This document discusses evidence of lateral transfer of a group IE intron between fungal and red algal small subunit rRNA genes. It finds that a group IE intron inserted at position 989 in the nuclear SSU rRNA gene of the red alga Hildenbrandia rubra is closely related to similar fungal IE introns, providing evidence the intron was laterally transferred rather than vertically inherited. Phylogenetic analysis of intron sequences and comparisons of intron secondary structures support a relationship between the red algal intron and fungal introns, making lateral transfer the most likely explanation for the intron's presence in H. rubra.

1. EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL AND
RED ALGAL SMALL SUBUNIT rRNA GENES1
Kirsten M. Mu¨ller2
, Darlene W. Ellenor
Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada
Alison R. Sherwood
Department of Botany, University of Hawaii, Honolulu, Hawaii 96822, USA
Robert G. Sheath
Provost’s Office, California State University San Marcos, San Marcos, California 92096, USA
and
Jamie J. Cannone and Robin R. Gutell
Institute of Cellular and Molecular Biology and the Section of Integrative Biology, University of Texas at Austin,
Austin, Texas 78712, USA
A previous study of the North American bioge-
ography of the red algal genus Hildenbrandia noted
the presence of group I introns in the nuclear small
subunit (SSU) rRNA gene of the marine species
H. rubra (Sommerf.) Menegh. Group IC1 introns have
been previously reported at positions 516 and 1506
in the nuclear SSU RNA genes in the Bangiales and
Hildenbrandiales. However, the presence of an un-
classified intron at position 989 in a collection of
H. rubra from British Columbia was noted. This in-
tron is a member of the IE subclass and is the first
report of this intron type in the red algae. Phyloge-
netic analyses of the intron sequences revealed a
close relationship between this IE intron inserted at
position 989 and similar fungal IE introns in posi-
tions 989 and 1199. The 989 IE introns formed a
moderately to well-supported clade, whereas the
1199 IE introns are weakly supported. Unique
structural helices in the P13 domain of the 989
and 1199 IE introns also point to a close relation-
ship between these two clades and provide further
evidence for the value of secondary structural char-
acteristics in identifying homologous introns in ev-
olutionarily divergent organisms. The absence of
the 989 IE intron in all other red algal nuclear SSU
rRNA genes suggests that it is unlikely that this in-
tron was vertically inherited from the common an-
cestor of the red algal and fungal lineages but rather
is the result of lateral transfer between fungal and
red algal nuclear SSU rRNA genes.
Key index words: fungi; group IE intron; Hi-
ldenbrandia; lateral transfer; small subunit rRNA
gene; Rhodophyta
Abbreviations: LSU, large subunit; MP, maximum
parsimony; NJ, neighbor joining; SSU, small sub-
unit
All group I introns have the same chemical reactions
to splice the intron out of the exon; at the reactive sites,
group I introns are also similar in shape and confor-
mation. Although a subset of these group I introns ex-
cises from the exon without the presence of proteins,
the remainder needs various proteins to facilitate their
excision (Cech 1990). The core of the group I intron
structure contains base pairings, helices, and loops that
are common to all group I introns. In contrast, the
structural elements beyond this core adopt various
forms (Burke et al. 1987, Michel and Westhof 1990).
These different structural arrangements have been
categorized into 12 subclasses (IA1-IA3, IB1-IB4,
IC1-IC3, ID, and IE), based on conserved primary
and secondary structural elements (Michel and West-
hof 1990, Suh et al. 1999). Introns interrupt many
different genes, including the small subunit (SSU) and
large subunit (LSU) nuclear and mitochondrial rRNA
genes in fungi and protists and other nuclear, mi-
tochondrial, and chloroplast genes of plants and photo-
synthetic protists (Belfort 1991, Wilcox et al. 1992,
Van Oppen et al. 1993, Bhattacharya et al. 1994, 1996,
2001, De Jonckheere 1994, Yamada et al. 1994, Hib-
bett 1996, Johansen et al. 1996, Takashima and
Nakase 1997, Suh et al. 1999, Cannone et al. 2002).
Group IC1 introns have been reported to interrupt
the nuclear SSU rRNA genes of several genera within
the Rhodophyta. These introns have only been
1
Received 12 August 2003. Accepted 9 December 2004.
2
Author for correspondence: e-mail kmmuller@sciborg.uwaterloo.
ca.
380
J. Phycol. 41, 380–390 (2005)
r 2005 Phycological Society of America
DOI: 10.1111/j.1529-8817.2005.03146.x

2. observed in positions 516 and 1506 (Escherichia coli
numbering) of the bangialean genera Bangia and
Porphyra (Stiller and Waaland 1993, Oliveira and
Ragan 1994, Oliveira et al. 1995, Mu¨ller et al. 1998,
2001) as well as at position 1506 of the florideophyte
Hildenbrandia rubra (Sommerfelt) Meneghini (Ragan
et al. 1993, Sherwood and Sheath 1999). In their
biogeographic and systematic study of the genus Hi-
ldenbrandia in North America, Sherwood and Sheath
(1999) noted the presence of an additional intron in
position 989 in the nuclear SSU rRNA gene of Hi-
ldenbrandia rubra collected from British Columbia,
Canada. Our analysis of this intron in the 989 position
revealed sequence and structural features characteris-
tic of the IE subclass, first described by Suh et al.
(1999) from an analysis of an intron in position 989
in the nuclear SSU rRNA of the ascomycete fungus
Cryptendoxyla hypophloia Malloch et Cain. Suh et al.
(1999) also characterized other intron sequences be-
longing to the subclass IE that were previously noted
in the Chlorophyta (green algae) (Kranz et al. 1995,
Krienitz et al. 1996), numerous ascomycete and bas-
idiomycete fungi (Suh and Sugiyama 1994, Haase
et al. 1995), and the fungal endosymbiont of a lichen
(DePriest and Been, 1992).
Although group IE introns contain the structural
elements P, Q, R, and S observed in all group I introns,
the consensus sequences for these regions differ con-
siderably from those of the other subclasses (Cech
1990, Suh et al. 1999). In addition, IE introns are
shorter and contain a unique P13 domain thought to
be involved in the tertiary interactions with the P9.1a
domain necessary for excising the intron out of the
rRNA gene (Cech 1990, Suh et al. 1999). This is the
first report of a IE intron in a red alga; no IE introns
have been reported in the more than 900 nuclear en-
coded SSU or LSU rRNA gene sequences in the Rho-
dophyta (all known orders) that are publicly available
from GenBank (http://www.ncbi.nlm.nih.gov/) and the
Comparative RNA (CRW) Site (Cannone et al. 2002,
http://www.rna.icmb.utexas.edu).
There are two possible explanations for the pres-
ence of the IE intron in H. rubra. First, the 989 IE in-
tron was present in the nuclear SSU rRNA gene of the
common ancestor of the red algae and was vertically
inherited by H. rubra and subsequently lost in all other
red algal lineages. A second explanation is the lateral
transfer of the 989 intron from another organism to
position 989 in the nuclear SSU rRNA gene of
H. rubra. Lateral transfer of introns between organ-
isms as well as organelles has been noted previously
and appears to be much more widespread than initially
hypothesized (Turmel et al. 1995, Bhattacharya 1998,
Nishida et al. 1998, Watanabe et al. 1998, Suh et al.
1999, Friedl et al. 2000, Bhattacharya et al. 2001). The
present study addresses the origin of the IE intron in-
serted at position 989 of the nuclear SSU rRNA gene
of H. rubra using phylogenetic analyses of introns and
SSU rRNA coding regions and structural analysis of
the intron sequence.
MATERIALS AND METHODS
Sequences of the group I introns and the nuclear SSU
rRNA genes used in the present study are listed in the Ap-
pendix. The nuclear SSU rRNA genes including the IC1 and
IE introns from H. rubra (BC2) from British Columbia (upper
tide pool in mid section of rocky headland, Snickett Park,
0.14 km west of Ocean Avenue and Boulevard, Sechelt, B.C.,
Canada) were amplified and sequenced according to the proto-
cols outlined in Sherwood and Sheath (1999), and the Gen-
Bank accession numbers for these sequences are also noted in
the Appendix.
Sequence alignment and secondary structure models. The
rRNA and intron sequences were aligned manually with
the alignment editor ‘‘AE2’’ (developed by T. Macke, Larsen
et al. 1993) that runs on the Solaris operating system on SUN
Microsystems workstations (SUN Microsystems, Santa Clara,
CA, USA). Nucleotides in the different rRNA and intron se-
quences that map to the same locations in the secondary and
tertiary structure models are aligned in the same column in
the alignment with the alignment editor AE2. Regions of the
rRNA and intron sequences with significant amounts of sim-
ilarity can be aligned with only the nucleotide sequence in-
formation. However, sequences with considerable variation
can only be aligned accurately and confidently when other
information such as secondary and/or tertiary structure in-
formation is included. The rRNA and intron secondary struc-
ture models were initially predicted with covariation analysis
(Gutell et al. 1985, 1992, Michel and Westhof 1990). Ap-
proximately 97%–98% of the 16S and 23S rRNA base pairs
predicted with comparative analysis are present in the high-
resolution crystal structures from the 30S and 50S ribosomal
subunits (Gutell et al. 2002).
The secondary structure diagrams for the H. rubra nuclear
SSU rRNA gene were templated from the other Eukarya nu-
clear SSU rRNA genes (Cannone et al. 2002), whereas the sec-
ondary structure diagrams for the group IC1 and IE introns
were determined from the original IC1 (Michel and Westhof
1990) and IE (Suh et al. 1999) structure models and the larger
collection of group I intron structure models (Damberger and
Gutell 1994, Cannone et al. 2002). The 18S rRNA and group I
intron secondary structure diagrams were drawn with the in-
teractive secondary structure program XRNA (developed by
B. Weiser and H. Noller, University of California, Santa Cruz).
This version of the program was written in C programming
language to run under the Solaris operating system on SUN
Microsystems workstations. However, this version of XRNA
has been replaced with a platform-independent version devel-
oped in Java (http://rna.ucsc.edu/rnacenter/).
An alignment with representative group IE and IC1 se-
quences that contain the major forms of IE and IC1 intron
structure and sequence variation was prepared. These introns
occur at several IE insertion sites, three in the SSU rRNA and
four in the LSU rRNA. The four IC1 introns are evenly split,
with two occurring in the SSU rRNA and two in the LSU rRNA.
This alignment, with 51 sequences, was used for the structural
and phylogenetic analyses in this study. Although there are
more than 200 IE introns available in GenBank, phylogenetic
analyses used representative sequences (based on secondary
structure). Future analyses will analyze all IE introns; however,
this will require considerable additional time. The alignment
and secondary structure diagrams generated for this project
are available online at http://www.rna.icmb.utexas.edu/
PHYLO/HILDINT/. A sampling of the phylogenetically and
structurally diverse SSU rRNA and group I intron secondary
structure models are available at the Comparative RNA website
(http://www.rna.icmb.utexas.edu, Cannone et al. 2002).
Test for mutational saturation and phylogenetic analyses. Be-
fore performing the phylogenetic analyses with the intron
LATERAL TRANSFER OF IE INTRON 381

3. sequences, the data were analyzed to determine whether they
encoded a significant phylogenetic signal. To do this, the data
set of 51 group IC1 and IE intron sequences was used to es-
tablish the extent of superimposed substitutions. Uncorrect-
ed distances were plotted against those corrected with the
simple DNA substitution model HKY-85 (Hasegawa et al.
1985) to detect mutational saturation with respect to transi-
tions and transversions (Daugbjerg and Andersen 1997,
Lopez et al. 1999). Phylogenetic analyses of both the nucle-
ar SSU rRNA gene and group I introns were carried out us-
ing only well-aligned (homologous) regions of the sequences.
The group I intron alignment consisted of 25 group IC1 and
26 IE intron sequences, resulting in an alignment with a total
length of 2296 characters. The considerable length of this
alignment is due to the inclusion of gaps to correctly align
homologous regions. For example, the group IC1 intron in
Porphyra spiralis var. amplifolia Oliveira Filho et Coll has a
large insertion (approximately 430 nt) not observed in any of
the remaining sequences in the alignment. Hence, gaps
needed to be added to all other sequences to account for
this nonhomologous region and to align homologous regions
properly. The nonhomologous regions are considered au-
tapomorphies and hence are excluded from phylogenetic
analyses, which were based on a final alignment of 391 char-
acters. This alignment was subjected to a pair-wise distance
analysis using PAUP, version 4.0 beta 10 (Swofford 2003), and
incorporating the HKY-85 model (Hasegawa et al. 1985) with
equal rates of change across sites and a transition/transver-
sion ratio of 2. The resulting distance matrix was used to
build a neighbor-joining (NJ) tree. The data were then sub-
jected to bootstrap resampling (1000 replicates).
Modeltest, version 3.06 (Posada and Crandall 1998) was also
used to examine 56 possible models of DNA substitution and
identify the model that best fit the intron data set. The model
selected was the general time-reversible model GTR þ I þ G
(Rodrı´quez et al. 1990) that calculated the base frequencies
(A 5 0.2268, C 5 0.2688, G 5 0.2999, T 5 0.2045) and the
gamma distribution shape parameter (a 5 0.6025). This mod-
el was used to calculate maximum likelihood trees and boot-
strap values (1000 replicates) for groups identified by the HKY-
85 distance tree and provided support for different groups
within the intron tree using an evolutionary model that was
parameter rich. Parsimony analysis was also carried out using
PAUP with a heuristic search under the constraints of random
sequence addition (100 replicates), steepest descent, and tree-
bisection-reconnection branch swapping, with bootstrap re-
sampling (1000 replicates). Phylogenetic trees for the intron
data sets were all mid-point rooted because of the absence of a
suitable outgroup. PAUP was used to search for the best max-
imum likelihood tree with and without constraint conditions,
and likelihood values for all trees were then compared using
the Kishino-Hasegawa test (Kishino and Hasegawa 1989) to
test for significant (Po0.05) differences among them.
Phylogenetic analyses of the nuclear SSU rRNA gene from a
diverse group of eukaryotes were similar to those described
above for the IE and IC1 introns. The nuclear SSU rRNA gene
alignment consisted of 41 sequences, resulting in an alignment
with a total length of 2639 characters, including alignment
gaps. However, this alignment was reduced to 1660 characters
because some sequences were not complete and nonhomolo-
gous regions were excluded. Subsequently, this alignment was
subjected to a pair-wise distance analysis, using the HKY-85
model with equal rates across sites and a transition/transversion
ratio of 2. The resulting distance matrix was used to build a NJ
tree. The data were then subjected to bootstrap resampling
(1000 replicates). The Modeltest program was again used to
identify the model that best fit this data set. The model selected
was the general time-reversible model GTR þ I þ G that calcu-
lated the base frequencies to be A 5 0.2652, C 5 0.2051,
G 5 0.2683, T 5 0.2615. Bootstrap analysis (1000 replicates)
under this model was also used to calculate support for the
groups noted in the HKY-85 NJ tree. The nuclear SSU rRNA
gene alignment was also analyzed using maximum parsimony
(MP) under the same criteria noted previously for the intron
alignment. The data were then subjected to bootstrap resam-
pling (1000 replicates). The nuclear SSU rRNA gene sequenc-
es from Homo sapiens Linnaeus and Xenopus laevis Daudin were
used as outgroups for these analyses.
RESULTS
Analyses of the intron structures. The secondary
structure of the intron at position 989 (E. coli num-
FIG. 1. Comparative secondary structure models for the group
IE intron at position 989 in the nuclear SSU rRNA gene of Hi-
ldenbrandia rubra (BC2). The 50
and 30
splice junctions are marked
with arrows. G:C and A:U base pairs are connected with short lines;
G:U base pairs with ‘‘ Á ’’; other noncanonical (non-Watson Crick)
base pairs with a ‘‘ .’’ The P2.1 domain is shaded (see Fig. 2).
KIRSTEN M. MU¨LLER ET AL.382

4. bering) in the nuclear SSU rRNA gene of H. rubra
(Fig. 1) is similar to the IE secondary structure
diagrams available at the Comparative RNA website
(Cannone et al. 2002, http://www.rna.icmb.utex-
as.edu/). Group IE introns have only been observed
in the rRNA genes. A total of 242 (as of July 2004) of
these has been identified in the SSU rRNA genes,
whereas 19 occur in the LSU rRNA genes. They are
distributed among 10 unique rRNA insertion sites, 6
in the SSU rRNA gene and 4 in the LSU rRNA gene.
Approximately 41% of the IE introns occur at SSU
rRNA position 516, another 41% at SSU rRNA posi-
tion 1199, 9% at position 989, and the remaining 9%
occur at seven other different SSU and LSU rRNA
positions. Of the 261 known IE introns, 84% (219)
occur in fungi, with 91% of these being in the As-
comycota, 7% in the Basidiomycota, and 2% in the
Chytridiomycota. The remaining 16% (42) are dis-
tributed among six phylogenetic groups, with most
being in the Chlorophyta (Cannone et al. 2002). All
24 IE introns at position 989 (except for H. rubra)
occur in the ascomycete fungi. Ninety-two of the 104
IE introns at position 1199 are also in ascomycete
fungi, 10 are in basidiomycete fungi, and 2 are in
Chytridiomycota fungi.
A visual examination of the alignment and second-
ary structure diagram of the IE intron in H. rubra and
other IE introns reveals more similarity between the
989 SSU introns and most of the 1199 SSU rRNA in-
trons than between the 989 introns and the remainder
of the IE introns (not shown). In addition, the H. rubra
IE intron shares more secondary structural character-
istics with all the ascomycete fungi IE introns that in-
terrupt the SSU rRNA at position 989 and the
ascomycete and basidomycete fungi IE introns at po-
sition 1199. The most striking characteristic is the set of
three helices, P2.1a, P2.1b, and P2.1c, flanking the P13
helix in the 989 and 1199 SSU rRNA introns (Fig. 2,
b–e), which differs considerably from that of the other
IE introns (Fig. 2a, represented by Metarhizium an-
isopliae (Metschnikoff) Sorokin). The first helix, P2.1a,
is situated between the P2.1 helix and the third insert-
ed helix, P2.1c, whereas the second inserted helix,
P2.1b, caps this structural element. Helix P2.1a is con-
served in length, with 7 bp in the 989 and 1199 IE
structures (Fig. 2, b–e). The second inserted helix,
FIG. 2. Gallery revealing similarities and differences in the P2.1 domains (shaded in Fig. 1) in secondary structure models of four
group IE introns. Each helical region is labeled; only the 50
half of P13 is shown. (a) Metarhizium anisopliae var. anisopliae LSU 2066
(Fungi); (b) Cryptendoxyla hypophloia SSU 989 (Fungi); (c) Exophiala nigra SSU 989 (Fungi); (d) Hildenbrandia rubra SSU 989 (red algae); (e)
alignment of the P2.1 domain of eight representative group IE introns. Helices are boxed and connected by lines; only the 50
half of P13
is shown. Parentheses enclose hairpin loops. Species names are abbreviated, and the intron positions in the rRNA genes are shown in
parentheses. Crypt.hypo., Cryptendoxyla hypophloia; Cryph.paras., Cryphonectria parasitica; Gaeum.gram., Gaeumannomyces graminis var. tritici;
Exo.nigra, Exophiala nigra; Hild.rubra., Hildenbrandia rubra; Metar.anis., Metarhizium anisopliae var. anisopliae; Scyta.dimi., Scytalidium
dimidiatum; Skele.pseu., Skeletonema pseudocostatum; Tille.flav., Tilletiopsis flava.
LATERAL TRANSFER OF IE INTRON 383

5. P2.1b, is also conserved in length, 4 bp (Fig. 2, b–e),
with the 50
half of the helix typically consisting of
purines (primarily G residues). The 50
half of the P13
helix follows the P2.1b helix and the 30
half of the P13
helix caps the P9.1a helix (Fig. 1). This helix ranges in
length from 5 to 8 nt within the IE introns, although 7
bp is the typical length. Of the three inserted helices,
P2.1c is the most variable in length, ranging from 15 to
36 bp. Thus far, these helices are only present in the IE
introns inserted into the nuclear SSU rRNA gene at
positions 989 and 1199. Thus, this structural element
is common to the IE introns at position 989 and 1199
and distinguishes them from other IE introns. These
‘‘signature’’ secondary structural elements are homo-
logous among the 989 IE introns in Hildenbrandia and
various fungi.
Phylogenetic analyses. Before conducting phyloge-
netic analyses, the intron data were assessed for mu-
tational saturation to ensure they encoded significant
signal. In this graphical analysis, curvature of the line
at higher uncorrected distances indicates mutational
saturation (Moritz et al. 1992). For transversions cur-
vature was observed at a distance of 0.2, indicating a
minor level of mutational saturation, whereas for
transitions it was 0.15, thereby indicating an even
lower level of mutational saturation (not shown). This
suggests that group I introns encode a significant
amount of phylogenetic signal that can be used to
estimate evolutionary relationships.
Parsimony analysis of 246 phylogenetically inform-
ative characters of the group IC1 and IE introns re-
sulted in 108 most-parsimonious trees (not shown)
with a length of 1749 and a consistency index of
0.35. These 108 trees differed in topology because of
unresolved relationships among clades of introns from
different insertion sites as well as relationships among
intron sequences within an insertion site. Despite the
low resolution, MP of the group IC1 and IE intron se-
quences is similar in some respects to the distance
analyses and hence only the NJ tree is shown, with
parsimony bootstrap values (Fig. 3). Maximum likeli-
hood trees (not shown) were computed for the best
tree without constraints as well as the best tree with an
enforced or constrained topology (forcing the IE in-
tron in Hildenbrandia as a sister group to the IE introns
in position 989 and forcing the IE intron in Hi-
ldenbrandia to group with the IC1 introns in position
1506 in H. rubra). Maximum likelihood analyses with-
out the constrained topology resulted in two trees with
a likelihood value of 4123.77. These placed the 989 IE
in H. rubra basal to the other 989 IE introns in the
fungi, as in the first constrained topology noted above,
and the likelihood values for the constrained analysis
were therefore identical to the nonenforced topology.
However, maximum likelihood analysis (likelihood val-
ue 5 4223.99) of the enforced topology of the IE in-
tron in H. rubra and the 1506 introns in the same
species were significantly different from the noncon-
strained likelihood tree. The Kishino-Hasegawa
test showed a significant difference (Po0.05) between
the two trees, with the nonconstrained tree being the
favored hypothesis.
Figure 3 depicts the NJ tree derived from the dis-
tance analysis of a representative set of group IE and
IC1 introns from nuclear SSU and LSU rRNA genes
used in the present study. The IC1 introns form a
moderately to weakly supported clade (83% bootstrap
[HKY-85], 67% bootstrap [GTR þ I þ G], ando50%
bootstrap [MP]) and the IE introns form a separate
weakly supported clade (64% bootstrap [HKY-85],
o50% bootstrap [GTR þ I þ G], and o50% bootstrap
[MP]). The introns within both of the subgroups (IE
and IC1) appear to form moderately to well-supported
clades based on the insertion position within the SSU
rRNA or LSU rRNA genes. For example, the IC1 in-
trons inserted at position 943 of the SSU rRNA gene
form a clade that is well supported by HKY-85 boot-
strap analysis (100%) and parsimony bootstrap (96%),
although it is less so with GTR þ I þ G bootstrap anal-
ysis (76%). Similarly, the IE intron in H. rubra (BC2),
which is inserted at position 989 of the nuclear SSU
rRNA gene, forms a moderate to well-supported clade
(100% bootstrap support [HKY-85], 64% bootstrap
support [GTR þ I þ G], and 99% bootstrap support
[MP]) with the IE introns inserted at position 989 in
the ascomycete fungi Exophiala nigra (Issatschenko)
Haase et de Hoog, Cryptendoxyla hypophloia, Cordyceps
coccidiicola Kobayasi, and Xylaria polymorpha (Persoon)
Greville (Fig. 3). Within this clade, the H. rubra (BC2)
intron is most closely associated with the 989 intron in
E. nigra with weak support (60% [HKY-85] and 72%
[MP]). The IE introns inserted at positions 989 and
1199 of the nuclear SSU rRNA gene form a clade
moderately supported by HKY-85 bootstrap analysis
only (77%). Interestingly, all the IE introns at position
1199 occur in the nuclear SSU rRNA genes of different
fungal lineages. Although the IE intron in H. rubra is
strongly associated with other IE introns inserted at
position 989 (see above), it is clearly distinct from the
well-supported clade of H. rubra IC1 introns at position
1506 of the nuclear SSU rRNA genes (92% bootstrap
[HKY-85] and 63% bootstrap [GTR þ I þ G]) (Fig. 3).
Phylogenetic analysis of the nuclear SSU rRNA
genes in red algae and other eukaryotes (Fig. 4) shows
two distinct and well-supported clades. The Rho-
dophyta, Plasmodiophorida, Alveolata, Chlorophyta,
and vascular plants (Viridiplantae) form one moder-
ately supported clade (72% bootstrap support [HKY-
85], 71% bootstrap support [GTR þ I þ G], and 75%
bootstrap support [MP]) that is a sister group to the
FIG. 3. Neighbor-joining tree constructed using corrected
distances (HKY-85 model) of IE and IC1 introns inserted in
the nuclear rRNA genes of different eukaryotes. Numbers above
branches represent distance bootstrap values (1000 replicates)
using the HKY-85 model (first number), distance bootstrap val-
ues using the GTR þ I þ G model (second number), and boot-
strap values using maximum parsimony (third number).
Branches with an asterisk or lacking a value had less than 50%
support in that analysis. The insertion site in the LSU or SSU
rRNA genes are indicated to the right of the taxa.
KIRSTEN M. MU¨LLER ET AL.384

6. LATERAL TRANSFER OF IE INTRON 385

7. solid fungal clade (100% bootstrap in HKY-85,
GTR þ I þ G, and MP) (Fig. 4). Recent and more ex-
tensive phylogenetic analyses suggest that the red al-
gae share a most recent common ancestry with green
algae and land plants (Burger et al. 1999, Moreira
et al. 2000). These analyses of the host nuclear SSU
rRNA gene phylogeny suggest it is unlikely that this
intron was vertically inherited from the common an-
cestor of the red algae and fungi because these lineages
only share a very distant evolutionary relationship, and
this would require multiple losses of the intron over
many distinct phylogenetic groups. This is further
supported by the limited distribution of the intron in
each of the host nuclear SSU rRNA gene lineages.
DISCUSSION
The secondary structure and phylogenetic analyses
of the intron sequences in the present study strongly
suggest that the group IE intron inserted at 989 in the
nuclear SSU rRNA gene of H. rubra is homologous to
the fungal IE introns inserted at position 989 in the
SSU rRNA gene. In addition, our analysis revealed a
close relationship between the IE introns inserted at
positions 989 and 1199. The IE introns at these two
positions form moderate to well-supported clades that
are closely associated in all phylogenetic analyses and
contain structural signatures in the P2.1 domain.
Similar ‘‘signature’’ structural characteristics have
been noted in other group I introns and are indicative
of homology. For example, Mu¨ller et al. (2001) noted
secondary structure elements characteristic of the
group IC1 intron inserted at position 516 and 1506
(nuclear SSU rRNA gene) in members of the Bangiales.
The P5b domain contains a signature bifurcated helix
that distinguishes the 516 IC1 introns in the Bangiales
from all other group IC1 introns. The conserved P8
domain was variable in length within the 1506 IC1 in-
tron in the Bangiales, which also differentiated these
introns from those in the 516 position in the Bangiales
and from all other IC1 introns (Mu¨ller et al. 2001).
Mu¨ller et al. (2001) concluded that these structural and
sequence signatures provided further evidence that the
IC1 introns in positions 516 and 1506 of the nuclear
SSU rRNA gene in the Bangiales were probably the
result of a single lateral transfer event and subsequent
vertical inheritance. Bhattacharya et al. (2001) also not-
ed the bifurcated helix in the P5b domain of a group
IC1 intron inserted at position 516 (nuclear SSU rRNA
gene) of the alga Aureoumbra lagunensis D. A. Stockwell,
DeYoe, Hargraves et P. W. Johnson (Pelagophyceae).
Based on this secondary structure element and phylo-
genetic analyses, they concluded that the 516 IC1 intr-
ons in the Bangiales and Aureoumbra are specifically
related, although their host cells are not (Bhattacharya
et al. 2001). Mu¨ller et al. (2001) postulated that these
structural signatures might provide a means for deter-
mining lateral transfer events of group I introns across
a wide phylogenetic range.
In addition, introns that occur at the same insertion
site within the rRNA gene are usually more similar to
one another than they are to introns at other positions
in the rRNA genes (Tan 1997). Hence, introns at the
same insertion site are thought to have a common an-
cestry. These introns may be traced back to one or
more events in which they were inserted into the rRNA
and vertically inherited or lost (Bhattacharya et al.
1994, Tan 1997, Mu¨ller et al. 2001). For example,
Bhattacharya et al. (1994) noted that bootstrap analy-
ses yielded little support for the single origin of all
group I introns at different insertion sites within rRNA
in the green algal order Zygnematales. Mu¨ller et al.
(2001) noted that the group IC1 introns inserted at
positions 516 and 1506 (nuclear SSU rRNA gene)
within the Bangiales formed two separate yet distinct
clades. In the protist Acanthamoeba, high sequence di-
vergence among introns in four different insertion po-
sitions in the rRNA genes suggests that the intron
acquisition occurred independently at these four sites
after divergence of the taxa within the tree (Schroeder-
Diedrich et al. 1998). High sequence similarity be-
tween introns would be consistent with descent from
an ancestral intron that was initially acquired and ver-
tically inherited (Schroeder-Diedrich et al. 1998).
Hence, because of similarities in sequences and speci-
fic structural elements in the IE introns inserted at po-
sitions 989 and 1199, these two intron families may be
specifically related. It has been postulated that lateral
transfer and vertical inheritance have contributed
extensively to the evolution of group I introns (Sogin
and Edman 1989, Tan 1997).
The distribution of group I introns is 1) not uniform
across the nuclear, chloroplast, and mitochondrial gen-
omes; 2) present in a wide range of phylogenetically
distant taxa; and 3) within many different exons.
Hence, group I introns appear to be very successful
at invading and maintaining their sequences among a
diverse group of eukaryotic organisms (Burke 1988,
Dujon 1989, Bhattacharya et al. 1994, 2001, Turmel
et al. 1995, Tan 1997, Bhattacharya 1998, Nishida
et al. 1998, Watanabe et al. 1998, Suh et al. 1999,
Friedl et al. 2000, Mu¨ller et al. 2001, Cannone et al.
2002). However, group I introns are relatively rare in
the bacterial phylogenetic group. Most of these, all be-
longing to the IC3 subgroup, occur in select tRNA
genes of the cyanobacteria (Xu et al. 1990, Rudi et al.
2002). In addition, group I introns occur in three bac-
terial rRNAs: Simkania negevensis K. D. Everett, R. M.
Bush et A. A. Andersen (Everett et al. 1999); Coxiella
burnetii (Derrick) Philip (Seshadri et al. 2003); and
Thermatoga naphthophila Y. Takahata, M. Nishijima, T.
FIG. 4. Neighbor-joining tree constructed using corrected
distances (HKY-85 model) of the nuclear SSU rRNA gene se-
quences from different eukaryotes. Numbers above branches
represent distance bootstrap values (1000 replicates) using the
HKY-85 model (first number), distance bootstrap values using
the GTR þ I þ G model (second number), and bootstrap values
using maximum parsimony (third number). Branches with an
asterisk or lacking a value had less than 50% support in that
analysis.
KIRSTEN M. MU¨LLER ET AL.386

8. LATERAL TRANSFER OF IE INTRON 387

9. Hoaki et T. Maruyama (Nesb and Doolittle 2003). In
sharp contrast, there are approximately 1025 docu-
mented introns that are associated with approximately
2000 known fungal nuclear encoded nuclear SSU
rRNA genes (Cannone et al. 2002). The mechanism
of lateral transfer between organisms is an important
process that is still poorly known (Woodson 1996,
Friedl et al. 2000). It has been suggested that the lat-
eral transfer of group I introns between distantly re-
lated organisms may be facilitated by viruses, because
group I introns that have high sequence similarity oc-
cur at the same position in the rRNA genes (Yamada
et al. 1994, Bhattacharya et al. 1996, Nishida et al.
1998, Nozaki et al. 1998, Friedl et al. 2000). Close
cell-to-cell contact occurring in a mutualistic or para-
sitic relationship may enable the viral-mediated trans-
fer of group I introns to occur between distantly
related organisms. For example, Neuveglise et al.
(1997) suggested that most fungi with rRNA genes
containing introns are mutualistic or symbiotic so the
presence of introns can be used to identify these par-
ticular fungi. In addition, Friedl et al. (2000), in a study
of group I introns in the lichen-forming chlorophyte,
Trebouxia, suggested that the close cell-to-cell contact
during lichenization has facilitated the transfer of
group I introns at least three times between the two
symbionts.
Besides lichens, there are numerous associations
between algae and fungi (mycophycobioses), including
symbioses, parasitisms, and endophyses (Kohlmeyer
and Kohlmeyer 1979, Sherwood and Sheath 1999)
that could facilitate the lateral transfer of group I in-
trons. For example, Kohlmeyer and Hawkes (1983)
reported an obligate symbiotic union between the as-
comycete Mycophycias apophlaeae (Kohlmeyer) Kohlm-
eyer et Volkmann-Kohlmeyer and the red alga
Apophlaea sp., which according to current systematic
understanding is a member of the Hildenbrandiales
(Saunders and Bailey 1999). In addition, endophytic
fungi have been isolated from H. rubra, but because no
fruiting bodies have been observed, a positive identi-
fication has not been made (unpublished data). Thus,
the precedent of lateral gene transfer in organisms liv-
ing in close proximity with these recent observations
about H. rubra and other members of the Hilden-
brandiales make lateral intron transfer between a fun-
gus and a red alga a possibility. Based on the close
phylogenetic relationships and the signature structural
characteristics among the group IE introns inserted in
the nuclear SSU rRNA gene at positions 989 and 1199,
we suggest that the 989 IE intron in H. rubra originated
from a lateral transfer from an ascomycete fungus; it
was not vertically inherited through the Rhodophyta
since the 989 intron is absent from the large number
(4600) of Rhodophyte nuclear SSU rRNA genes
sequenced.
This research was supported by CFI, OIT, and NSERC (RGP
238619) grants to K. M. M., NSERC grant RGP 0183503 to R.
G. S., NSF grant MCB-0110252 and NIH grants GM 48207
and GM067317 to R. R. G, and NSERC PGSA/PGSB to A. R.
S. Technical assistance in DNA sequencing by Angela Holliss
is gratefully acknowledged.
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APPENDIX
Nuclear SSU rRNA. Arxula terrestris AB000663; Au-
douinella arcuata AF079786; Bangia fuscopurpurea
AF342745; Batrachospermum gelatinosum AF026045;
Beauveria bassiana AB079125; Bensingtonia sakaguchii
AB001746; Bensingtonia yamatoana AF101826; Co-
mpsopogon coeruleus AF342748; Cordyceps capitata
AB027318; Cordyceps coccidiicola AB031195; Cordyceps
inegoensis AB027322; Cordyceps kanzashiana
AB027325; Cryphonectria parasitica L42441; Cry-
phonectria radicalis L42442; Dendryphiopsis atra
AF053731; Graphiola phoenicis D63928; Graphium pen-
icillioides AB007681; Hildenbrandia rubra AF076995;
Hildenbrandia rubra (AK1) AF108399; Hildenbrandia
rubra (CA1) AF108401; Hildenbrandia rubra (CT1)
AF108408; Hildenbrandia rubra (MA1) AF108409; Hi-
ldenbrandia rubra (NS1) AF108412; Hildenbrandia ru-
bra (BC2) to be submitted; Homo sapiens M10098;
Nadsoniella nigra X91896; Nectria ochroleuca
AB012952; Oryza sativa X00755; Oxytricha nova
M14601; Plasmodiophora brassicae U18981; Porphyra
spiralis var. amplifolia L26177; Rhodella maculata
U21217; Rhodochaete parvula AF139462; Scenedesmus
costatus AB037090; Scytalidium dimidiatum AF258604;
Stichococcus bacillaris AB055864; Tilletiopsis flava
AB045703; Trichocoma paradoxa AB003944; Xenopus
laevis X04025; Xylaria polymorpha AB014043; Zea mays
K02202.
IC1 introns. Chlorophyta: Stichococcus bacillaris
AB055864 (SSU 1506). Fungi: Arthrobotrys cladodes
U51945 (SSU 1506); Beauveria bassiana AB079125
(SSU 943); Bensingtonia yamatoana AF101826 (SSU
1506); Bionectria ochroleuca AB012952 (SSU 943);
Cenococcum geophilum Z48521 (SSU 1506); Cordyceps
coccidiicola AB031195 (SSU 943); Cordyceps kanzashi-
ana AB044639 (LSU 2563); Cordyceps prolifica
AB044640 (LSU 1921); Graphium penicillioides
AB007681 (SSU 943); Metarhizium anisopliae
AF197122 (LSU 1921), AF197124 (LSU 2449); Till-
etiopsis flava AB045703 (SSU 1506); Venturia inaequalis
AF065831 (SSU 1506); Xylaria polymorpha AB014043
(SSU 943). Rhodophyta: Bangia fuscopurpurea
AF342745 (SSU 1506); Hildenbrandia rubra (1)
L19345 (SSU 1506); Hildenbrandia rubra (2)
AF076995 (SSU 1506); Hildenbrandia rubra (AK1)
AY571898 (SSU 1506); Hildenbrandia rubra (CA1)
AY571897 (SSU 1506); Hildenbrandia rubra (CT1)
AY571896 (SSU 1506); Hildenbrandia rubra (MA1)
AY571894 (SSU 1506); Hildenbrandia rubra (NF1)
AY571899 (SSU 1506); Hildenbrandia rubra (NS1)
AY571895 (SSU 1506); Porphyra spiralis var. amplifolia;
L26177 (SSU 1506).
IE introns. Chlorophyta: Stichococcus bacillaris
AB055864 (SSU 516). Fungi: Beauveria bassiana
AB079125 (SSU 1199); Bensingtonia sakaguchii
AB001746 (SSU 1199); Bionectria ochroleuca
AB012952 (SSU 1199); Candida albicans X74272
(LSU 1923); Candida dubliniensis AF405231 (LSU
1923); Cordyceps capitata AB027318 (SSU 516); Cord-
yceps coccidiicola AB031195 (SSU 989); Cordyceps in-
egoensis AB027322 (SSU 1199); Cordyceps prolifica
AB044640 (LSU 2066); Cryphonectria parasitica
L42441 (SSU 1199); Cryptendoxyla hypophloia
AF015912 (SSU 989); Exophiala nigra X91897 (SSU
989); Fusarium solani f. robiniae AF150489 (SSU 1199);
Gaeumannomyces graminis U17160 (LSU 2563); Met-
arhizium anisopliae AF197121 (LSU 2066); Roccella
canariensis AF110343 (SSU 516); Rotaliella elatiana
X78521 (LSU 1926); Scytalidium dimidiatum
AF258604 (SSU 1199); Tilletiopsis flava AB045703
(SSU 516); Trichocoma paradoxa AB003944 (SSU
516); Xylaria polymorpha AB014043 (SSU 989). Rho-
dophyta: Hildenbrandia rubra (BC2) AY571893 (SSU
989). Stramenopile: Skeletonema pseudocostatum
Y11512 (LSU 1926). Plasmodiophorida: Plasm-
odiophora brassicae U18981 (SSU 516).
KIRSTEN M. MU¨LLER ET AL.390