Phylogenetically Close Group I Introns with Different Positions among
Paramecium bursaria Photobionts Imply a Primitive Stage of Intron
Diversification
Ryo Hoshina* and Nobutaka Imamura *Department of Biomedical Science, College of Life Sciences, Ritsumeikan University, Shiga, Japan; and Department of Pharmacy,
College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, Japan
Group I introns are a distinct RNA group that catalyze their excision from precursor RNA transcripts and ligate the
exons. Group I introns have a sporadic and highly biased distribution due to the two intron transfer mechanisms of
homing and reverse splicing. These transfer pathways recognize assigned sequences even when introns are transferred
beyond the species level. Consequently, introns at homologous gene sites between different host organisms are more
related than those at heterologous sites within an organism. We describe the subgroup IE introns of two Chlorella species
that are symbiotic green algae (photobionts) of a ciliate, Paramecium bursaria. One strain Chlorella sp. SW1-ZK (Csw.)
had two IE introns at S651 and L2449, and the other strain Chlorella sp. OK1-ZK (Cok.) had four IE introns at S943,
L1688, L1926, and L2184 (numbering reflects their homologous position in Escherichia coli rRNA gene: S 5 small
subunit rRNA, L 5 large subunit rRNA). Despite locating on six heterologous sites, the introns formed a monophyletic
clade independent of other groups. Phylogenetic and structural analyses of the introns indicated that Csw.L2449 has an
archaic state, and the other introns are assumed to be originated from this intron. Some of the introns shared common
internal guide sequences, which are necessary for misdirected transfer (i.e., transposition) via reverse splicing. Other
introns, however, shared similar sequence fragments further upstream, after the insertions. We propose a hypothetical
model to explain how these intron transpositions may have occurred in these photobionts; they transposed by
a combination of homing-like event requiring relaxed sequence homology of recognition sequences and reverse splicing.
This case study may represent a key to describe how group I intron explores new insertion sites.
Introduction
Group I introns are a distinct RNA group that function
as enzymes, splicing themselves out of precursor RNA transcripts, and ligating exons (Kruger et al. 1982; Cech 1985).
To date, more than 2,000 group I introns have been identified from nuclear, mitochondrial, and plastid DNA
(Comparative RNA Web [CRW] site [http://www.rna.ccbb.
utexas.edu/]; Cannone et al. 2002). Group I introns have
a wide but sporadic and highly biased distribution in nature
and are found in seven (i.e., Opisthokonts, Amoebozoa,
Plants, Cercozoa, Alveolates, Heterokonts, and Discicristates) of the eight eukaryotic supergroups classified by
Baldauf (2003) and Bacteria and viruses (e.g., Kuhsel
et al. 1990; Yamada et al. 1994; Edgell et al. 2000; Cannone
et al. 2002; Jackson et al. 2002).
Group I introns have various lengths (e.g., 200–1,500 nt),
but primary and secondary structures, especially enzymatic
core regions, are surprisingly well conserved, which suggests that they derived from a common origin (Burke
1988; Cech 1988). In contrast, peripheral elements of the
core have various forms; these were used to subdivide introns into four major subgroups, namely IA, IB, IC, and ID
(Michel and Westhof 1990). Subsequently, another independent subgroup, IE, was created from a subset of subgroup IB introns (Suh et al. 1999). Nuclear encoding
introns belong to either subgroup IC or IE, and for unknown
reasons, they are restricted to ribosomal RNA genes
(rDNA); this differs from organelles, in which introns
are found in rRNA, tRNA, and protein-coding regions
(see CRW).
Key words: degenerative homing, intron diversification, intron
transposition, Paramecium bursaria symbiotic algae, reverse splicing.
E-mail: [email protected].
Mol. Biol. Evol. 26(6):1309–1319. 2009
doi:10.1093/molbev/msp044
Advance Access publication March 11, 2009
Ó The Author 2009. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
A distinctive character of group I introns is their mobility. Some similar introns are found in distantly related organisms. Some introns within a lineage have phylogenetic
relationships that do not correspond to their host phylogeny.
This may be due to the lateral transfer (duplication) of introns
beyond the species level. That is, group I introns are mobile
genetic elements that specialize in invading genomes of different species. They may spread by homing (e.g., Dujon
1989; Jurica and Stoddard 1999; Chevalier and Stoddard
2001) or reverse splicing (Woodson and Cech 1989; Roman
and Woodson 1995, 1998). In homing, an intron encodes
a homing endonuclease (HE) gene located at the terminus
of a peripheral helix (e.g., the tip of P1, P2, or P8). HE recognizes 14–40 specific nucleotide rows of double-stranded
DNA and then cleaves the target site, typically resulting
in a 3# single-stranded overhang. As one side tries to repair
its loss, the intron (with the HE gene) serves as the template
for repair synthesis. Reverse splicing is the inverse process
of intron splicing at the RNA level. Because additional steps
of cDNA synthesis and recombination are necessary, and
rDNA, for example, is a multicopy gene, this type of movement likely hinders intron fixation in a population or species
(Bhattacharya et al. 2005; Haugen, Simon, and Bhattacharya
2005). Recent intron phylogenetic analyses have provided
strong evidence for lateral transfer events via reverse splicing
(Bhattacharya et al. 1996, 2002, 2005). Consequently, the
highly sporadic distributions of group I introns in presentday organisms may be explained by a combination of ancestral gains, vertical inheritance with continual loss, and new
gains by horizontal transfer (e.g., Bhattacharya et al. 2005;
Haugen, Simon, and Bhattacharya 2005). However, this explanation further complicates what is known about intron
evolution. As mentioned above, group I introns are found
in diverse lineages of organisms and at extremely unbalanced
frequencies. Most nuclear-encoded group I introns are particularly dense in fungi, red algae, and chlorophyte algae
(Haugen, Simon, and Bhattacharya 2005).
1310 Hoshina and Imamura
Paramecium bursaria is a ciliate that maintains a symbiotic relationship with Chlorella-like photobionts (for reviews on Chlorella taxonomy, see Huss et al. 1999;
Krienitz et al. 2004). Although two exceptional photobionts
have been reported (Chlorella vulgaris and Coccomyxa sp.,
see Hoshina and Imamura 2008a), all other algae whose
phylogenetic placements are known belong to ‘‘American’’
(‘‘Southern’’) or ‘‘European’’ (‘‘Northern’’) groups (Hoshina
et al. 2005; Summerer et al. 2008). Both the American and
European groups belong to the Chlorellaceae (Trebouxiophyceae) and the phylogenetic relationships within this
family are still unclear; however, the rather larger genetic
dissimilarities between American and European groups
compared with their host Paramecium suggest that they
are derived from different origins as paramecian photobionts (Hoshina and Imamura 2008a). Symbiotic Chlorella
spp. demand organic nitrogen compounds (Reisser 1984;
Kamako et al. 2005) and are sensitive to Chlorella viruses,
which are abundant in natural freshwater (Van Etten et al.
1991; Yamada et al. 1991). Moreover, they have never been
collected from nature as free-living Chlorella. In other
words, both the American and European groups comprise
distinct species inhabiting the cells of P. bursaria.
We previously introduced an American group alga,
Chlorella sp. NC64A collected in NY, which was particularly
intron-rich, containing eight group I introns (Cnc. introns:
four subgroup IC and four IE) in the nuclear rDNA (Hoshina
and Imamura 2008b). These had five new insertion sites
(S1367, L200, L1688, L2183, and L2437; numbering reflects
their homologous position in Escherichia coli rRNA gene:
S 5 small subunit [SSU] rRNA, L 5 large subunit [LSU]
rRNA) and one occupied a unique site (S943) as subgroup
IE. Phylogenetic analyses revealed that the Cnc. IC introns
formed a clade with chlorophyte introns with insertions at
nine heterologous sites and all Cnc. IE introns made a robust
clade with an intron of S651 (Chlorella sp. AN 1-3) independent of other chlorophyte introns. These data could not be
rationally explained in terms of evolutionary processes.
Therefore, in the present study, we sequenced rDNA
of two strains of P. bursaria photobionts: Chlorella sp.
SW1-ZK (isolated from German P. bursaria) belonging
to European group and Chlorella sp. OK1-ZK (isolated
from Japanese P. bursaria) belonging to American group.
Our aim is to answer the following questions: 1) Does the
SW1-ZK have some introns in the LSU rDNA region?
European algae contain one intron in their SSU rDNA
(Hoshina et al. 2005; Gaponova et al. 2007; Hoshina and
Imamura 2008a), but no analysis on this intron has been
published, 2) How does the SW1-ZK introns phylogenetically relate to the Cnc. introns or other introns?, and 3)
Does the OK1-ZK also possess the same types of introns
as Chlorella sp. NC64A? This last question would help address whether these introns are stable among the algal group
even if rather distant habitats (e.g., over 10,000 km). Although group I introns, especially their enzymatic functions, are well understood (see Cech 2002 and references
therein), their roles from the perspective of evolutionary biology and the mechanisms of their diversification are poorly
understood. We introduce a set of subgroup IE introns that
cannot be explained by existing theories of intron spread
and discuss their evolutionary context.
Materials and Methods
Establishment of Algal Strains and Cultures
Photobiont strains of Chlorella sp. SW1-ZK and OK1ZK were obtained as follows. Cells of P. bursaria SW1
(German strain; see Hoshina et al. 2005) and OK1 (Japanese
strain; see Hoshina et al. 2004) were physically disrupted,
and the suspension was spread onto C medium (Ichimura
1971) agar plates. Algal microcolonies that formed were
transferred to C þ bacto-peptone (1 g l1) agar plates. These
grew into small colonies, which were subsequently transferred to C liquid medium with 2.3 mM casamino acid.
The photobionts were maintained under fluorescent illumination (16:8 h light/dark, 100 lmol photons m2 s1)
at 25 °C.
DNA Extraction, Amplification, and Sequencing
For DNA extraction, we used a DNeasy plant mini kit
(Qiagen, GmbH, Düsseldorf, Germany) according to the
manufacturer’s directions. SSU rDNA (of Chlorella sp.
SW1-ZK) was amplified by polymerase chain reaction
(PCR) with the three primer pairs SR-1/SR-5, SR-4/SR-9,
and SR-8/SR12, and LSU rDNA was amplified with the
following five primer pairs: INT4F/HLR3R, HLR0F/
LR5, HLR5F/LR8, HLR7F/HLR9R, and HLR9F/LR12k.
The PCR products were confirmed via agarose gel electrophoresis, purified via polyethylene glycol precipitation, and
then sequenced directly. A fragment of the posterior
HLR9F/LR12k product of Chlorella sp. SW1-ZK could
not be read by direct sequencing, and thus, we reamplified
this region with the primer pair HLR10F/LR12k, which was
subcloned into pGEM-T Easy Vector (Promega, Madison,
WI). The subcloning procedure defined several polymorphic sequences with indels at nucleotide position L2629,
indicating that the sequence was made unreadable by
one of the nucleotide indels. PCR and the sequencing primers are described by Hoshina et al. (2004) and Hoshina
and Imamura (2008b).
Structure Diagrams of Csw. Introns
We found two group I introns in the rDNA of Chlorella sp. SW1-ZK (Csw.) and eight in Chlorella sp. OK1-ZK
(Cok.). Secondary structure diagrams of Csw. introns were
prepared according to previously reported models (e.g.,
Lehnert et al. 1996; Haugen et al. 2002). The central catalytic intron cores (e.g., from P3 to P7) were determined
and then the remaining helices predicted by the Mfold
(Mathews et al. 1999; Zuker 2003) version 3.2 Web server
(http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/
rna-form1.cgi/) were appended to the core.
Phylogenetic Analyses
We used several group I introns belonging to subgroups IC and IE (to which nuclear group I introns belong)
to show the rough phylogenetic positions of the Csw.
introns. The secondary structure diagrams of all these
Primitive Stage of Group I Intron Spread 1311
FIG. 1.—Insertion sites of group I introns on nuclear ribosomal RNA genes of Chlorella sp. SW1-ZK and Chlorella sp. OK1-ZK. Subgroup IE
introns are in bold. The numbering reflects their homologous positions in the Escherichia coli rRNA gene.
comparative introns are published (e.g., Suh et al. 1999;
Mavridou et al. 2000; Müller et al. 2005) or are available
at CRW. We manually aligned the highly conserved (ribozymatic core) regions of the P, Q, R, S, and P3 helix elements and appended two Csw. introns and some Cok.
introns. Phylogenetic analyses were conducted with these
alignment data (supplementary fig. S1, Supplementary Material online) using MEGA version 4 (Tamura et al. 2007).
An unrooted tree was obtained by the minimum evolution
(ME) method of equally weighted P distance and then bootstrap analyses were calculated (1,000 replicates) using the
same evolutionary model.
Only subgroup IE introns were used for the posterior
analyses. The intron sequences were aligned manually, taking into account the secondary structures, and in total, 268
aligned positions (200 parsimony informative) were selected (supplementary fig. S2, Supplementary Material online). These positions contained not only the catalytic cores
of P3–P7 but also some peripheral elements. For accurate
alignment, we excluded ambiguous regions, such as the
highly diverged P9.2 region and thereafter, additional
branching elements of P2.1c (seen in S989 and S1199
introns, see Müller et al. 2005) and P1, the sequences of
which highly depend on insertion sites. Unrooted trees were
constructed using the ME method of equally weighted
P distance and the Neighbor-Joining (NJ) method of Jukes
and Cantor in MEGA and maximum likelihood (ML)
method in PAUP 4.0b10 (Swofford 2003). The best-fit evolutionary models for the ML analysis were determined via
Modeltest 3.6 (Posada and Crandall 1998) and then the best
likelihood score (ln L 5 5647.84) was obtained under the
general time reversible (GTR) þ G þ I evolutionary model
with the following parameters: nucleotide frequencies of
A 5 0.229676, C 5 0.234538, G 5 0.301781, and T 5
0.234005; substitution rate matrix of AC 5 0.8384134,
AG 5 3.9786037, AT 5 2.8529331, CG 5 1.3359373,
CT 5 6.1273343, and GT 5 1; an invariable proportion
of sites (0.090251); variable site rates assumed to follow
a gamma distribution with a shape parameter of
0.892388; and four rate categories. The bootstrap probabilities in each method were computed for 1,000 replicates.
Results and Discussion
Ribosomal DNA Sequences and Intron Detection
We established two P. bursaria photobiont strains:
the European-type Chlorella sp. SW1-ZK (Csw.) and the
American-type Chlorella sp. OK1-ZK (Cok.). For the
Chlorella sp. SW1-ZK, we obtained a 6,210-bp (or
6,209, see Materials and Methods) product between primers
SR1 and LR12k, including SSU rDNA, internal transcribed
spacer (ITS) 1, 5.8S rDNA, ITS2, and LSU rDNA
(AB437244–56). The SSU rDNA–ITS region was identical
to the uncultured algal DNA sequence (AB206547) that we
previously obtained directly from P. bursaria SW1 (Hoshina
et al. 2005). This sequence included an intron-like (324 nt)
insertion at S651. In the LSU rDNA region of the algal isolate SW1-ZK, we detected another long (330 nt) insertion at
L2449 (fig. 1). Chlorella sp. OK1-ZK had eight group I
introns with three in the SSU rDNA region (S943,
S1367, and S1512; AB162912) and five in the LSU rDNA
region (L200, L1688, L1926, L2184, and L2437;
AB437257). These insertion positions and sequences are
virtually identical to those of Chlorella sp. NC64A
(Cnc.), an authentic American strain, reported by Hoshina
and Imamura (2008b). The only difference between them
was in the L200 introns; the 89th residue (P3–P4 junction)
of Cok.L200 was A, whereas that of Cnc.L200 was a double
composition of A and C.
Structure Models of Csw. Introns
To confirm the two Csw. insertion identities, we performed a Fasta similarity search on each sequence in the
DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.
ac.jp/), which defined their similarities to some group I
introns belonging to subgroup IE. Figure 2 shows the secondary structure diagrams of Csw.S651 and Csw.L2449.
They were constructed by tracing the well-known core region (as ribozyme) and adding physically folded peripheral
loops. The structures contained helices P1–P10 and the P13
loop–loop contact segment (thought to be involved in the
tertiary interaction necessary for excising the intron from
rRNA, Cech 1990; Suh et al. 1999). Of these, the shorter
P5 helices are typical for subgroup IE introns.
Rough Phylogenetic Positions of Csw. Introns
A ME tree constructed using only the core regions of
60 nt (49 parsimony informative) sites strongly suggested
that the Csw. insertions were subgroup IE introns; sets of IE
and IC introns were clearly separated (fig. 3).
The distinctive structural difference between nuclear
rDNA introns, that is, subgroups IC and IE, is the possession (IC) or lack (IE) of an extended P5 domain. However,
recent studies have reported derived (inherited for a long
period) IC introns with shortened P5 and/or P9 domains
(Haugen, Joseph, and Bhattacharya 2004; Simon et al.
2005), resembling IE intron structure. Csw. introns had
structures that are typical of the IE subgroup (fig. 2) and
1312 Hoshina and Imamura
FIG. 2.—Structure diagrams for the Chlorella sp. SW1-ZK rDNA introns (Csw.S651 and Csw.L2449), folded according to previously reported
models (e.g., Lehnert et al. 1996; Haugen et al. 2002). Capital letters indicate intron sequences, and lowercase letters indicate flanking exon sequences.
Arrows point to the 5# and 3# splice sites. Pairing segments of P1–P10 and P13 loop–loop contact (this domain is thought to be involved in tertiary
interactions necessary for splicing from rRNA; Cech 1990; Suh et al. 1999) locations are indicated.
were phylogenetically strongly suggested to be affiliated
with the IE group. The Metarhizium anisopliae L2449 intron (Man.L2449) has an extended P5abc helix (Mavridou
et al. 2000) typical of subgroup IC introns; however, some
studies have treated it as an IE intron (e.g., Bhattacharya
et al. 2005; Simon et al. 2005). Our tree indicated that
Man.L2449 was located in an intermediate position between IE and IC. With or without Man.L2449, however,
fungal L2449 introns have commonly emerged at the base
of IE or as IC in previous reports (Nikoh and Fukatsu 2001;
Haugen, Wikmark, et al. 2005; Müller et al. 2005; Simon
et al. 2005), in agreement with our results. We also confirmed that there is no link between Csw.L2449 and
Man.L2449.
In IE, although some phylogenetic branches had lower
bootstrap support, fungal S516 and foraminiferous L1926
diverged first and then S989 of rhodophyte and fungi
branched off. The Csw. introns were included in a moderately supported (71%) clade including L1926 of diatoms,
fungal L2066, chlorophyte S516, and Cok.S943 (only
one IE Cok. intron was used in this analysis).
Phylogenetic Relationships of Csw., Cok., and Other
Introns
The second tree was constructed of only IE introns,
separate from IC and IC-like introns, to more deeply resolve
the relationships among closely related IE introns. To execute the comparison, we included wide structural elements
of the introns that might include phylogenetic information
to the alignment. The IC introns, whose structure varies,
were hinder when attempting to align with IE introns with
accuracy. These efforts were fully rewarded as the data set
included as many as 268 (200 parsimony informative) sites
of P2–P9.1 (see supplementary fig. S2, Supplementary
Material online).
Phylogenetic trees were constructed by ME (fig. 4), NJ
(supplementary fig. S3, Supplementary Material online),
Primitive Stage of Group I Intron Spread 1313
The Structural Characters of Clade A
FIG. 3.—Phylogenetic relationships of subgroup IE and IC inferred
from 60 nt sites across highly conserved sequence elements of the P, Q, R,
S, and P3 segments. An unrooted tree was constructed by the ME method
of equally weighted P distance. IE introns of Chlorella sp. SW1-ZK (Csw.)
and Chlorella sp. OK1-ZK (Cok.) are in bold. The bootstrap probabilities of
the ME analyses are given; only values above 50% support are shown.
Letters in brackets indicate taxonomic affiliations: Vc, Chlorophyceae; Vk,
Klebsormidiophyceae; Vt, Trebouxiophyceae; A, ascomycetes; B, basidiomycetes; D, diatoms; F, foraminiferans; and R, rhodophytes. The inserted
positions of rRNA sites are given after brackets.
and ML (supplementary fig. S4, Supplementary Material
online) methods. ME and NJ trees shared the nearly same
topology in that discussed below; however, ML tree had
some different branches from those in ME and NJ. For short
and highly divergent sequences, like our intron alignment,
the parameter-rich ML analyses may give incorrect topologies (Nei et al. 1998). The ‘‘simple’’ models such as the
single parameter ME or NJ methods are thought to be potentially useful for these cases (Bruno and Halpern 1999;
Piontkivska 2004). We, therefore, place more emphasis
on simple distance trees rather than parameter-rich ML tree.
The ME tree (fig. 4) showed a large but slightly supported
clade as a sister to the S989–S1199 clade (not supported in
ML, supplementary fig. S4, Supplementary Material online). We termed this ‘‘Clade A,’’ which contained three
partially to highly supported clades of chlorophyte S516
(not supported in ML, supplementary fig. S4, Supplementary Material online), fungal L2066 with L1926 of diatoms,
and ‘‘Clade B’’ composed of Csw. introns, Cok. introns, and
Chlorella sp. AN 1-3 intron (Can.S651) with six heterologous insertion sites. Cok.L1926 did not seem to have a direct relationship to the L1926 of diatoms or foraminiferans.
The tree revealed some evolutionary contexts in Clade B:
Csw.L2449 diverged first (not supported in ML, supplementary fig. S4, Supplementary Material online) and then
Cok.L2184 branched off from the other five introns of
Cok.S943, Cok.L1688, Cok.L1926, Csw.S651, and
Can.S651.
In general, distinct but similar characters, whether morphological or molecular, are indicative of a closely related
group. The same is true of introns. Group I introns that occur
at the same insertion sites (i.e., position family, as described
below) usually share similar structures (Tan 1997; Müller
et al. 2005), whereas introns in different positions but with
similar structures are often specifically related. In the case of
IE introns, S989 and S1199 members commonly possess
a branched P2.1 helix (Li and Zhang 2005; Müller et al.
2005), indicative of monophyly (Nikoh and Fukatsu
2001; Bhattacharya et al. 2001; Müller et al. 2005; fig. 4).
The IE intron analyses defined a large clade consisting of
chlorophyte S516, fungal L2066, L1926 of diatoms,
Can.S651, and the P. bursaria photobiont introns (Clade
A; fig. 4). This clade would correspond to the minor subgroup IE2 sensu Li and Zhang (2005), although the ML analysis did not support (supplementary fig. S4, Supplementary
Material online). Although we excluded the P9.2 helix region
from the phylogenetic analyses, this section contained a surprisingly consensual sequence in Clade A members; they
shared a GAGGAAAUGC (or highly similar) terminal loop
(fig. 2) with the only exceptions being the S516 introns of
Heterochlorella luteoviridis and Watanabea reniformis.
The monophyly of these two exceptions was partially indicated within the closely related S516 insertion position family, and thus, this common sequence of the P9.2 termini is
considered a symplesiomorphic character of Clade A. Such
high character conservation may be necessary for ribozyme
stability. Heterochlorella luteoviridis and W. reniformis are
phylogenetically close taxa (Huss et al. 1999; Krienitz et al.
2004), suggesting that an evolutionary change in the P9.2
terminus occurred in their common ancestor, and thereafter,
the intron with a mutated P9.2 was vertically inherited.
Six Heterologous Introns in Clade B
Subgroup IE introns have only been observed in nuclear rRNA genes, and only about 300 sequences are listed
in the CRW. Except for those in P. bursaria photobionts,
they have extremely biased insertion position distributions;
almost 90% of the introns are in S516, S989, and S1199.
Conversely, the introns of P. bursaria photobionts are rich
in original insertion positions: L1688 and L2184 are specific to American photobionts, each insertion at S943
and L2449 is specific to American or European alga as
IE subgroup, and the S651 site is only seen in European
alga and in Chlorella sp. AN 1-3, all of which formed
a clade with robust bootstrap support (Clade B in fig. 4).
This result is unusual and is discussed in detail below. Assuming that they were truly monophyletic, we measured the
variation in their primary and secondary structures. Wikmark
et al. (2007) studied fungal S1389 introns that were assumed to have diverged from a common origin; they constructed a consensus secondary structure of six S1389
introns. In the core regions of PQRS and P3, the S1389 introns had 33 invariants and 27 over-50% conserved positions out of 62 juxtaposed positions. Clade B members,
on the other hand, had higher consistency: 42 invariants
and 20 over-50% conserved positions out of the same 62
1314 Hoshina and Imamura
FIG. 4.—Unrooted ME (equally weighted P distance) tree of selected IE introns. Introns of Chlorella sp. SW1-ZK (Csw.) and Chlorella sp. OK1ZK (Cok.) are in bold. Bootstrap values above the internal nodes were inferred from ME (left) and NJ of Jukes and Cantor (right) analyses, whereas ML
bootstrap analyses of GTR þ I þ G evolutionary models selected via Modeltest 3.6 (Posada and Crandall 1998) are shown below the nodes; only values
above 50% support are shown. Letters in brackets indicate taxonomic affiliations: Vc, Chlorophyceae; Vk, Klebsormidiophyceae; Vt,
Trebouxiophyceae; A, ascomycetes; B, basidiomycetes; D, diatoms; F, foraminiferans; and R, rhodophytes. Intron insertion positions are given on
the right side of the vertical bars.
positions (data not shown). In addition, the P6 region of
Clade B members had highly different structures. Some
IE introns had a longer P6 with a GNRA tetraloop at the
termini. GNRA tetraloops are frequently observed in group
I introns and other large self-assembling RNA molecules
(e.g., Woese et al. 1990; Bhattacharya et al. 2001), and
the tertiary interactions of those that reside at the P5 terminus of IC introns are well studied (e.g., Jaeger et al. 1994;
Cate et al. 1996; Costa and Michel 1997). Longer P6 regions were seen in fungal and chlorophyte S516, L1926
of diatoms, and Csw.L2449 (fig. 5). However, Cok.L2184
had a slightly shorter P6, and the other Clade B members
had a much shorter conserved P6. Moreover, some termini
substituted and lost the GNRA motif (Cok.L2184 and
CokL1926). Although the tertiary contact region of the
P6 GNRA tetraloop among IE introns is not yet known, this
may represent a loss event of a tertiary interaction and, further, may imply the ancestral status of Csw.L2449 among
Clade B members. If so, how did the ancestral Csw.L2449
spread into six different positions, as seen in Clade B?
Position Family, the Theory of Intron Spread
With increasing data on group I intron sequences in
GenBank, many phylogenetic analyses have been conducted to better understand intron evolution. One important
Primitive Stage of Group I Intron Spread 1315
FIG. 5.—Structural variations in P6 helices. Fungal and chlorophyte S516, L1926 of diatoms, and Csw.L2449 share longer helices with a GNRA
tetraloop (boxed), whereas others have shorter helices with or without the GNRA tetraloop. Csw. and Cok. are abbreviations for Chlorella sp. SW1-ZK
and Chlorella sp. OK1-ZK, respectively.
discovery is that introns at homologous gene sites are related (i.e., belong to the same position family), even among
distantly related host organisms (Bhattacharya et al. 1994,
1996; Hibbett 1996; Nikoh and Fukatsu 2001). This phenomenon is linked to intron spreading mechanisms.
Lateral transfer of group I introns may be due to homing
or reverse splicing. Despite a lack of any direct evidence of
intron movement via reverse splicing from genetic crosses,
there is no other explanation for transposition (except among
neighboring sites; see Haugen, Reeb, et al. 2004), due to sequence recognition differences. HEs require a longer sequence of 14–40 nt, which strongly prevents transfer
among heterologous sites, whereas reverse splicing may occur with small sequences of 4–6 nt. This region is called the
internal guide sequence (IGS); it constructs the P1 helix (fig.
2), which is spliced from rRNA, andis a targetfor lateral transfer (with or without transposition) via reverse splicing. Most
of the 5# flanking sequences of S989 and S1199, which
formed a clade in the present study (fig. 4), shared a common
IGS of –CAGGT– (see the group I intron sequence and structure Database [GISSD], http://www.rna.whu.edu.cn/gissd/;
Zhou et al. 2008). This is in agreement with previous studies
(Bhattacharya et al. 1996, 2002, 2005), indicating that they
may have been derived from a common ancestor and currently reside at two heterologous positions, caused by reverse
splicing events with transposition.
Among Clade B members with six heterologous insertion sites, L2184 and L1926 shared a common IGS of –
CTCTT–and L1688 and S943 appeared to share a shorter
IGS of –CGT– (see Cnc. introns; Hoshina and Imamura
2008b). A part of Clade B introns are, consequently,
thought possibly to be incorrect duplications via reverse
splicing. However, the others did not have a common
IGS. Therefore, reverse splicing alone is not enough to explain the entire intron lineage of Clade B.
Scheme of Group I Intron Evolution
It is not clear why introns appear to have evolved into
five major subgroups. And after the primal diversification,
introns have spread into many genic loci (multichannelization). In IE introns, for example, more than 10 (exclusive of
the introns of P. bursaria photobionts’) insertion sites have
been found (see CRW) and of those most heterologous introns do not share a common 5# flanking sequence. Studies
on the evolution of group I introns have been hard to put to
reply how the introns gained such various insertion positions (e.g., Nikoh and Fukatsu 2001; Bhattacharya et al.
2002, 2005; Wikmark et al. 2007). Introns with many insertion positions can be understood to have undergone the
stages shown in figure 6. In stage I, an ancestral group I
intron diversified into the five subgroups currently
1316 Hoshina and Imamura
FIG. 6.—Schematic model of group I intron evolution with implied
insertion position diversification, showing seven insertion positions.
L2184 originated from L2449 via an unknown pathway, and L1926 is
thought to be an incorrect duplication via reverse splicing. S287
(Bhattacharya et al. 2002, 2005) and S989 (this study) are also attributed
to transpositional movements via reverse splicing, starting from S1199.
recognized. In stage II, each intron identified acceptable
gene insertion sites (e.g., S516, S1199, and L2449 of rRNA
genes); it is possible that some younger introns originated in
this stage from older introns (e.g., Csw.L2449 might generate Cok.L2184). Finally, in stage III, incorrect duplication, though rare, occurred via reverse splicing (S1199
spread into S287 [Bhattacharya et al. 2002, 2005] and also
into S989, as described above; Cok.1926 might be derived
from Cok.L2184). Intron spreading (homing or reverse
splicing) without transpositional events is rather ordinary,
helping to consolidate position families.
Based on the above scheme, Clade B can be explained by
stage II and III diversifications. Csw.L2449 explored position
L2184 by an unknown mechanism. Cok.L2184 then duplicated within the genome and explored position L1926 via
reverse splicing and other sites by an unknown mechanism.
Possibility of Degenerative Homing
No recognized pathway can explain how introns gain
new insertion sites or transposed heterologous sites (e.g.,
stage II in fig. 6), although Belfort and Perlman (1995) reported an important possibility that allows for illegitimate
recombination, namely, intron transposition by a degenera-
tive homing event requiring relaxed sequence homology.
When considering Csw.L2449 and Cok.L2184 from this
perspective, although they do not share a common IGS,
we unexpectedly detected similar sequence blocks of –
GRAGCT–, –GGYGYCRG–, and –AAA(T/-)TACCAC–
in upstream regions (fig. 7). Likewise, similar sequence
blocks (–TAGAAYAA– and –GYCGGYRAAAT–) were
seen between L2184 and L1688. A shorter region
(–GG(A/-)AGTCGGC–), but with higher homology, was
also found between Cok.L1688 and Csw.S651. That is,
a specific six-nucleotide-long sequence emerges stochastically once every 4 kb, and a seven-nucleotide-long sequence emerges once per 16 kb. Such similar sequence
blocks occurring at almost the same position relative to intron insertions are therefore unlikely to be coincidental.
More noteworthy is that three (or more) nucleotides following insertions were identical. These data suggest the possibility of degenerative homing events.
HE genes promote intron homing, and HE proteins are
classified into four major families: LAGLIDADG, His-Cys
box, GIY-YIG, and HNH (Jurica and Stoddard 1999 and
references therein). The GIY-YIG endonuclease has the
most flexible sequence recognition, that is, I-TevI (T4 phage
GIY-YIG endonuclease) allows for 15% degenerative substrate (Jurica and Stoddard 1999). Further, the cleavage and
intron insertion sites are well separated, resulting in a long
sequence recognition (not palindromic) up to 40 bp (Bryk
et al. 1993). If an HE has promoted the transpositional
transfer of Csw.L2449 into Cok.L2184 and subsequent
spreads, it might analogize GIY-YIG type endonuclease
with further flexible sequence recognition.
The alternation of sequence recognition with HE genes
or mutations has been partially simulated (Chevalier et al.
2003; Haugen and Bhattacharya 2004; Haugen, Reeb, et al.
2004). Each member of the HE family, thought to have
evolved from a common origin, has a variety of recognition
sites. For example, the His-Cys box family is exclusively
associated with nuclear rDNA group I introns (more than
20 introns reported) and possesses five different recognition
sites (S516, S943 and nearby sites, S1199, S1506, and
L1926 and nearby sites), depending on each HE-containing
intron (Haugen, Reeb, et al. 2004). This indicates that HE
has evolved by dramatically changing its sequence recognition and may involve intron diversification at an early
stage.
HE genes are selfish genetic elements that invade noncritical regions of introns and regions between introns
(Haugen, Reeb, et al. 2004; Haugen, Wikmark, et al.
2005). Moreover, the introns that include the HE gene were
FIG. 7.—Sequences near intron insertion sites. Similar sequence fragments (highlighted) are found further upstream and after insertions. Identical
nucleotides are marked with asterisks. The sequences of these regions are common in Chlorella sp. SW1-ZK and Chlorella sp. OK1-ZK.
Primitive Stage of Group I Intron Spread 1317
FIG. 8.—Evolutionary context of subgroup IE introns found in
Paramecium bursaria photobionts (outline characters: Chlorella sp.
SW1-ZK; solid characters: Chlorella sp. OK1-ZK) with six heterologous
insertion sites. This scheme is based on hypothetical transposition
pathways of degenerative homing (DH) and reverse splicing (RS).
once fixed in the population; the HE genes no longer have
active functions and are ultimately lost (Goddard and Burt
1999; Haugen, Reeb, et al. 2004). Only a tiny minority of
introns contain the HE gene. Therefore, the fact that Clade
B members do not contain any HE gene-like sequences
does not contradict the hypothesis. Rather, Cok.L2184
and Cok.L1926 have long (more than 130 and 180 nt) insertions at P1 and P8 (see Cnc.L2184 and Cnc.L1926,
Hoshina and Imamura 2008b). Such long sequence insertions (.50) at peripheral helices can be regarded as HE
gene remnants (Haugen, Reeb, et al. 2004).
Conclusion
Most group I introns that share a common insertion
site are phylogenetically more closely related than are
the hosts of these introns. However, we identified a deviant
group in which six heterologous subgroup IE introns in two
algal photobionts (European-type Chlorella sp. SW1-ZK
and American-type Chlorella sp. OK1-ZK) of P. bursaria
and one in Chlorella sp. AN 1-3 formed an independent
clade. This result is of essential importance in understanding the evolution of group I introns. These heterologous introns were likely partly duplicated by a primitive
mechanism of intron spread, that is, degenerative homing
with a succession of recognition sequences. The introns
share similar sequence fragments upstream of the insertions
and a few residues after insertions, which could be ideal
recognition sequences for HE. Although the introns of photobionts do not contain intact HE or HE-like gene sequences, some retain unusual extra sequences (might be HE gene
wreckage). These findings represent the first strong evidence for the multichannelization strategy of group I intron
insertion positions (fig. 6).
The above hypothesis and the phylogenetic relationships suggest the following scenario for their evolutionary
context, as shown in figure 8: an ancestral group I intron
infected a highly acceptable L2449 site (Jackson et al.
2002; Simon et al. 2005) of European alga via an unknown
pathway; the L2449 intron laterally transferred into the
novel site L2184 of American alga via degenerative homing; this L2184 intron was copied and transferred within the
genome via degenerative homing (into L1688) and via reverse splicing (into L1926); finally, the L1688 intron was
copied to the American S943 site via reverse splicing and
laterally back to the European genome via degenerative
homing as a novel intron, S651. Neither European nor
American algae live freely in natural water sources due
to their nutritional requirement and the viral sensitivity
(as mentioned above). However, the completion of the hereditary line from L2449 to S651, including two transfer
events beyond the species barrier, might have needed a special situation where two algae have frequently contacted
each other. It is just conceivable that there was a period during which the two photobionts lived sympatrically, simultaneously in the P. bursaria cell (double-photobiont
association in P. bursaria has been known to occur; Nakahara
et al. 2004). Such a situation where cell–cell contact within
a small space may accelerate the lateral transfer of group
I introns (Bhattacharya et al. 1996; Friedl et al. 2000). In
the future, these introns may be duplicated into other organisms via mechanisms such as homing or reverse splicing.
Indeed, Can.S651 may be a first step in the formation of
a new position family of introns.
Group I intron has yet to be found in P. bursaria genome. Intron transfer is rather ordinary among ingroup taxa
(e.g., among green algae); however, intron transfer straddling different Kingdoms is extremely rare. Lichen photobionts (e.g., Trebouxia species, Trebouxiophyceae) are
a treasury of group I introns, and these introns have been
suggested their frequent lateral transfer (due to cell–cell
contact, Friedl et al. 2000). Although such transmissions
have continuously undergone in the small space of filamentous tissue of lichen-forming fungi, any algae-derived intron
(in phylogenetic sense) has not been discovered from the
host fungi. These facts imply that existing introns are hard
to transfer beyond higher biological ranks in nature.
Then how have the organisms gained initial introns?
Viruses possibly play a part in the intron transfer (Yamada
et al. 1994; Bhattacharya et al. 1996; Nishida et al. 1998;
Friedl et al. 2000). A few kinds of viruses possess group I
intron, of which only eukaryotic virus is Chlorella virus
(see GISSD). This virus, surprisingly, infects only the photobionts of P. bursaria. The intron sequences of Chlorella
virus are phylogenetically related to eukaryotic introns assigned to subgroup IC (Yamada et al. 1994; Nishida et al.
1998); however, the introns are not close to IC introns of
American-type photobiont (contains four IC introns, mentioned above) or of other green algae (Hoshina and Imamura
2008b). IE-type intron has not been documented from viral
genomes. Whether or not Chlorella virus truly mediates intron transfer remains to be seen.
1318 Hoshina and Imamura
Supplementary Material
Supplementary figures S1–S4 are available at Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
We sincerely thank Mr T. Nishimura (Nagahama Institute of Bio-Science and Technology) for preparing
E. coli-competent cells, Dr T. Sasaki (Tokyo Institute of
Technology) for kindly providing many copies of articles,
and Dr S-i. Kamako (Ritsumeikan University) for help in
establishing algal cultures.
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Charles Delwiche, Associate Editor
Accepted February 27, 2009