Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.
Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.
Over thirty years ago, the discovery that eukaryotic genes were split, interrupted by
non-coding DNA (e.g. [1], [2], [3]), caused a revolution in biology. The functional and
evolutionary consequences of this unexpected gene structure were immediately
foreseen by W. Gilbert in a famous and short article, in which he coined the terms
intron and exon [4]. Since that time, the existence of introns in nuclear
genes has been the source of a long-standing -and in some aspects, still lasting-
debate. That debate concerns whether introns were present at the very beginning of
life (the so-called “intron-early theory”), or were inserted much later
in previously uninterrupted coding sequences (intron-late). Predictions of both
theories have been tested using increasingly available data, although with sometimes
opposite results. For instance, predictions of the intron-early theory regarding
intron phase distribution (an excess of phase 0 introns) were confirmed for ancient
genes, but contradictory interpretations were given by different authors, according
to the analytical models they used [5], [6], [7]. For intron-late supporters, the age of the first introns
was previously thought to be rather recent, given the initial lack of known introns
in mitochondria-lacking eukaryotes (named Archezoa [8]). Much progress in the
debate has been brought by the general effort of genome sequencing of a number of
eukaryotes and prokaryotes, which has shown that (1) all sequenced prokaryotes lack
spliceosomal introns and the elements of the splicing machinery, and (2) nearly all
eukaryotes sequenced to date have at least a few spliceosomal introns, and all have
elements of the spliceosome [9]. This demonstrates, according to many authors, that
introns have been inserted in eukaryotes, at a very early stage of their evolution,
so that all extant eukaryotes stem from an intron-bearing, and potentially
intron-rich ancestor [10], [11], [12]. Potentially intron spread was tightly linked to the
still mysterious origin of eukaryotes (e.g ref. [13]), maybe concomitant to it, and
introns may have been a powerful booster of evolutionary novelties through
exon-shuffling, alternative splicing, surveillance of mRNA integrity, promoting and
favoring recombination [14], [15], [16], [17], [18]. Indeed, as Lynch and Richardson said [16], “it is
likely that few, if any, of today's eukaryotes could survive without
introns”.The focus of the debate have shifted today to understanding intron gains and losses
in the course of the history of eukaryotes, and estimating intron density in the
genome of the last eukaryotic common ancestor. Are intron losses more frequent than
intron gains, and if so, how long has this been the case? Or, in contrast, have
gains been a high-frequency phenomenon at some time in the past, and thereafter have
decreased to become rare events at more recent periods? Are rates of gains and
losses correlated? Are rates of gains and losses lineage dependent? Genome data
increasingly show significant levels of corresponding intron positions in conserved
orthologous genes between remote eukaryotes, such as plants and animals [10], [19], [20], [21]. The extent to
which these coincidental positions reflect true orthologous introns (i.e. present in
the common ancestor) or parallel gains has been estimated by several workers, but
remains controversial (reviewed in [22]).A particular point that remains to be clarified is the gene-dependence of intron
dynamics. Many authors have emphasized the importance of introns in the functions of
a number of genes, for example because of the presence of regulatory information
within introns (e.g. [23]), or because of the size of introns, which would simply
act as timers for the proper temporal expression in the embryo development [24], or because of
their role in mRNA surveillance through the nonsense mediated decay (NMD) process
[17]. If
introns differ significantly in their functional roles, then genomic studies of
introns will tend to pool introns upon which selective forces act differently, and
differences in functional fitness consequences of intron dynamics (i.e. gain or
loss) will be overlooked.Investigating intron dynamics in a single gene (or gene family) in various species
may be valuable in this respect [25]. We compared intron-exon
structures in alpha-amylase genes of animals, to illustrate the diversity of intron
dynamics in genes with identical function in various organisms. Alpha-amylases form
a multigene family in most organisms, including animals [26], [27], [28], [29], [30], but all copies have virtually
the same function of degrading starch and related polysaccharides (http://www.cazy.org
[31]). The amino
acid sequence is poorly conserved among kingdoms [32], so that comparisons of
intron-exon structures with plants or fungi are unreliable. Although it is possible
to perform structural alignments owing to the conservation of the three-dimensional
structure, such alignments remain ambiguous and limited to small parts of the
sequence. Since studying intron gains and losses, through the conservation of intron
positions, requires unambiguous alignments, we restricted our investigation to
animal-type amylases in Bilateria, which align well to each others. No animal-type
amylase (subfamily GH13_15/24 [33]) was found to date in non-bilaterian Metazoa, i.e.
Porifera and Cnidaria [34] and Placozoa (unpublished). Instead, Fungus-type (GH13_1)
alpha-amylase, also called Dictyostelium-type, seems to be the
common and ancestral type in Unikonts, excluding Bilateria. Regarding the focus of
the present work, it is interesting to note that the animal-type alpha-amylase
studied here may be considered a “recent” gene, because it is assumed to
be of bacterial origin through horizontal transfer [34]. Thus, the ancestral structure
should have been intronless. As detailed below, the intron-exon structures are
highly diverse among animal species, even, for example, within insects. Importantly,
neither alternative splicing nor regulatory information within Amy
introns have been reported to date in amylases. Thus, amylase genes will be
considered free of this kind of constraints, so that the observed structural
differences may be interpreted more easily: intron positions, sizes, losses or gains
may be more likely related to factors linked to splicing mechanisms and
requirements, and recognition of introns and exons, or to more extrinsic factors,
such as life cycle and population size. Repeated intron losses in amylase genes have
been reported in Drosophila and other Diptera [35], [36]. This study extends the
investigation to the Bilateria.
Materials and Methods
Data collection
The animal species used in this study are listed in Table 1. To our knowledge, amylase genes are
absent in the following bilaterian species whose genome has been deciphered: the
louse Pediculus humanus, the flatworms Schmidtea
mediterranea and Schistosoma mansoni, the leech
Helobdella robusta, the aphid Acyrtosiphon
pisum. Alpha-amylase genes were either determined experimentally or
obtained from databases. In the latter case, the genome data, generally as draft
releases, were searched with TBLASTN or BLASTP [37] using an animal sequence
as a query. Given the similarity among animal amylases, we found that any animal
Amy sequence could be used. For raw traces archives data,
the best BLAST hits were assembled manually, with attention to the fact that
several gene copies may occur. For experimental work, DNAs were extracted with
classical methods. Polymerase chain reaction (PCR) amplifications were performed
using combinations of a set of primers spanning a large part of the coding
sequence, listed in Table S1. Amplification reactions were
performed with increased elongation time to allow correct elongation, even in
the presence of a further 1–2 kb of intronic DNA, in addition to the
expected spliced length. For Asterias rubens (DNA supplied by
A. van Wormhoudt, Station Marine de Concarneau), a partially amplified
Amy gene was used as a probe for screening a mini-library
The same method was applied to Ceratitis capitata and
Apis mellifera (for the latter, a genomic library was
kindly screened by M. Solignac in our laboratory). A genome walking strategy
(Universal Genome Walking kit, Clontech) was applied to Bibio
marci, Musca domestica, Spodoptera
frugiperda (DNA from Sf9 cells), Blaps mucronata,
Periplaneta americana, Dysdera crocata,
Lithobius forficatus, Corbicula fluminea,
Cerastoderma edule, Mytilus edulis,
Patella vulgata.
Table 1
Alphabetical list of the species used in this study.
: sum of several fragments belonging to several copies;
: several fragments generated using specific primers;
: personal communication of D. Chourrout and A. van Wormhoudt;
**: More detailed accession numbers may be found in Table
S4.
Lab: experimental data;DB: data from public databases;*: number of copies considered for this study;: sum of several fragments belonging to several copies;: several fragments generated using specific primers;: personal communication of D. Chourrout and A. van Wormhoudt;**: More detailed accession numbers may be found in Table
S4.
Intron identification
Putative introns were identified by translating the DNA sequences in the three
frames and comparing the results with a manual alignment of known amylase
proteins. Discrepancies and premature stops indicated the presence of introns,
the boundaries of which were marked by finding donor and acceptor sites, which
led to an in frame coding sequence. There were a few ambiguous cases in variable
regions of the protein, where the alignment was uncertain. When possible, EST
databases were used to check the predicted intron positions. Searches for
remnants of transposable elements or duplicated neighboring exons within introns
were performed using BLASTX (the introns of a single gene against Genbank, and
against the coding sequence of the same gene, respectively). Intron losses or
gains were inferred by parsimony, taking into account the phylogenetic
relationships between the species harboring an intron at a given position.
Introns were numbered, choosing intron 1 as the most widespread, conserved
position among Bilateria.
Results
Number of introns, phylogenetic distribution
We obtained 79 genomic sequences of partial or complete amylase genes from 55
Bilaterian taxa. The extent of sequence available for each gene is shown on
Figure 1. Seventy-four
intron positions were found. We did not consider introns which may be found in
the extra C-terminal domain present in some species (JLDL, unpublished). All the
positions were mapped onto the pigpancreaticamylase protein sequence (Fig. 2). Figure S1
shows the mapping of intron positions onto a protein alignment, showing the
conservation of the amylase sequence at each inferred position. Figure 1 shows the high
diversity of intron-exon structures of Amy genes among animals:
from no intron in Drosophila melanogaster to 14 introns in one
gene of Daphnia pulex. In some documented cases, there is also
a variation between gene copies of a single species: in D.
pulex, we studied three copies, Amy1,
Amy2 and Amy3, with a total of 25
positions, 21 of which were specific of one of the three copies. In the Annelide
Capitella teleta, three copies harbored a total of 7 intron
positions, four of which were copy-specific. In the amphioxusBranchiostoma floridae, there were a total of 14 positions,
among which 5 were found only in the AmyC copy. In contrast, in
vertebrates, all the intron positions were shared by all gene copies within a
species. It was for example the case in Xenopus tropicalis,
Tetraodon nigroviridis, and human (not shown).
Figure 1
Distribution and nomenclature of introns along the amylase sequences
for each gene and species of the sample.
Sequence length available for each gene was shaded in grey. Introns were
numbered according to their position, with intron 1 being the most
widespread. When structures of tandem duplicated genes were similar
(e.g. Tetraodon nigroviridis), only one was retained
for the figure. Pink circles: phase zero introns; green circles: phase
one introns; blue circles: phase two introns. The black circle in
B. floridae AmyB indicates a dubious position,
which was excluded from the study.
Figure 2
Positions of the identified intron on the typical pig amylase protein
sequence (AF064742).
Pink lines: phase zero introns; green lines: phase one introns; blue
lines: phase two introns. The signal peptide is shaded. Secondary
structures of the (β/α)8 barrel in the domain A of
the protein are indicated as orange (β strands) or red (α
helices). *: position −6 cannot be placed with accuracy, due
to the high variability of the signal sequence (see text).
Distribution and nomenclature of introns along the amylase sequences
for each gene and species of the sample.
Sequence length available for each gene was shaded in grey. Introns were
numbered according to their position, with intron 1 being the most
widespread. When structures of tandem duplicated genes were similar
(e.g. Tetraodon nigroviridis), only one was retained
for the figure. Pink circles: phase zero introns; green circles: phase
one introns; blue circles: phase two introns. The black circle in
B. floridaeAmyB indicates a dubious position,
which was excluded from the study.
Positions of the identified intron on the typical pig amylase protein
sequence (AF064742).
Pink lines: phase zero introns; green lines: phase one introns; blue
lines: phase two introns. The signal peptide is shaded. Secondary
structures of the (β/α)8 barrel in the domain A of
the protein are indicated as orange (β strands) or red (α
helices). *: position −6 cannot be placed with accuracy, due
to the high variability of the signal sequence (see text).The diversity of intron/exon structures is highest among arthropods, with
D. pulex and D. melanogaster being
representative of the two extremes. The Lepidoptera seem to have many introns in
their Amy genes, whereas Diptera have fewer. The case of
Diptera Amy genes has been detailed in [36]. This high diversity
may be not specific to arthropods, but our sample is biased; arthropods,
especially insects, are largely represented here. More comprehensive data from
other phyla might give similar results. For example, the tunicate Ciona
intestinalis has numerous introns but not
Oïkopleura, a distant Urochordate with a compact
genome [38]. On
the contrary, all vertebrates have many introns in their Amy
genes, but to our knowledge, there is almost no variation in number: 8 in
teleost fishes and 9 in other vertebrates.
The case of tandemly arranged gene copies
As mentioned above, in some species, the same intron/exon structure is shared by
the whole Amy gene family, whereas other species show striking
differences between copies. The copies that share the same structure are
generally physically close to each others, like in vertebrates. In the
amphioxus, AmyC, which has several specific introns, is
isolated, whereas the clustered AmyA and AmyB
share most of their intron positions. Divergence in structure is correlated with
divergence in sequence: AmyA and AmyB share
78% identity with each other at the protein level, but with
AmyC, only 64% (A/C) or 55% (B/C). In the
flour beetle Tribolium castaneum, four Amy
copies are tandemly arranged, all of which have the same gene structure
according to the genome annotation. Interestingly, in a previous cloning of two
tandem copies [GenBank: U04271], both copies had been found to lack
intron 5. In the whole genome sequenced, the four copies have this intron. This
suggests a polymorphic presence/absence of this intron in T.
castaneum, which has been rarely reported [39], [40], [41]. Another example is the
wasp Nasonia vitripennis: two tandem copies
Amy2 and Amy3, very close to each other,
share the same structure, whereas a third copy Amy1, found on a
different contig, has a different structure. Thus, the conservation of
intron/exon structure between gene copies, like the coding sequence, may be
linked to their physical vicinity, as expected if the duplication events are
more recent than with more distant copies. Additionally, there may be some
concerted evolution (likely gene conversion was observed between
Amy1 and Amy1' of Daphnia
pulex). However, although the intron positions are similar in
tandem copies, it is also frequent that the sizes and sequences of homologous
introns are very different (e.g. Amphioxus, Xenopus, Tetraodon). In the
centipede Lithobius forficatus, there are two close tandem
Amy copies. Intron 1 sequences (data not available for
other introns in Amy2) are of different sizes (1417 bp vs. 1130
bp) and share no similarity at all. In the tandem Amy genes of
Nasonia vitripennis, the coding sequences are 90%
identical, whereas there is no similarity in introns, except the most distal
(3′) intron 63, which is highly conserved.
Widely conserved introns and phylum-specific introns
Figure 1 shows the intron
positions for each of the sequences studied. It is immediately clear that only a
few positions are widely shared among the sample: introns 1 and 11 are shared by
various Protostomes and Deuterostomes; intron 5 may be also considered as widely
shared if it is related to the vertebrate-specific intron 4 through intron
sliding. All other intron positions are more restricted, but some are shared by
several species of a single taxonomic entity. Two positions may be considered
Protostome-specific (15 and 33); positions 17, 21, 37, 57, and 63 are specific
to Arthropods. In addition, position 42 is insect-specific; position 12 is
Coleoptera-specific; position 24 is Diptera-specific; position 46 is
Lepidoptera-specific (moths only are represented). According to our data,
mollusks-specific positions are scarce: position 62, which is also shared by the
Annelide Capitella, and position −6, which is the only
intron found in the region encoding the signal peptide. Owing to low sequence
conservation of this region, orthology of this position among species is not
sure (see Fig. 2). In
Deuterostomes, only position 13 may be considered Deuterostome-specific, i.e.
shared by several phyla. Six out of nine vertebrate intron positions are
vertebrate-specific: introns 4 (assuming this does not represent an intron
sliding event), 30, 35, 41, 50 and 55. Echinodermata are represented by a sea
urchin, a sea star and an ophiura, which share positions −4 and 6. The
Hemichordate S. kowalevskii shares with them position −4,
and potentially its specific position 6b which could be misplaced due to
ambiguous alignment. Hemichordates are the likely sister group of Echinodermata
[42].Clearly, these conclusions rely on the sampling, and could be changed with
increasing data set. For instance, a number of positions are specific to
Caenorhabditis, but other nematode species would be
necessary to clarify the phylogenetic spread of these positions within
Nematodes. More intron positions are bound to be found within most of the phyla
represented here in the future. However, the collected data already allow to
analyse some aspects of intron dynamics in Amy genes.
Phases and insertion sites of introns
All Amy introns found so far are of the usual GT-AG type. Of the
74 intron positions, 25 are in phase zero (between two codons), 27 are in phase
one (between the first et second base of a codon) and 22 are in phase two
(between the second and third base of a codon). This distribution is not
significantly different from a 1∶1∶1 proportion
(χ2 = 0.513, ns), and is different from the
usual bias observed toward phase 0 introns [6], [7]. We divided the sequence (Fig. 2) in five equal parts of
102 amino acids. The global intron distribution among these parts is not
significantly uneven (χ2 = 5.09, ns).
Intron-rich genes show a spread of the introns along the whole sequence. In
contrast there is no clear trend for intron-poor genes (one or two introns) to
have their introns located at the beginning of the sequence. However in some
cases the pattern of distribution does appear unusual: in a number of Molluscan
and Annelide sequences, most introns are concentrated in the 5′ part of
the gene, with the exception of a single 3′ one.We also studied intron insertion sites. Exonic nucleotides flanking intron
positions are not random: with observed preference for a consensus
AG/(intron)/GT [43], the so-called “protosplice site”. To
what extent this represents biases in the sites of intron creation, versus
post-insertional selection, for instance for splicing efficiency, remains
debated. For each intron position, we compared flanking exonic sequences across
genes which did and did not contain an intron – “occupied”
sites and “empty” sites (introns lost or never inserted) (Fig. 3 and Table S2).
Occupied sites showed a stronger protosplice consensus, except for phase 1
introns, for which a G/G minimal consensus was common to occupied and empty
sites, perhaps because they were inside conserved glycine codons. For phase 0
introns, the classical protosplice consensus AG/GT was found when introns were
present. This sequence was also particularly clear for the three presumably
oldest sites, i.e. positions 1, 5, 11, for which an absence of intron, supposed
to be unambiguously due to a loss, was related to a weaker consensus (Fig. 3E). This suggests a
relaxation of constraints after intron loss at these positions. We also checked
for a few “recently gained” intron positions (25a, 27, 29, 53b
pooled) whether the surrounding sequence showed a consensus motif. There was no
consensus when the intron was absent, assumed to be the ancestral state (232
occurrences, not shown), nor in the four cases of insertion (Table S2).
In these few cases, insertion was not linked to the presence of a preferential
sequence. These results are consistent with the notion that the positions of the
de novo intron insertions are largely unbiased, and that
selection drives the emergence of protosplice-like sequences following intron
insertion [44]; in addition, the finding that protosplice sites are
observed for very old introns but not for much younger ones, may suggest that
the transition to protosplice sites is a slow process.
Figure 3
Conservation of the sequences surrounding intron positions in animal
Amy genes.
Positions −2, −1, +1 and +2 relative to the introns
are shown. Intron positions in badly alignable regions were not used.
Data are from Table S2. n: number
of positions used; occupied: sites currently with an
intron; empty: homologous sites devoid of intron. A:
all intron positions; B: phase 0 positions; C: phase 1 positions; D:
phase 2 positions; E: old positions, considered as ancestrally filled
with an intron (pos. 1, 5, 11). Diagrams were made with Weblogo
(http://weblogo.berkeley.edu/).
Conservation of the sequences surrounding intron positions in animal
Amy genes.
Positions −2, −1, +1 and +2 relative to the introns
are shown. Intron positions in badly alignable regions were not used.
Data are from Table S2. n: number
of positions used; occupied: sites currently with an
intron; empty: homologous sites devoid of intron. A:
all intron positions; B: phase 0 positions; C: phase 1 positions; D:
phase 2 positions; E: old positions, considered as ancestrally filled
with an intron (pos. 1, 5, 11). Diagrams were made with Weblogo
(http://weblogo.berkeley.edu/).
Intron losses and gains; ancestral and recent introns
Investigating the rates or frequencies of intron losses and gains in
Amy genes is the main goal of this study. Relating the
presence/absence factual data to the loss/gain interpretation is mainly a matter
of sampling. From our data set, we have been able to identify numerous intron
losses at various periods in the past, ancient losses basal to a whole phylum,
or more recent, phylogenetically more limited losses. For example, Figure 1 shows that intron 1,
the most common one, is certainly ancestral in Bilateria, and has been lost
several times, in some dipteran genes, in Echinoderms, in the tunicate
Oïkopleura and in Caenorhabditis. We
focused on Drosophila Amy genes more thoroughly by PCR and we
confirmed that intron 1 was lost independently in several lineages within the
last 30 million years, but only in the subgenus Sophophora, in
which the number of gene copies is more than one (excluding the old paralog
Amyrel), whereas no intron loss was recorded in the
subgenus Drosophila, in which Amy seems to be
generally single-copy. It is possible that increased intron losses could be
related to the number of gene copies. Intron position 11 is also considered
ancestral, but would have been lost, according to the data, in Mollusks, in
Echinoderms, and in Diptera, Coleoptera and Hymenoptera. Intron 5 is also
widespread and old, since it is shared throughout Protostomes. It is also
present in the amphioxus, but since it is absent from other Deuterostomes, it is
not clear whether it is an ancestral position or represents a case of parallel
insertion, unless one considers intron 4 (vertebrates) as the result of a 5
bp-sliding from position 5, a hypothesis for which there is no evidence.The tree in Figure 4
summarizes the proposed status of lost or gained introns, according to their
phylogenetic distribution. Inferences of gains or losses on the tree reflect
parsimony reconstructions; other interpretations are plausible for a number of
positions (Table
S3) because the data suggest either massive losses or parallel gains,
which is a common issue in this kind of investigations. For instance, in Figure 4, introns 15 and 33,
which are Protostome-specific, have been considered each as cases of independent
gains. The Deuterostome-specific intron 13 was considered as two independent
gains rather than ancestral to Deuterostomes. As another example, position 20 is
shared by vertebrates and one gene of the worm Capitella. This
is more suggestive of parallel gains. Indeed, some of these uncertain cases
could be solved with an increased sample. Some cases are however clear: intron
35 has been lost in fishes since it is present in the lamprey P.
marinus and the tetrapods; intron 34 has been lost in C.
elegans, since it is present in C. briggsae,
C. brenneri, C. remanei and
Pristionchus pacificus (not shown). Other examples may be
found in Figure 1.
Figure 4
A scenario for intron gains and losses along the bilaterian
phylogeny.
Phylogenetic tree of the Bilateria species included in this study,
consensus drawn from refs. [42], [110],
[111], [112], [113],
[114], [115]. Intron
positions were placed at nodes corresponding to the putative losses (red
numbers) or gains (blue numbers), deduced from the phylogenetic
distribution of Fig.
1. Alternative possibilities are proposed on Table
S3. For clarity of the figure, the repeated losses in
Protostomes of the positions shared with Amphioxus (question mark) were
not reported. Color code for taxonomical groups is the same as in Fig. 1.
A scenario for intron gains and losses along the bilaterian
phylogeny.
Phylogenetic tree of the Bilateria species included in this study,
consensus drawn from refs. [42], [110],
[111], [112], [113],
[114], [115]. Intron
positions were placed at nodes corresponding to the putative losses (red
numbers) or gains (blue numbers), deduced from the phylogenetic
distribution of Fig.
1. Alternative possibilities are proposed on Table
S3. For clarity of the figure, the repeated losses in
Protostomes of the positions shared with Amphioxus (question mark) were
not reported. Color code for taxonomical groups is the same as in Fig. 1.Cases of gains are also likely, but most often less directly conclusive, and
there is no obvious case of “recent” gain. The seemingly high number
of gains on terminal branches of the tree (Fig. 4) does not mean that these gains are
truly recent. Intron gain cases were inferred when the phylogenetic distribution
of the intron was scarce or limited to a clade, given a correct sampling in
related taxa or phyla (see paragraph Widely conserved introns and
phylum-specific introns above). The assignment of intron losses and
gains is thus dependent on sampling. The extreme case is for introns limited to
one species or one gene in the data set. For instance, one can infer an intron
gain for position 25a in the Nematocere A. sarothamni because
the dipteran sampling is good; intron 53b may have been gained in the wasp
N. vitripennis, by comparison with other Hymenoptera
sequences. In Bivalves, the oyster and scallop sequences each show a specific
intron (27 and 40, respectively), which could be examples of species-specific
intron gains. However, this interpretation is weakened because we do not know
all Amy copies of each species. It is thus possible that these
introns are present in other copies of the other species. The most striking
example of possible gains is that of Daphnia pulex. This
species has the most split Amy gene (14 introns), and 14
species-specific positions, which can be considered gained. Indeed, many likely
paralog-specific gains are inferred. Considering Amy1 and
Amy2, which are related to each other, since they are
clustered in an animal Amy gene tree (not shown), there are
only two common positions (not shared with Amy3 nor any other
sequence), but six additional positions which are unique to
Amy1, reflecting either gains in Amy1 or
losses in Amy2. The structure of Amy3 is less
derived, sharing five introns with other Arthropods: 15, 21, 37, 52, 57, 63.
This suggests a complex structural history after gene duplications in this
lineage. However, though we are confident that these positions are gains, the
sampling for Branchiopoda is limited to this species, so that the exact
antiquity of these introns is unknown. They could also be shared by other,
unsampled Crustacean clades. Overall, we did not find conclusive cases of intron
gains more recent than 150–200 million years.In contrast, phylum-specific positions, corresponding to ancient gains stemming
from the origin of the phyla, perhaps during the Cambrian or late Precambrian
explosion, seem to be frequent. It is visually clear on Figure 1 for Deuterostomes, but also in
Protostomes. In contrast, there are few positions shared by several Protostome
phyla, or by several Deuterostome phyla. Regarding Deuterostomes, at most one
intron position is shared exclusively by them (and it may be a parallel gain).
Surprisingly, in the amphioxus, 7 of 13 positions are shared with Protostomes,
but not with other Deuterostomes (5, 31, 37, 48, 49, 54, 58).
Intron sliding
Intron sliding is suspected to have occurred when two intron positions are very
close to each other between two gene copies or two species. The phylogenetic
sample must be sufficient enough to rule out parallel insertions. In our study,
some positions are conspicuous: in D. pulex, positions −2
and −1, one base pair apart, harbored by two paralogs
Amy2 and Amy1, respectively, create each
with intron 1, a very small exon of 8 bp and 7 bp, respectively. This was
confirmed by ESTs (http://wfleabase.org/genomics/est/) and our experimental
resequencing of this gene region. Introns 8 (Amy3) and 9
(Amy2) are also one base pair apart. Rather than
independent gains in paralogous copies, both cases strongly suggest 1 bp intron
sliding, the most frequently encountered, according to some authors [20], [45]. However,
the intron sequences are very different, so that the ultimate evidence, intron
sequence similarity, is lacking. Other putative candidates for intron sliding
may be introns 44–45, or 46–47 (Fig. 2) but the phylogenetic distribution of
these positions is rather in favor of independent gains. The case of introns
4–5 was mentioned above, but cannot be solved with our data.
Discussion
The origin of animal amylases remains enigmatic. Up to now, animal-type
alpha-amylases (i.e. GH13_15/24 [33]) were found only in bilaterian metazoa. The age and the
origin of the ancestral bilaterian amylase are not well established (discussed in
ref. [34]). Recent
estimates suggest the origin of Bilateria to be rather close to the basal Cambrian
[46], [47], [48]. We have
proposed that Amy arose by horizontal transfer from a bacterium
after the split of Cnidaria [34]. This implies a massive and not so old colonization by
introns. Basu et al. [49] have shown that genes transferred to the nucleus from the
plastid precursor cyanobacterium were quickly colonized by introns. Nuclear genes of
mitochondrial origin were also shown to be colonized quickly [50]. But these events are
probably much older. Importantly, the bilaterian Amy genes cannot
be regarded as “ancient genes” such as those included in clusters of
orthologous genes (COG) and used in comparative genomic studies (e.g. [7], [10], [19], [21], [51], [52]).The simple observation of the variety of structures among the holometabolous insects
was an invitation to try reconstituting the history of intron movements, not only in
insect amylases, but also in other animals. Such data could help understanding more
general rules of intron dynamics. Focusing on this single gene, either as a
single-copy or, most often in animals, duplicated, we have identified dozens of
intron positions. It is clear that new positions will still be found by searching in
taxonomic groups not studied yet.An important result of this study is that few intron positions can be identified as
certainly ancestral, but it does not mean that the last bilaterian common ancestor
had only these relic introns. On the other hand, numerous positions may stem from
the origins of individual phyla or sub-phyla, in the late Proterozoic or during the
Cambrian, and be concomitant to the genome novelties that accompanied new bauplans
(what Babenko et al. [53] called “transitional periods of evolutionary
history”). The ancestral Amy structure would have been partly
reset in most phyla. After a burst of intron movements, especially gains, basal to
Bilateria, losses would have become predominant until now, at various rates. In
Amy genes, intron gains and losses seem to have occurred in a
temporally irregular manner. The uneven nature of these rates has been already
reported in comparative genomic studies [51], [53], [54]. This picture is akin to the
one depicted in genomic studies [22], [53] or in single gene studies [55], [56]. A majority of comparative
genomic studies have suggested excess of losses over gains over the last 500 MY (e.g
[49], [57], [58], [59], except in Fungi
[60].
However, increasing data suggest that intron acquisition is still ongoing (reviewed
in [61]). Case
studies have often shown frequent intron losses too, e.g. [35], [36], [39], [62], [63], [64], [65], [66], [67]. But in some cases, where
the sample was phylogenetically large with known divergence dates, intron gains were
found to be dominant [68].
Intron resets in Deuterostomes and the amphioxus conundrum
At the genome level, vertebrates share many more intron positions than expected
with the sea anemone Nematostella vectensis, a non-bilaterian
animal [69]
and also with the polychaete annelid Platynereis dumerilii in a
genome fragment of 30 contiguous genes [70] (we did not confirm this in
the Polychaeta Capitella teleta). This suggests that
vertebrates could have conserved ancestral exon-intron structures, while most
other phyla would show derived patterns. However, this reasoning does not hold
regarding Amy since we posit that the bilaterian
Amy gene originated from bacteria, and then was devoid of
introns in the early Bilateria. Genome data indicate that 85% of intron
positions in the amphioxusB. floridae are shared with
vertebrates [71]. Thus it was unexpected to find that seven positions
out of 13 in the amphioxus Amy genes were shared with
Protostomes, and with Protostomes only, whereas few intron positions are shared
among Deuterostomes. This situation is intriguing. This suggests either repeated
parallel gains, or retention of ancestral positions. Parallel gains are
possible: other Deuterostomes share about one Amy intron
position with Protostomes. But seven parallel gains in this single species raise
questions. On the other hand, retention of ancestral positions implies that the
last ancestor between Protostomes and Deuterostomes had Amy
gene(s) with a Protostome-like structure, which, in Deuterostomes, was retained
solely in the amphioxus; but there is no clear pattern of conservation of the
amphioxus introns with those of a subset of Protostomes. Instead, these
coincident positions are scattered among various protostome species. Many losses
are needed for this scenario. Thus, there is still little evidence for this
hypothesis. A mixed model is also likely. In any hypothesis, the observed
pattern implies numerous intron losses and gains in vertebrates, urochordates,
echinoderms. In many genes of the urochordate Oïkopleura
dioica, introns have strikingly moved even after the split from
Ciona intestinalis, so that most of them are
species-specific [72], [73]. More generally, the numerous losses and gains basal
to most phyla, which are necessary to explain the observed patterns, suggest a
positive correlation between the rates of gain and loss.
Patterns and mechanisms of intron gains and losses in amylase genes
A good phylogenetic coverage is crucial for inferring true gains. In this
respect, our best data are for insects. Recent cases of losses and gains have
been documented in the genus Drosophila, showing that losses
were eight times as frequent as gains [74]. Accordingly,
we have identified several independent losses of the same intron in
Amy genes from various Drosophila species probably less
than 20 MY ago [35], [36], [75], [76]. Regarding gains, the most recent datable gains in
insect amylases are not younger than 200 MY, given the divergence times and
origin of the holometabolous orders [77]. The gain of intron 25a
in A. sarothamni may be younger, but it is not possible to date
it. Insect Amy genes have rather undergone mostly intron losses
(Fig. 4).In contradiction with Qiu et al. [7], who assume constant rates for a given gene across all
the phyla, intron dynamics in Amy genes has been quite
different among the studied lineages, e.g. arthropods vs.
vertebrates: in vertebrates, almost no intron movement occurred since the split
of lampreys and jawed vertebrates 500 MY ago [78]. We noticed only one loss
in teleost fishes. This low variability in intron positions is considered
typical of vertebrate genomes [79], [80], although recent works suggest that changes occurred
in some gene families [81]. During the same period of time, and even shorter
regarding insects, arthropods amylases evolved a wealth on intron-exon
structures. What is the origin of such a diversity? What are the intrinsic or
extrinsic factors involved in intron losses and gains and their fixation?The most frequently assumed mechanism of intron loss considers a cDNA
intermediate produced by endogenous reverse transcriptases [51], [74], [79], [82], [83]. The cDNA is
most often a truncated, 3′ part of the spliced gene. Thus, 3′ biased
losses are expected, and intron richness should be higher by the 5′ part
of the gene [51], [84]. Our data seem to be roughly in agreement with such a
mechanism, in that there are no widely shared introns in the 3′ half of
the Amy genes, suggesting that recurrent losses may have
restricted most 3′ introns to particular clades. However, “orphan
introns” (those found only in one species or in a restricted group) also
frequently exist in the 5′ part of the gene, and in several species, a
proximal, clearly old intron was lost (e.g. intron 1), but not other, more
distal ones. This shows that the mechanism may be more complex, or use more
5′ partial retrotranscripts, with internal priming. In our study, genomic
deletions seem not to be responsible for the observed losses, since all intron
losses are accurate, removing or adding no coding sequence, contrary to what
occurred for example in the jingwei gene of Drosophila
teissieri
[40] or in
pufferfishes [80].To be transmitted to the progeny, an intron loss or gain must occur in the
germline, as pointed out earlier [63], [67], [85]. In our context of a
retrotranscript intermediate, this implies that Amy genes
experiencing intron losses should be transcribed in the germline, at least at a
basal level. Differential levels of germline expression among species could
account for the differential variability of intron-exon structure. For example,
to explain the intron-rich structures of Amy genes, with little
variability in the Lepidoptera studied here, since their divergence from each
other over 75 MY ago (dates from fossil records [77]), as compared to Diptera,
we may hypothesize an absence of germline transcription. Unfortunately, there
are little data about Amy expression in the germline for the
species studied here, except for D. melanogaster, C.
elegans and mammals (GEO profiles: www.ncbi.nlm.nih.gov/sites/entrez?db=geo; flyatlas.org). Nonetheless, supporting this hypothesis, the
comparison between Amy and its paralog Amyrel
within flies shows that while the Amy intron (intron 1) was
lost several times, there was almost no case of loss in Amyrel
(intron 17) in more than 200 species studied, diverged from 0.5 to over 80 MY
[36],
[86]. The
Flyatlas data suggest indeed that in D. melanogaster,
Amy is transcribed in testis whereas
Amyrel is not (nor in ovaries).Another factor may influence the final intron-exon pattern and intron dynamics.
The efficiency of the splicing machinery to splice out introns is linked to its
ability to recognize and identify exons or introns properly. The exon definition
model [87]
suggests strong constraints on the size of exons in vertebrates, with a critical
upper size, beyond which an exon could be misrecognized and skipped. This
implies that intron losses (and also sliding, if mediated by a loss and gain
mechanism [88], [89]) would be often counterselected. This would explain
the low variability in intron-exon structure of vertebrate genes, including
Amy. This model might apply to other species with short
exons and long introns of our sample, such as Lepidoptera or the shrimp
L. vannamei. In some other species, in contrast, introns
are numerous but very short, e.g. in D. pulex, C.
elegans and C. briggsae. In these species, the
genome of which has been sequenced, short introns are a general feature
(wfleabase.org; [90]). This could be linked to mechanistic requirement for
proper intron recognition [91]. Some species harbor Amy genes with
both short and long introns, short and long exons, probably requiring mixed
splicing recognition mechanisms. Daphnia pulex deserves
particular mention for its 7 bp and 8 bp exons. Such small exons are rare in
animals [92],
[93],
[94], [95]. Their
correct splicing might require strenghtened splicing sites and splice enhancers
[56], or
else they could be skipped. The corresponding introns −2 and −1 are
bounded by GCG/GA and CGG/TG, respectively, which are not strong, canonical
protosplices, but unknown signals may lie inside the introns surrounding these
short exons.A correlation between the intron number or length and the level of expression of
amylase could be expected as an optimization to lower the cost of transcription
in highly expressed genes [96], [97]. But in many
Drosophila species, amylase is highly expressed, irrespective of intron presence
and size (no intron in some species, one intron longer than 1,2 kb in D.
phalerata). In mammals, nine long introns are present, yet amylase
is produced at a high level. In A. thaliana, Knowles et al.
[98] also
found no relationship between intron gain or loss and gene expression.Intron richness of organisms may be influenced by generation time and cell cycle
duration, as mentioned and discussed earlier [38], [54], [72], [99]. This view assumes that fast
reproducing animals would have less and/or shorter introns, as a genome
compaction. This has been examplified in the urochordate Oïkopleura
dioica, whose life cycle is four days long [72]. However, our data on
amylase genes seem not to show a clearcut discrimination such as long-cycle
animals with many introns vs. short-cycle animals with few
introns. For instance, C. elegans probably has dozens of
generations a year, and a six-intron Amy gene (although introns
are short). The same observation applies to the moth S.
frugiperda, which has about 12 generation per year in the wild, and
yet, its amylase gene has six long introns, increasing the gene size by
150%.The influence of population size on shaping gene structures has been proposed as
a major evolutionary force [16], [100], [101]. It is assumed that many introns, considered
slightly deleterious, could have been fixed by genetic drift in the putatively
small populations occurring at the early times of eukaryote evolution,
explaining that many unicellular eukaryotes, with large population sizes,
experienced intron losses driven by purifying selection, whereas multicellular
organisms with small population sizes retained their introns, and then remained
intron-rich. Such effects may be difficult to test in Metazoa, owing to the
order of magnitude of the differences in population sizes between species, which
is not as high as between unicellular and multicellular organisms. However, it
is likely that effective population sizes are comparable, for instance, between
moths and flies, which have very different Amy gene structures.
This suggests that demographic factors had little influence in our case.Several mechanisms of intron gains have been proposed. But since intron sequences
evolve rapidly, the origin and the insertion mechanism of an intron cannot be
identified, unless the intron gain is recent enough. Indeed, we never found the
origin of gained introns in our data. We found no case of introns created by
insertion of transposable elements, although a few introns contained transposon
fragments (Table
S5). However, retrotransposable elements may act through the
synthesis of reverse transcriptase (RT) by Pol genes. As for
intron loss, RT would produce partial cDNAs, which would be reinserted in the
genome. Internal duplications at the DNA level could also be a source of introns
if correct splicing sequences were present at the duplicate ends. We found
neither trace of such intronization of duplicated exons in the putatively most
recently gained introns nor evidence of intron gain through (recent) intron
transposition between two genes or within the same gene by reverse splicing. Our
ability to detect intron transposition may be scrambled (see [99], [102]) because
sequence similarity may be shared by different introns without any direct
relationship. For example, in the moth S. frugiperda, a part of
the intron 5 of one Amy copy was a repeated element, which was
also found in introns of other genes in various Lepidoptera (not shown). Recent
works suggest that DNA repair through nonhomologous end joining could generate
introns from any template, explaining that one rarely discover a
“parental” sequence. This mechanism creates short direct repeats at
the insertion site [61]. This was found in Daphnia for very recent introns
[103].
However, these repeats may diverge quickly as time goes on, so that our data are
unable to show such traces.The protosplice is defined as a preferential target sequence for insertion,
involving the spliceosome machinery [43]. Such sequences have been
shown to be “active” as potential targets for intron insertion [104], [105].
Insertions could alternatively occur at random, with subsequent elimination of
inserts located in an environment not suitable for proper splicing, or
adaptation of the surrounding sequences to improve splicing efficiency; this
hypothesis has been infirmed by [52]. Our data show a global
preference for Amy introns to be surrounded by sequences
matching the conservative protosplice sequence AG/G; interestingly, the
surrounding bases −2 to +2 fitted the protosplice far less well in
“empty” sites (Fig.
3, Table S2). Oldest introns (positions 1, 5, 11) showed a good fit to
the sequence AG/GT, whereas we found gained intron positions for which the
surrounding sequence was completely different (Table S2).
This contrasts with ref. [20], [44] who suggested that old introns are surrounded by
sequences deviating from the protosplice consensus, contrary to more recent
introns. It may be due to the shorter time scale of our study.The multigene nature of Amy genes should not be omitted in this
discussion, since it has been proposed that intron gains are more frequent in
paralogous genes, partly due to relaxation of selective constraints on the
duplicates ([53], discussed in ref. [106]). The conspicuous case of
D. pulex could illustrate this trend. On the other hand, we
also observed more losses in Drosophila Amy genes, in species
that had several gene copies. At a rather short time scale, significant numbers
of gains and losses were found between paralogous genes in A.
thaliana
[98]. In
addition, it has been suggested that, in the case of tandemly arranged genes,
introns tend to diverge in length and sequence to prevent illegitimate
recombination [72]. We have observed such a trend for example in
vertebrates, in the amphioxus, in L. forficatus, in N.
vitripennis.In this single gene study, we have shown that contrasted intron patterns occur
even in the absence of selection for informational content in introns or
alternative splicing, but might depend on mechanistic requirements. Our data
suggest that intron dynamics is a various and changing story, which depends on
both the lineage, even at an intra-phylum scale, and the gene considered, and
also probably depends on the intron position. Hence, we share the wise
conclusion of Jeffares et al. [54]. Additional complete genomes covering much better the
eukaryotes will increasingly enable to draw a much more correct estimate of
intron dynamics at a broader time scale. In addition, comparative genomics of
related species, such as the 12 Drosophila genomes [107], [108], [109], and even
at the intraspecific level (ref. [103] shows an extraordinary snapshot of ongoing intron
gains in waterfleas) bring valuable data at a small time scale, which is the
best way of estimating current rates and mechanisms of intron gain and loss.Mapping of intron positions on a protein alignment of animal
alpha-amylases performed using CLUSTALW
[
and manually adjusted for additional sequences. Intron positions
were mapped on top of the figure, except position −6. Color code: pink
lines: phase zero introns; green lines: phase one introns; blue lines: phase
two introns. Amino acid colors are RasMol default colors. The alignment was
edited with Geneious v.5.3.6.(TIF)Click here for additional data file.List of the the most useful PCR primers used for partial amplification
of alpha-amylase genes in animals. Position is relative to the
D. melanogaster sequence. *: specific for
Drosophila Amy gene amplification excluding the
Amyrel paralog.(DOC)Click here for additional data file.Sequences surrounding intron sites. Color code is as in
Black: no intron. For unalignable positions, no corresponding intronless
site could be indicated in other genes (e.g. position 10).(XLS)Click here for additional data file.Alternative scenarii to the intron gains/losses shown on(DOC)Click here for additional data file.Accessions of(DOC)Click here for additional data file.Results of BLASTX searches in long introns of the data set. RT: reverse
transcriptase.(DOC)Click here for additional data file.
Authors: Jérôme Collemare; Henriek G Beenen; Pedro W Crous; Pierre J G M de Wit; Ate van der Burgt Journal: PLoS One Date: 2015-06-05 Impact factor: 3.240
Authors: Leandro Rodríguez-Viera; Erick Perera; Juan Antonio Martos-Sitcha; Rolando Perdomo-Morales; Antonio Casuso; Vivian Montero-Alejo; Tsai García-Galano; Gonzalo Martínez-Rodríguez; Juan Miguel Mancera Journal: PLoS One Date: 2016-07-08 Impact factor: 3.240
Figure 1
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Figure 4