; Martin and
Koonin 2006). Recently, this hypothesis received a strong
boost from the resolved structure of a Group II intron that
showed extensive similarities to the structures of spliceosomal
snRNAs and the ends of introns themselves (Toor et al. 2008).
It has been further proposed that the invasion of the
ancestral eukaryotic genome by Group II introns was
triggered by the mitochondrial endosymbiosis (Martin and
Koonin 2006). Under this hypothesis, the a-proteobacterial
ancestor of the mitochondria contained multiple Group II
introns (this is compatible with the relatively high
abundance of these elements in some a-proteobacteria
[Robart and Zimmerly 2005]) that became unleashed, in
part, because of recurrent release of the symbiont DNA into
the host cell. This scenario of intron invasion does not
depend on the nature of the organism that hosted the
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Journal of Heredity 2009:100(5)
Figure 2. Mean and median intron lengths in extant
eukaryotic genomes and inferred mean and median lengths of
introns in a hypothetical pre-LECA eukaryote. For each taxon,
the genome with the highest values of mean and median
lengths was chosen. The data are from the National Center for
Biotechnology Information genome division (http://
www.ncbi.nlm.nih.gov/Genomes/).
Figure 3. Approximate fraction of the genome occupied by
introns in selected extant eukaryotic taxa and in a hypothetical
pre-LECA eukaryote. For each taxon, the genome with the
highest intron content (as in Figure 2) was chosen. The data are
from the National Center for Biotechnology Information
genome division (http://www.ncbi.nlm.nih.gov/Genomes/).
mitochondrial endosymbiont, that is, whether it was a typical
archaeon as posited by the symbiotic hypotheses of
eukaryogenesis (Embley and Martin 2006; Martin and
Koonin 2006) or a distinct protoeukaryotic form as
suggested by the archezoan hypothesis (Kurland et al.
2006; Poole and Penny 2007).
As suggested by the above estimates, the invasion of
Group II introns led to a fairly high density of introns over
the supposedly brief time interval that separated the
acquisition of the mitochondrial endosymbiont from the
advent of LECA. Let us assume that the intron invasion was
instantaneous on the evolutionary scale, that is, rapid enough
to disregard intron loss and deterioration. This assumption
appears credible if the invasion was the direct consequence of
endosymbiosis (Martin and Koonin 2006). The implications
for the genome architecture of the immediate predecessors of
LECA seem to be striking. Group II introns are complex
elements that encode a large protein containing a reverse
transcriptase domain and several accessory domains; accordingly, these elements have a (nearly) uniform size of
approximately 2.5 kb (Lambowitz and Zimmerly 2004).
Thus, under the (near) instantaneous invasion scenario,
that is, assuming that intron invasion occurred faster than
substantial loss of intronic sequences, the median size of the
introns in the (pre)LECA genome would be considerably
greater than in any extant genomes, and the mean size
would be greater than that in any modern forms, with
the exception of mammals and some other vertebrates
(Figure 2). In modern bacteria, Group II introns reside,
mostly, in intergenic regions (and therefore should be more
properly regarded as retroelements rather than bona fide
introns), presumably, because, if an intron invades a functionally important protein-coding sequence, it is rapidly
weeded out by the highly efficient purifying selection that
affects large prokaryotic populations (Robart and Zimmerly
2005). By contrast, protein-coding genes of endosymbiotic
organelles, namely, fungal and plant mitochondria and plant
and algal chloroplasts, often carry bona fide, self-splicing
introns (Toro et al. 2007). The latter situation could be the
model for the events that transpired during the mitochondrial endosymbiosis except that the original intron invasion
of protein-coding regions that quickly reached the inferred
high intron density must have been a much more dramatic
event, a virtual genome catastrophe that could realize only
under the conditions of a major population bottleneck
(see Implications for Eukaryogenesis: Intron Invasion Was
Accompanied by a Major Population Bottleneck That
Enabled Key Eukaryotic Innovations). All known genomes
of nonparasitic prokaryotes possess short intergenic regions
that amount to, at most, 10–15% of the genomic sequence
and a uniform mean gene size of about 1000 nucleotides
(Koonin and Wolf 2008). Assuming that the prokaryotic
host of the mitochondrial endosymbiont and, accordingly,
the emerging eukaryote at the earliest stages of eukaryogenesis possessed the typical prokaryotic genome architecture and that Group II introns inserted randomly into
the coding and noncoding sequences, one can estimate the
fraction of its genome allotted to introns. The estimated
intron density of 2 introns per kilobase of the coding
sequence and the mean intron length of 2.5 kb yield: 5/6 0.85 5 0.71, that is, more than 70% of the genome of the
pre-LECA eukaryotes would be occupied by introns, by far
the greatest intron content compared with modern
eukaryotic genomes (Figure 3). Thus, the ancient eukaryotic
forms might have had literally intron-dominated genomes so
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Koonin Intron-Dominated Genomes
that the rest of eukaryotic evolution, with some notable
exceptions like the expansion of introns in mammals
and, possibly, some short episodes of substantial intron
gain (Carmel et al. 2007), could be a story of intron loss
and shrinkage.
It is worth noting that the above inference of the
extremely high fraction of intron sequences in the genomes
of primordial eukaryotes is predicated on the scenario under
which the primary invasion of introns antedated the
emergence of the spliceosome (Martin and Koonin 2006).
In principle, the reverse order of events is conceivable
whereby the spliceosome that, clearly, preceded LECA
(Collins and Penny 2005) evolved prior to intron invasion,
perhaps, performing a different function, and was available
to catalyze the excision of the invading Group II introns.
However, considering the apparent homology between the
catalytic snRNAs of the spliceosome and the ribozyme part
of Group II introns (Toor et al. 2008), the invasion-first
scenario appears distinctly more plausible.
Implications for Eukaryogenesis: Intron
Invasion Was Accompanied by a Major
Population Bottleneck That Enabled Key
Eukaryotic Innovations
Nonfunctional, in particular, intronic DNA is subject to
purifying selection that purges nonfunctional sequences from
genomes in large populations where the selection pressure is
strong. Nonfunctional sequences can be fixed and survive for
extended time intervals only in small populations with weak
selective pressure. According to population-genetic theory, the
condition for the fixation of introns is Ngnu , , 1 where Ng
is the effective number of genes per locus (related to the more
traditional effective population size), n is the number of
nucleotides that are required for intron splicing, and u is
mutation rate per nucleotide per generation (Lynch 2007).
Conservatively, it can be assumed that splicing requires,
approximately, 25 nucleotides/intron (Lynch 2002). We first
consider an extreme, ‘‘worst case’’ scenario under which all
the introns are acquired by the pre-LECA eukaryote, literally,
in one coup. Assuming that the organism in question
possessed, approximately, 5000 genes (a reasonable size after
the putative fusion of 2 prokaryotic genomes) with the intron
density of ;2 introns/gene (see above), we get ;10 000
introns per genome and n 2.5 105.Using a conservative
estimate of u 5 10 9 (Lynch 2007), the condition for
intron fixation after rapid invasion is Ng , , 1/nu 5 1/2.5
105 5 10 9 or Ng , , 103. According to this
estimate, the stage of eukaryogenesis evolution between the
mitochondrial endosymbiosis and the advent of LECA was
characterized by an extremely severe population bottleneck
(Figure 4). Of course, the literally simultaneous invasion of all
introns assumed in this scenario is unlikely, so this should be
regarded as an absolute low bound of Ng during eukaryogenesis. The most conservative, ‘‘best case’’ scenario would
have the introns invading one by one, each new intron
arriving only after the fixation of the preceding one. Then Ng
Figure 4. A putative major population bottleneck on the
path of evolution from mitochondrial endosymbiosis to LECA.
, , 1/nu 5 1/25 5 10 9 or Ng , , 107. Taken
literally, this scenario, with the successive invasion of all
;10 000 introns, is unlikely as well, so the actual characteristic
Ng values of pre-LECA eukaryotes, in all likelihood, lie in the
range between these estimates, presumably, below Ng ; 106,
a characteristic value in unicellular eukaryotes that prevents
massive fixation of introns (Lynch 2007).
This bottleneck that enabled the fixation of numerous,
large introns also would have been the key condition for
the other dramatic innovations associated with eukaryogenesis including extensive duplication of many key genes
(Makarova et al. 2005) and the emergence of a variety of
eukaryote-specific cellular structures. Within the framework
of the symbiotic scenario of eukaryogenesis, it appears likely
that the symbiosis directly caused the bottleneck (Martin
and Koonin 2006). Conceivably, then, the course of early
history of eukaryotes, in terms of population dynamics, can
be represented as transition from a typical prokaryotic
population, with Ng ; 109, to the bottleneck with, perhaps,
Ng ; 104 – 105 to a partial rebound reaching Ng ; 106,
a typical value for unicellular eukaryotes (Figure 4).
Conclusion
Although the introns-early theory in its original form does
not seem to be viable, self-splicing introns, in all likelihood,
existed even at the earliest stages of the evolution of life
and invaded the emerging eukaryotic genomes, giving rise
to spliceosomal introns, shortly after the mitochondrial
endosymbiosis (Koonin 2006; Koonin et al. 2006). As a result
of the invasion of self-splicing introns, the early eukaryotic
ancestor might have an unusual genome that consisted of
;70% of intronic DNA. Such a heavy burden of (at least,
originally) nonfunctional DNA could be sustained only under
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Journal of Heredity 2009:100(5)
conditions of a population bottleneck that would also
precipitate other innovations associated with eukaryogenesis.
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Funding
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Acknowledgments
I thank Igor Rogozin for help with data collection for Figures 2 and 3 and
Michael Lynch for most helpful discussions and critical reading of the
manuscript.
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Corresponding Editor: Michael Lynch
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