Gutell 093.jphy.2005.41.0380


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Gutell 093.jphy.2005.41.0380

  1. 1. EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL ANDRED ALGAL SMALL SUBUNIT rRNA GENES1Kirsten M. Mu¨ller2, Darlene W. EllenorDepartment of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, CanadaAlison R. SherwoodDepartment of Botany, University of Hawaii, Honolulu, Hawaii 96822, USARobert G. SheathProvost’s Office, California State University San Marcos, San Marcos, California 92096, USAandJamie J. Cannone and Robin R. GutellInstitute of Cellular and Molecular Biology and the Section of Integrative Biology, University of Texas at Austin,Austin, Texas 78712, USAA previous study of the North American bioge-ography of the red algal genus Hildenbrandia notedthe presence of group I introns in the nuclear smallsubunit (SSU) rRNA gene of the marine speciesH. rubra (Sommerf.) Menegh. Group IC1 introns havebeen previously reported at positions 516 and 1506in the nuclear SSU RNA genes in the Bangiales andHildenbrandiales. However, the presence of an un-classified intron at position 989 in a collection ofH. rubra from British Columbia was noted. This in-tron is a member of the IE subclass and is the firstreport of this intron type in the red algae. Phyloge-netic analyses of the intron sequences revealed aclose relationship between this IE intron inserted atposition 989 and similar fungal IE introns in posi-tions 989 and 1199. The 989 IE introns formed amoderately to well-supported clade, whereas the1199 IE introns are weakly supported. Uniquestructural helices in the P13 domain of the 989and 1199 IE introns also point to a close relation-ship between these two clades and provide furtherevidence for the value of secondary structural char-acteristics in identifying homologous introns in ev-olutionarily divergent organisms. The absence ofthe 989 IE intron in all other red algal nuclear SSUrRNA 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 ratheris the result of lateral transfer between fungal andred algal nuclear SSU rRNA genes.Key index words: fungi; group IE intron; Hi-ldenbrandia; lateral transfer; small subunit rRNAgene; RhodophytaAbbreviations: LSU, large subunit; MP, maximumparsimony; NJ, neighbor joining; SSU, small sub-unitAll group I introns have the same chemical reactionsto 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 theirexcision (Cech 1990). The core of the group I intronstructure contains base pairings, helices, and loops thatare common to all group I introns. In contrast, thestructural elements beyond this core adopt variousforms (Burke et al. 1987, Michel and Westhof 1990).These different structural arrangements have beencategorized into 12 subclasses (IA1-IA3, IB1-IB4,IC1-IC3, ID, and IE), based on conserved primaryand secondary structural elements (Michel and West-hof 1990, Suh et al. 1999). Introns interrupt manydifferent genes, including the small subunit (SSU) andlarge subunit (LSU) nuclear and mitochondrial rRNAgenes 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 andNakase 1997, Suh et al. 1999, Cannone et al. 2002).Group IC1 introns have been reported to interruptthe nuclear SSU rRNA genes of several genera withinthe Rhodophyta. These introns have only been1Received 12 August 2003. Accepted 9 December 2004.2Author for correspondence: e-mail Phycol. 41, 380–390 (2005)r 2005 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2005.03146.x
  2. 2. observed in positions 516 and 1506 (Escherichia colinumbering) of the bangialean genera Bangia andPorphyra (Stiller and Waaland 1993, Oliveira andRagan 1994, Oliveira et al. 1995, Mu¨ller et al. 1998,2001) as well as at position 1506 of the florideophyteHildenbrandia rubra (Sommerfelt) Meneghini (Raganet al. 1993, Sherwood and Sheath 1999). In theirbiogeographic and systematic study of the genus Hi-ldenbrandia in North America, Sherwood and Sheath(1999) noted the presence of an additional intron inposition 989 in the nuclear SSU rRNA gene of Hi-ldenbrandia rubra collected from British Columbia,Canada. Our analysis of this intron in the 989 positionrevealed 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 989in the nuclear SSU rRNA of the ascomycete fungusCryptendoxyla hypophloia Malloch et Cain. Suh et al.(1999) also characterized other intron sequences be-longing to the subclass IE that were previously notedin the Chlorophyta (green algae) (Kranz et al. 1995,Krienitz et al. 1996), numerous ascomycete and bas-idiomycete fungi (Suh and Sugiyama 1994, Haaseet al. 1995), and the fungal endosymbiont of a lichen(DePriest and Been, 1992).Although group IE introns contain the structuralelements 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 (Cech1990, Suh et al. 1999). In addition, IE introns areshorter and contain a unique P13 domain thought tobe involved in the tertiary interactions with the P9.1adomain necessary for excising the intron out of therRNA gene (Cech 1990, Suh et al. 1999). This is thefirst report of a IE intron in a red alga; no IE intronshave 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 availablefrom GenBank ( and theComparative RNA (CRW) Site (Cannone et al. 2002, 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 thecommon ancestor of the red algae and was verticallyinherited by H. rubra and subsequently lost in all otherred algal lineages. A second explanation is the lateraltransfer of the 989 intron from another organism toposition 989 in the nuclear SSU rRNA gene ofH. rubra. Lateral transfer of introns between organ-isms as well as organelles has been noted previouslyand appears to be much more widespread than initiallyhypothesized (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). Thepresent study addresses the origin of the IE intron in-serted at position 989 of the nuclear SSU rRNA geneof H. rubra using phylogenetic analyses of introns andSSU rRNA coding regions and structural analysis ofthe intron sequence.MATERIALS AND METHODSSequences of the group I introns and the nuclear SSUrRNA genes used in the present study are listed in the Ap-pendix. The nuclear SSU rRNA genes including the IC1 andIE introns from H. rubra (BC2) from British Columbia (uppertide 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 inthe Appendix.Sequence alignment and secondary structure models. TherRNA and intron sequences were aligned manually withthe alignment editor ‘‘AE2’’ (developed by T. Macke, Larsenet al. 1993) that runs on the Solaris operating system on SUNMicrosystems 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 andtertiary structure models are aligned in the same column inthe alignment with the alignment editor AE2. Regions of therRNA and intron sequences with significant amounts of sim-ilarity can be aligned with only the nucleotide sequence in-formation. However, sequences with considerable variationcan only be aligned accurately and confidently when otherinformation 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 pairspredicted with comparative analysis are present in the high-resolution crystal structures from the 30S and 50S ribosomalsubunits (Gutell et al. 2002).The secondary structure diagrams for the H. rubra nuclearSSU 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 intronswere determined from the original IC1 (Michel and Westhof1990) and IE (Suh et al. 1999) structure models and the largercollection of group I intron structure models (Damberger andGutell 1994, Cannone et al. 2002). The 18S rRNA and group Iintron secondary structure diagrams were drawn with the in-teractive secondary structure program XRNA (developed byB. Weiser and H. Noller, University of California, Santa Cruz).This version of the program was written in C programminglanguage to run under the Solaris operating system on SUNMicrosystems workstations. However, this version of XRNAhas been replaced with a platform-independent version devel-oped in Java ( alignment with representative group IE and IC1 se-quences that contain the major forms of IE and IC1 intronstructure and sequence variation was prepared. These intronsoccur at several IE insertion sites, three in the SSU rRNA andfour 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 structuraland phylogenetic analyses in this study. Although there aremore than 200 IE introns available in GenBank, phylogeneticanalyses used representative sequences (based on secondarystructure). Future analyses will analyze all IE introns; however,this will require considerable additional time. The alignmentand secondary structure diagrams generated for this projectare available online at A sampling of the phylogenetically andstructurally diverse SSU rRNA and group I intron secondarystructure models are available at the Comparative RNA website(, Cannone et al. 2002).Test for mutational saturation and phylogenetic analyses. Be-fore performing the phylogenetic analyses with the intronLATERAL TRANSFER OF IE INTRON 381
  3. 3. sequences, the data were analyzed to determine whether theyencoded a significant phylogenetic signal. To do this, the dataset 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 thesimple 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 and26 IE intron sequences, resulting in an alignment with a totallength of 2296 characters. The considerable length of thisalignment is due to the inclusion of gaps to correctly alignhomologous regions. For example, the group IC1 intron inPorphyra spiralis var. amplifolia Oliveira Filho et Coll has alarge insertion (approximately 430 nt) not observed in any ofthe remaining sequences in the alignment. Hence, gapsneeded to be added to all other sequences to account forthis nonhomologous region and to align homologous regionsproperly. The nonhomologous regions are considered au-tapomorphies and hence are excluded from phylogeneticanalyses, which were based on a final alignment of 391 char-acters. This alignment was subjected to a pair-wise distanceanalysis using PAUP, version 4.0 beta 10 (Swofford 2003), andincorporating the HKY-85 model (Hasegawa et al. 1985) withequal rates of change across sites and a transition/transver-sion ratio of 2. The resulting distance matrix was used tobuild 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 alsoused to examine 56 possible models of DNA substitution andidentify the model that best fit the intron data set. The modelselected 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 thegamma 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 groupswithin the intron tree using an evolutionary model that wasparameter rich. Parsimony analysis was also carried out usingPAUP with a heuristic search under the constraints of randomsequence addition (100 replicates), steepest descent, and tree-bisection-reconnection branch swapping, with bootstrap re-sampling (1000 replicates). Phylogenetic trees for the introndata sets were all mid-point rooted because of the absence of asuitable 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 usingthe Kishino-Hasegawa test (Kishino and Hasegawa 1989) totest for significant (Po0.05) differences among them.Phylogenetic analyses of the nuclear SSU rRNA gene from adiverse group of eukaryotes were similar to those describedabove for the IE and IC1 introns. The nuclear SSU rRNA genealignment consisted of 41 sequences, resulting in an alignmentwith a total length of 2639 characters, including alignmentgaps. However, this alignment was reduced to 1660 charactersbecause some sequences were not complete and nonhomolo-gous regions were excluded. Subsequently, this alignment wassubjected to a pair-wise distance analysis, using the HKY-85model with equal rates across sites and a transition/transversionratio of 2. The resulting distance matrix was used to build a NJtree. The data were then subjected to bootstrap resampling(1000 replicates). The Modeltest program was again used toidentify the model that best fit this data set. The model selectedwas 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 thegroups noted in the HKY-85 NJ tree. The nuclear SSU rRNAgene alignment was also analyzed using maximum parsimony(MP) under the same criteria noted previously for the intronalignment. 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 wereused as outgroups for these analyses.RESULTSAnalyses of the intron structures. The secondarystructure of the intron at position 989 (E. coli num-FIG. 1. Comparative secondary structure models for the groupIE intron at position 989 in the nuclear SSU rRNA gene of Hi-ldenbrandia rubra (BC2). The 50and 30splice junctions are markedwith 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. 4. bering) in the nuclear SSU rRNA gene of H. rubra(Fig. 1) is similar to the IE secondary structurediagrams available at the Comparative RNA website(Cannone et al. 2002, Group IE introns have only been observedin the rRNA genes. A total of 242 (as of July 2004) ofthese has been identified in the SSU rRNA genes,whereas 19 occur in the LSU rRNA genes. They aredistributed among 10 unique rRNA insertion sites, 6in the SSU rRNA gene and 4 in the LSU rRNA gene.Approximately 41% of the IE introns occur at SSUrRNA 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 rRNApositions. 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 theChytridiomycota. The remaining 16% (42) are dis-tributed among six phylogenetic groups, with mostbeing in the Chlorophyta (Cannone et al. 2002). All24 IE introns at position 989 (except for H. rubra)occur in the ascomycete fungi. Ninety-two of the 104IE introns at position 1199 are also in ascomycetefungi, 10 are in basidiomycete fungi, and 2 are inChytridiomycota fungi.A visual examination of the alignment and second-ary structure diagram of the IE intron in H. rubra andother IE introns reveals more similarity between the989 SSU introns and most of the 1199 SSU rRNA in-trons than between the 989 introns and the remainderof the IE introns (not shown). In addition, the H. rubraIE intron shares more secondary structural character-istics with all the ascomycete fungi IE introns that in-terrupt the SSU rRNA at position 989 and theascomycete and basidomycete fungi IE introns at po-sition 1199. The most striking characteristic is the set ofthree helices, P2.1a, P2.1b, and P2.1c, flanking the P13helix in the 989 and 1199 SSU rRNA introns (Fig. 2,b–e), which differs considerably from that of the otherIE 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 IEstructures (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 fourgroup IE introns. Each helical region is labeled; only the 50half 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 50half of P13is shown. Parentheses enclose hairpin loops. Species names are abbreviated, and the intron positions in the rRNA genes are shown inparentheses. 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., Scytalidiumdimidiatum; Skele.pseu., Skeletonema pseudocostatum; Tille.flav., Tilletiopsis flava.LATERAL TRANSFER OF IE INTRON 383
  5. 5. P2.1b, is also conserved in length, 4 bp (Fig. 2, b–e),with the 50half of the helix typically consisting ofpurines (primarily G residues). The 50half of the P13helix follows the P2.1b helix and the 30half of the P13helix caps the P9.1a helix (Fig. 1). This helix ranges inlength from 5 to 8 nt within the IE introns, although 7bp is the typical length. Of the three inserted helices,P2.1c is the most variable in length, ranging from 15 to36 bp. Thus far, these helices are only present in the IEintrons inserted into the nuclear SSU rRNA gene atpositions 989 and 1199. Thus, this structural elementis common to the IE introns at position 989 and 1199and distinguishes them from other IE introns. These‘‘signature’’ secondary structural elements are homo-logous among the 989 IE introns in Hildenbrandia andvarious fungi.Phylogenetic analyses. Before conducting phyloge-netic analyses, the intron data were assessed for mu-tational saturation to ensure they encoded significantsignal. In this graphical analysis, curvature of the lineat higher uncorrected distances indicates mutationalsaturation (Moritz et al. 1992). For transversions cur-vature was observed at a distance of 0.2, indicating aminor level of mutational saturation, whereas fortransitions it was 0.15, thereby indicating an evenlower level of mutational saturation (not shown). Thissuggests that group I introns encode a significantamount of phylogenetic signal that can be used toestimate 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 of0.35. These 108 trees differed in topology because ofunresolved relationships among clades of introns fromdifferent insertion sites as well as relationships amongintron sequences within an insertion site. Despite thelow resolution, MP of the group IC1 and IE intron se-quences is similar in some respects to the distanceanalyses and hence only the NJ tree is shown, withparsimony bootstrap values (Fig. 3). Maximum likeli-hood trees (not shown) were computed for the besttree without constraints as well as the best tree with anenforced or constrained topology (forcing the IE in-tron in Hildenbrandia as a sister group to the IE intronsin position 989 and forcing the IE intron in Hi-ldenbrandia to group with the IC1 introns in position1506 in H. rubra). Maximum likelihood analyses with-out the constrained topology resulted in two trees witha likelihood value of 4123.77. These placed the 989 IEin H. rubra basal to the other 989 IE introns in thefungi, as in the first constrained topology noted above,and the likelihood values for the constrained analysiswere 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 samespecies were significantly different from the noncon-strained likelihood tree. The Kishino-Hasegawatest showed a significant difference (Po0.05) betweenthe two trees, with the nonconstrained tree being thefavored hypothesis.Figure 3 depicts the NJ tree derived from the dis-tance analysis of a representative set of group IE andIC1 introns from nuclear SSU and LSU rRNA genesused in the present study. The IC1 introns form amoderately to weakly supported clade (83% bootstrap[HKY-85], 67% bootstrap [GTR þ I þ G], ando50%bootstrap [MP]) and the IE introns form a separateweakly supported clade (64% bootstrap [HKY-85],o50% bootstrap [GTR þ I þ G], and o50% bootstrap[MP]). The introns within both of the subgroups (IEand IC1) appear to form moderately to well-supportedclades based on the insertion position within the SSUrRNA or LSU rRNA genes. For example, the IC1 in-trons inserted at position 943 of the SSU rRNA geneform 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 SSUrRNA gene, forms a moderate to well-supported clade(100% bootstrap support [HKY-85], 64% bootstrapsupport [GTR þ I þ G], and 99% bootstrap support[MP]) with the IE introns inserted at position 989 inthe ascomycete fungi Exophiala nigra (Issatschenko)Haase et de Hoog, Cryptendoxyla hypophloia, Cordycepscoccidiicola Kobayasi, and Xylaria polymorpha (Persoon)Greville (Fig. 3). Within this clade, the H. rubra (BC2)intron is most closely associated with the 989 intron inE. nigra with weak support (60% [HKY-85] and 72%[MP]). The IE introns inserted at positions 989 and1199 of the nuclear SSU rRNA gene form a clademoderately supported by HKY-85 bootstrap analysisonly (77%). Interestingly, all the IE introns at position1199 occur in the nuclear SSU rRNA genes of differentfungal lineages. Although the IE intron in H. rubra isstrongly associated with other IE introns inserted atposition 989 (see above), it is clearly distinct from thewell-supported clade of H. rubra IC1 introns at position1506 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 rRNAgenes in red algae and other eukaryotes (Fig. 4) showstwo 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 theFIG. 3. Neighbor-joining tree constructed using correcteddistances (HKY-85 model) of IE and IC1 introns inserted inthe nuclear rRNA genes of different eukaryotes. Numbers abovebranches 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 SSUrRNA genes are indicated to the right of the taxa.KIRSTEN M. MU¨LLER ET AL.384
  7. 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 greenalgae and land plants (Burger et al. 1999, Moreiraet al. 2000). These analyses of the host nuclear SSUrRNA gene phylogeny suggest it is unlikely that thisintron was vertically inherited from the common an-cestor of the red algae and fungi because these lineagesonly share a very distant evolutionary relationship, andthis would require multiple losses of the intron overmany distinct phylogenetic groups. This is furthersupported by the limited distribution of the intron ineach of the host nuclear SSU rRNA gene lineages.DISCUSSIONThe secondary structure and phylogenetic analysesof the intron sequences in the present study stronglysuggest that the group IE intron inserted at 989 in thenuclear SSU rRNA gene of H. rubra is homologous tothe fungal IE introns inserted at position 989 in theSSU rRNA gene. In addition, our analysis revealed aclose relationship between the IE introns inserted atpositions 989 and 1199. The IE introns at these twopositions form moderate to well-supported clades thatare closely associated in all phylogenetic analyses andcontain structural signatures in the P2.1 domain.Similar ‘‘signature’’ structural characteristics havebeen noted in other group I introns and are indicativeof homology. For example, Mu¨ller et al. (2001) notedsecondary structure elements characteristic of thegroup IC1 intron inserted at position 516 and 1506(nuclear SSU rRNA gene) in members of the Bangiales.The P5b domain contains a signature bifurcated helixthat distinguishes the 516 IC1 introns in the Bangialesfrom all other group IC1 introns. The conserved P8domain was variable in length within the 1506 IC1 in-tron in the Bangiales, which also differentiated theseintrons from those in the 516 position in the Bangialesand from all other IC1 introns (Mu¨ller et al. 2001).Mu¨ller et al. (2001) concluded that these structural andsequence signatures provided further evidence that theIC1 introns in positions 516 and 1506 of the nuclearSSU rRNA gene in the Bangiales were probably theresult of a single lateral transfer event and subsequentvertical inheritance. Bhattacharya et al. (2001) also not-ed the bifurcated helix in the P5b domain of a groupIC1 intron inserted at position 516 (nuclear SSU rRNAgene) 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 specificallyrelated, although their host cells are not (Bhattacharyaet al. 2001). Mu¨ller et al. (2001) postulated that thesestructural signatures might provide a means for deter-mining lateral transfer events of group I introns acrossa wide phylogenetic range.In addition, introns that occur at the same insertionsite within the rRNA gene are usually more similar toone another than they are to introns at other positionsin the rRNA genes (Tan 1997). Hence, introns at thesame insertion site are thought to have a common an-cestry. These introns may be traced back to one ormore events in which they were inserted into the rRNAand 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 allgroup I introns at different insertion sites within rRNAin the green algal order Zygnematales. Mu¨ller et al.(2001) noted that the group IC1 introns inserted atpositions 516 and 1506 (nuclear SSU rRNA gene)within the Bangiales formed two separate yet distinctclades. In the protist Acanthamoeba, high sequence di-vergence among introns in four different insertion po-sitions in the rRNA genes suggests that the intronacquisition occurred independently at these four sitesafter divergence of the taxa within the tree (Schroeder-Diedrich et al. 1998). High sequence similarity be-tween introns would be consistent with descent froman 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 bespecifically related. It has been postulated that lateraltransfer and vertical inheritance have contributedextensively to the evolution of group I introns (Soginand Edman 1989, Tan 1997).The distribution of group I introns is 1) not uniformacross the nuclear, chloroplast, and mitochondrial gen-omes; 2) present in a wide range of phylogeneticallydistant taxa; and 3) within many different exons.Hence, group I introns appear to be very successfulat invading and maintaining their sequences among adiverse group of eukaryotic organisms (Burke 1988,Dujon 1989, Bhattacharya et al. 1994, 2001, Turmelet al. 1995, Tan 1997, Bhattacharya 1998, Nishidaet 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 inthe bacterial phylogenetic group. Most of these, all be-longing to the IC3 subgroup, occur in select tRNAgenes 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); Coxiellaburnetii (Derrick) Philip (Seshadri et al. 2003); andThermatoga naphthophila Y. Takahata, M. Nishijima, T.FIG. 4. Neighbor-joining tree constructed using correcteddistances (HKY-85 model) of the nuclear SSU rRNA gene se-quences from different eukaryotes. Numbers above branchesrepresent distance bootstrap values (1000 replicates) using theHKY-85 model (first number), distance bootstrap values usingthe GTR þ I þ G model (second number), and bootstrap valuesusing maximum parsimony (third number). Branches with anasterisk or lacking a value had less than 50% support in thatanalysis.KIRSTEN M. MU¨LLER ET AL.386
  9. 9. Hoaki et T. Maruyama (Nesb and Doolittle 2003). Insharp contrast, there are approximately 1025 docu-mented introns that are associated with approximately2000 known fungal nuclear encoded nuclear SSUrRNA genes (Cannone et al. 2002). The mechanismof lateral transfer between organisms is an importantprocess 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, becausegroup I introns that have high sequence similarity oc-cur at the same position in the rRNA genes (Yamadaet al. 1994, Bhattacharya et al. 1996, Nishida et al.1998, Nozaki et al. 1998, Friedl et al. 2000). Closecell-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 distantlyrelated organisms. For example, Neuveglise et al.(1997) suggested that most fungi with rRNA genescontaining introns are mutualistic or symbiotic so thepresence of introns can be used to identify these par-ticular fungi. In addition, Friedl et al. (2000), in a studyof group I introns in the lichen-forming chlorophyte,Trebouxia, suggested that the close cell-to-cell contactduring lichenization has facilitated the transfer ofgroup I introns at least three times between the twosymbionts.Besides lichens, there are numerous associationsbetween algae and fungi (mycophycobioses), includingsymbioses, parasitisms, and endophyses (Kohlmeyerand 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 algaApophlaea sp., which according to current systematicunderstanding is a member of the Hildenbrandiales(Saunders and Bailey 1999). In addition, endophyticfungi have been isolated from H. rubra, but because nofruiting 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 observationsabout 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 closephylogenetic relationships and the signature structuralcharacteristics among the group IE introns inserted inthe nuclear SSU rRNA gene at positions 989 and 1199,we suggest that the 989 IE intron in H. rubra originatedfrom a lateral transfer from an ascomycete fungus; itwas not vertically inherited through the Rhodophytasince the 989 intron is absent from the large number(4600) of Rhodophyte nuclear SSU rRNA genessequenced.This research was supported by CFI, OIT, and NSERC (RGP238619) grants to K. M. M., NSERC grant RGP 0183503 to R.G. S., NSF grant MCB-0110252 and NIH grants GM 48207and GM067317 to R. R. G, and NSERC PGSA/PGSB to A. R.S. Technical assistance in DNA sequencing by Angela Hollissis gratefully acknowledged.Belfort, M. 1991. Self-splicing introns in prokaryotes: migrant fos-sils? Cell 64:9–11.Bhattacharya, D. 1998. The origin and evolution of protist group Iintrons. Protist 149:113–22.Bhattacharya, D., Cannone, J. J. Gutell, R. R. 2001. 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  11. 11. Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A. Shub, D. A. 1990. Bacterial origin of a chloroplast intron:conserved self-splicing group I introns in cyanobacteria.Science 250:1566–70.Yamada, T., Tamura, K., Aimi, T. Songsri, T. 1994. Self-splicinggroup I introns in eukaryotic viruses. Nucleic Acids Res.22:2532–7.APPENDIXNuclear SSU rRNA. Arxula terrestris AB000663; Au-douinella arcuata AF079786; Bangia fuscopurpureaAF342745; Batrachospermum gelatinosum AF026045;Beauveria bassiana AB079125; Bensingtonia sakaguchiiAB001746; Bensingtonia yamatoana AF101826; Co-mpsopogon coeruleus AF342748; Cordyceps capitataAB027318; Cordyceps coccidiicola AB031195; Cordycepsinegoensis AB027322; Cordyceps kanzashianaAB027325; Cryphonectria parasitica L42441; Cry-phonectria radicalis L42442; Dendryphiopsis atraAF053731; Graphiola phoenicis D63928; Graphium pen-icillioides AB007681; Hildenbrandia rubra AF076995;Hildenbrandia rubra (AK1) AF108399; Hildenbrandiarubra (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 ochroleucaAB012952; Oryza sativa X00755; Oxytricha novaM14601; Plasmodiophora brassicae U18981; Porphyraspiralis var. amplifolia L26177; Rhodella maculataU21217; Rhodochaete parvula AF139462; Scenedesmuscostatus AB037090; Scytalidium dimidiatum AF258604;Stichococcus bacillaris AB055864; Tilletiopsis flavaAB045703; Trichocoma paradoxa AB003944; Xenopuslaevis X04025; Xylaria polymorpha AB014043; Zea maysK02202.IC1 introns. Chlorophyta: Stichococcus bacillarisAB055864 (SSU 1506). Fungi: Arthrobotrys cladodesU51945 (SSU 1506); Beauveria bassiana AB079125(SSU 943); Bensingtonia yamatoana AF101826 (SSU1506); Bionectria ochroleuca AB012952 (SSU 943);Cenococcum geophilum Z48521 (SSU 1506); Cordycepscoccidiicola AB031195 (SSU 943); Cordyceps kanzashi-ana AB044639 (LSU 2563); Cordyceps prolificaAB044640 (LSU 1921); Graphium penicillioidesAB007681 (SSU 943); Metarhizium anisopliaeAF197122 (LSU 1921), AF197124 (LSU 2449); Till-etiopsis flava AB045703 (SSU 1506); Venturia inaequalisAF065831 (SSU 1506); Xylaria polymorpha AB014043(SSU 943). Rhodophyta: Bangia fuscopurpureaAF342745 (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 bacillarisAB055864 (SSU 516). Fungi: Beauveria bassianaAB079125 (SSU 1199); Bensingtonia sakaguchiiAB001746 (SSU 1199); Bionectria ochroleucaAB012952 (SSU 1199); Candida albicans X74272(LSU 1923); Candida dubliniensis AF405231 (LSU1923); Cordyceps capitata AB027318 (SSU 516); Cord-yceps coccidiicola AB031195 (SSU 989); Cordyceps in-egoensis AB027322 (SSU 1199); Cordyceps prolificaAB044640 (LSU 2066); Cryphonectria parasiticaL42441 (SSU 1199); Cryptendoxyla hypophloiaAF015912 (SSU 989); Exophiala nigra X91897 (SSU989); Fusarium solani f. robiniae AF150489 (SSU 1199);Gaeumannomyces graminis U17160 (LSU 2563); Met-arhizium anisopliae AF197121 (LSU 2066); Roccellacanariensis AF110343 (SSU 516); Rotaliella elatianaX78521 (LSU 1926); Scytalidium dimidiatumAF258604 (SSU 1199); Tilletiopsis flava AB045703(SSU 516); Trichocoma paradoxa AB003944 (SSU516); Xylaria polymorpha AB014043 (SSU 989). Rho-dophyta: Hildenbrandia rubra (BC2) AY571893 (SSU989). Stramenopile: Skeletonema pseudocostatumY11512 (LSU 1926). Plasmodiophorida: Plasm-odiophora brassicae U18981 (SSU 516).KIRSTEN M. MU¨LLER ET AL.390