A Structural and Phylogenetic Analysis of the Group IC1 Introns in the Order Bangiales (Rhodophyta)

Kirsten M. Müller, Jamie J. Cannone, Robin R. Gutell and Robert G. Sheath

Department of Botany and Dean's Office, University of Guelph, Guelph, Ontario, Canada; and
Institute of Cellular and Molecular Biology, University of Texas at Austin


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Our previous study of the North American biogeography of Bangia revealed the presence of two introns inserted at positions 516 and 1506 in the nuclear-encoded SSU rRNA gene. We subsequently sequenced nuclear SSU rRNA in additional representatives of this genus and the sister genus Porphyra in order to examine the distribution, phylogeny, and structural characteristics of these group I introns. The lengths of these introns varied considerably, ranging from 467 to 997 nt for intron 516 and from 509 to 1,082 nt for intron 1506. The larger introns contained large insertions in the P2 domain of intron 516 and the P1 domain of intron 1506 that correspond to open reading frames (ORFs) with His-Cys box homing endonuclease motifs. These ORFs were found on the complementary strand of the 1506 intron in Porphyra fucicola and P. umbilicalis (HG), unlike the 516 intron in P. abbottae, P. kanakaensis, P. tenera (SK), Bangia fuscopurpurea (Helgoland), and B. fuscopurpurea (MA). Frameshifts were noted in the ORFs of the 516 introns in P. kanakaensis and B. fuscopurpurea (HL), and all ORFs terminated prematurely relative to the amino acid sequence for the homing endonuclease I-Ppo I. This raises the possibility that these sequences are pseudogenes. Phylogenies generated using sequences of both introns and the 18S rRNA gene were congruent, which indicated long-term immobility and vertical inheritance of the introns followed by subsequent loss in more derived lineages. The introns within the florideophyte species Hildenbrandia rubra (position 1506) were included to determine relationships with those in the Bangiales. The two sequences of intron 1506 analyzed in Hildenbrandia were positioned on a well-supported branch associated with members of the Bangiales, indicating possible common ancestry. Structural analysis of the intron sequences revealed a signature structural feature in the P5b domain of intron 516 that is unique to all Bangialean introns in this position and not seen in intron 1506 or other group IC1 introns.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Many eukaryotic genes have their coding regions interrupted by intervening sequences or introns. Group I introns represent a family of RNA molecules with a specific higher-order structure and the ability to catalyze their own excision by a common splicing mechanism (Cech 1990Citation ). Group I introns are divided into 11 subgroups based on conserved primary- and secondary-structure elements (Michel and Westhof 1990Citation ) and have been reported in the 18S rRNA genes of numerous organisms (Johansen, Muscarella, and Vogt 1996Citation ), such as fungi (Takashima and Nakase 1997Citation ), amoebae (De Jonckheere 1994Citation ; Schroeder-Diedrich, Fuerst, and Byers 1998Citation ), and green algae (Wilcox et al. 1992Citation ; VanOppen, Olsen, and Stam 1993Citation ; Bhattacharya et al. 1994, 1996Citation ; Bhattacharya 1998Citation ). Group I introns have also been reported in several red algae, including Hildenbrandia rubra (Ragan et al. 1993Citation ) and particularly members of the order Bangiales (Porphyra spiralis var. amplifolia and Bangia fuscopurpurea [as Bangia atropurpurea] [Oliveira and Ragan 1994Citation ]; Porphyra miniata, P. purpurea, P. linearis [Oliveira et al. 1995Citation ]; Bangia spp. [Müller et al. 1998Citation ]). Stiller and Waaland (1993)Citation reported cryptic diversity in a number of species of Porphyra after finding large insertions in the nuclear SSU rRNA genes of a number of taxa. Oliveira et al. (1995)Citation speculated that these introns may be a means to differentiate among geographic entities within the genus Porphyra. During the course of a study of the biogeography of Bangia in North America, we reported the presence of two introns in positions 516 and 1506 (Escherichia coli numbering) in the nuclear SSU rRNA gene (Müller et al. 1998Citation ). Subsequent analysis of additional nuclear SSU rRNA gene sequences of Bangia and Porphyra revealed a rich source of these introns, which are also variable in occurrence. Variable occurrence of group I introns can be simply explained by two models: intron insertion or intron deletion (Burke 1988Citation ). The first model, intron insertion, is hypothesized to have begun with a gene devoid of introns followed by subsequent insertion of one or more introns (Burke 1988Citation ). The deletion model proposes a gene initially containing one or more introns, after which precise deletion of these introns occurs; that is, nonmobile introns are destined to be lost over time if they cannot reinfect homologous sites. Burke (1988)Citation proposed that the variable occurrence of introns in genes may be the result of a combination of these two models. High sequence similarity between introns is consistent with descent from an ancestral intron that was initially acquired and vertically inherited (Schroeder-Diedrich, Fuerst, and Byers 1998Citation ). In addition, congruent intron and rRNA gene phylogenies would provide support for this theory (Bhattacharya et al. 1994, 1996Citation ; Friedl et al. 2000Citation ). The present study provides a large data set that will allow us to test these hypotheses in context with the phylogeny of the rhodophyte order the Bangiales.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Collections of Bangia and Porphyra collected for molecular analyses and obtained from GenBank for the present study are listed in table 1 Filaments collected for DNA analysis were cleaned of visible epiphytes, bases were removed to prevent contamination, and the specimens were stored at-20°C. Samples were ground in liquid nitrogen, and the DNA was extracted according to the protocol outlined by Saunders (1993)Citation with modifications given in Vis and Sheath (1997)Citation . Amplification and sequencing of the nuclear SSU rRNA gene for both genera are as outlined in Müller et al. (1998)Citation .


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Table 1 Specimens of Porphyra and Bangia Utilized in the Present Study, Including GenBank Accession Numbers for 18S rRNA Gene and Intron Sequences

 

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Table 1 Continued

 
Intron Amplification
The intron in position 516 was amplified in the large fragment of the nuclear SSU rRNA gene as described in Müller et al. (1998)Citation using the primers G02.1 (5'-CGA TTC CGG AGA GGG AGC CTG-3') and G15.1 (5'-CTT GTT ACG ACT TCT CCT TCC-3'). These fragments were then sequenced in both directions using internal primers flanking the intron. The intron in position 1506 was amplified using primers GO6 (5'-GTT GGT GGT GCA TGG CCG TTC-3') and G07 (5'-TCC TTC TGC AGG TTC ACC TAC-3') from Saunders and Kraft (1994)Citation as follows: initial denaturation at 95°C for 2 min, 35 cycles of denaturation for 1 min at 93°C, primer annealing at 50°C for 1 min, and extension for 2 min at 72°C, followed by a final extension time of 3 min at 72°C. This intron was sequenced in both directions using an internal primer at the 5' end of the intron and the primer G07 (sequences for internal primers can be obtained from R.G.S.). All products were prepared for sequencing and sequenced using the protocols outlined in Müller et al. (1998)Citation .

RNA Extraction/cDNA Production in the Bangiales
RT-PCR was used to determine if introns were present in mature RNA or excised from all collections of Bangia and Porphyra listed in table 1 and the Bangia collections utilized in Müller et al. (1998)Citation . RNA was extracted following the procedure previously outlined for DNA extraction. Following this, 12 µl of extract was placed in a clean tube, to which 3 µl of 25 mM MgCl2 and 1 µl of DNAase was added. This mixture was then left at room temperature for 2 h, after which 5 µl was loaded in a 0.5% agarose gel to determine if all DNA had been digested within the extract. If this was the case, the extract was then immediately placed in the freezer at -20°C. RT-PCR using Titan by Boehringer Mannheim was used to amplify the RNA. The primers G02.1 and G09 (5'-ATC CAA GAA TTT CAC CTC TG-3') and G15.R (5'-GGA AGG AGG AGT CGT AAC AAG-3') and G07 were used to amplify the region containing the intron in positions 516 and 1506, respectively (Saunders and Kraft 1994Citation ; Müller et al. 1998Citation ). Following the protocols for RT-PCR outlined by Boehringer Mannheim, the reaction consisted of master mix 1 (1 µl of RNA template; 20 µM of each primer; 40 mM of dNTP; 2.5 µl DTT solution; dH2O) and master mix 2 (14 µl dH2O; 5 x RT-PCR buffer with MgCL2; 1 µl enzyme mix). These two master mixes were combined and placed in a 0.5-µl thin-walled PCR tube on ice. Amplification was performed as follows: initial denaturation at 94°C for 2 min; 10 cycles of denaturation at 94°C for 30 s, primer annealing at 50°C for 30 s, and extension for 2 min at 68°C; 25 cycles of denaturation at 94°C for 30 s, primer annealing at 50°C for 30 s, extension for 2 min at 68°C, plus cycle elongation for 5 s for each cycle; and a final extension of 7 min at 68°C. Sequencing of the amplified regions was carried out as previously outlined.

Alignment of Nuclear SSU rRNA Genes and Group IC1 Introns
The nuclear SSU rRNA gene and group IC1 intron sequences in the present study were incorporated into large alignments that spanned a large taxonomic range in order to ensure optimum alignment. The new sequences were manually aligned with the alignment editor AE2 (developed by T. Macke; see Larsen et al. 1993Citation ) on the basis of sequence similarity and a previously established eukaryotic secondary-structure model (Gutell 1993Citation ). The alignments were then subjected to a process of comparative sequence analysis (Gutell et al. 1985Citation ). This process consisted of searching for compensating base changes using computer programs developed within the Gutell Laboratory (University of Texas at Austin, http://www.rna.icmb.utexas.edu/; discussed in Gutell et al. 1985Citation ) and using the subsequent information to infer additional secondary-structural features. This refined alignment was reanalyzed and the entire process was repeated until the proposed structures were entirely compatible with the alignment. Secondary-structure diagrams were generated with the computer program XRNA (developed by B. Weiser and H. Noller, University of Santa Cruz). Individual secondary-structure diagrams will be available at http://www.rna.icmb.utexas.edu/, and alignments can be obtained from K.M.M.

Analysis of Nuclear SSU rRNA Gene and Group I Intron Sequences
All analyses on both the nuclear SSU rRNA gene and the group I introns were carried out using only well-aligned regions of the sequences. Parsimony analyses on the two introns were carried out with PAUP 3.1.1 (Swofford 1993Citation ) with a heuristic search under the constraints of random sequence addition (100 replicates), steepest descent, and tree bisection-reconnection (TBR) branch swapping. The data were then subjected to bootstrap resampling (1,000 replicates). Analyses of the introns were carried out as follows: (1) the alignment gaps were treated as missing data, and (2) the alignment gaps were coded as independent single evolutionary events (insertion or deletion) based on secondary-structure models (Damberger and Gutell 1994). Group IC1 introns in Chlorella ellipsoidea (GenBank accession number A: X63520, B: D13324) were used as outgroups in the phylogenetic analyses. Parsimony trees could not be determined for the nuclear SSU rRNA gene due to the large number of taxa and limited computational capacity; however, neighbor-joining trees were calculated for this data set. PHYLIP (Felsenstein 1993Citation ) was used to construct neighbor-joining trees for both the introns and the nuclear SSU rRNA genes using a matrix of distance values estimated according to the Kimura two-parameter model (Kimura 1980Citation ) with a transition/transversion ratio of 2.0 and a single-category substitution rate, as well as the Jukes and Cantor (1969)Citation model (intron data set only). These data sets were also subjected to bootstrap resampling. Maximum-likelihood analysis was carried out using the puzzle function in PAUP (version 4.0 beta 2a) as described by Strimmer and von Haeseler (1996)Citation with 1,000 puzzling steps. The nuclear SSU rRNA gene sequence analyses of the Bangiales were run using Erythrotrichia carnea (L26189) and Erythrocladia sp. (L26188) as outgroups. These taxa were determined to be basal to the Bangiales and have been previously used as outgroup taxa for this order (Ragan et al. 1994Citation ; Oliveira et al. 1995Citation ; Müller et al. 1998Citation ).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sequence data for all intron sequences were submitted to GenBank, and accession numbers are given in table 1 . Presence and absence of introns within the nuclear SSU rRNA gene Bangiales is depicted in figure 4 . Letters and numbers behind taxonomic names refer to various collections that are listed in table 1 , of which some represent standard state, provincial, or country codes, whereas others are arbitrary numbers or initials of the author of the particular sequence. Due to the difficulty in delineating species in Bangia (Müller et al. 1998Citation ) and the lack of a definitive global key for Porphyra species, identification of species that are used in the molecular analyses should be considered tentative. Thus, many of the analyses in the present study will focus more on the relationship among the intron sequences and nuclear SSU rRNA gene within the two genera than on morphological species within each genus, other than for characters that would not be disputed (e.g., monostromatic vs. distromatic). With respect to Bangia, based on the findings in Müller et al. (1998)Citation , all freshwater collections (AT22, BI12, GL, IR, IT, and NL) should be classified as B. atropurpurea, while collections from marine locations have provisionally been given the name B. fuscopurpurea until they can be differentiated morphologically.



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Fig. 4.—Presence (+) and absence (-) of group IC1 introns in positions 516 (first symbol) and 1506 (second symbol) mapped on an 18S rRNA gene neighbor-joining tree. Lengths of branches in tree correspond to sequence divergence calculated using the Kimura (1980)Citation two-parameter model

 
Using RT-PCR it was determined that neither intron is present in the mature rRNA, indicating that these introns are excised (not shown). Thirty-three nuclear SSU rRNA gene sequences from Bangia and 52 from Porphyra were examined for the presence of either intron in position 516 (intron 516) (E. coli numbering system) and position 1506 (intron 1506). Of these 52 Porphyra specimens, there were 39 complete sequences of the nuclear SSU rRNA gene; the remainder consisted of species for which the nuclear SSU rRNA gene could not be obtained (difficulty in obtaining sequences, or sample was not sequenced) but sequences of some introns were available. Among the 33 collections of Bangia, 21 were determined to have intron 516 (64%), and 13 were observed to have intron 1506 (39%). Only 10 (30%) collections contained neither intron, and 11 (33%) collections had both introns (fig. 4 ). Within Porphyra, 20 (40%) of 50 nuclear SSU rRNA gene sequences were determined to have intron 516, and 26 (52%) contained intron 1506. Only 14 (28%) Porphyra sequences had either intron, and 10 (20%) contained both introns (fig. 4) .

Structural Characteristics of Group IC1 Introns in the Bangiales
All of the rRNA introns in Bangia and Porphyra were determined to belong to the CI subgroup of the group I introns based on characteristic primary- and secondary-structure features (fig. 1 ) (Michel and Westhof 1990; Damberger and Gutell 1994). The lengths of these introns within the Bangiales varied considerably, ranging from 467 to 998 nt for intron 516 and ranging from 509 to 1,082 nt for intron 1506 (table 1 ). Figure 1 is a consensus diagram of all of the group IC1 introns in the Bangiales. Both introns were observed to have large insertions within the P1 (12–567 nt), P2 (62–553 nt), P5 (3–91 nt), P6b (10–84 nt), and P9.2 (5–53 nt) domains (fig. 1 ). The large insertions in the P1 and P2 domains were found in introns 1506 and 516, respectively (fig. 1 ), whereas the other insertions (P5, P6b, and P9) were similar in size between both the 516 and the 1506 introns. The majority of the base pairs in the P4 and P7 helices are highly conserved (>95%) between both sets of introns, whereas the remaining helices are less conserved. The single positions and base pair "signatures" that distinguish the 516 and 1506 introns are shown as square boxes and dashed lines in figure 1 and detailed in table 3 . Approximately half of these nucleotides and the 5' positions of the base pairs noted in table 3 (and depicted in fig. 1 ) are purines (A, G) in the 516 intron and are pyrimidines (C, U) in intron 1506 (positions 2, 25, 26, 97:277, 216:257, 217:256, 262:312, 322:326, and 335:364 [relative to Tetrahymena thermophila]). All of these positions are highly conserved (>95%) with the exception of base pair 97:277 (>90%, <95%). Position 172 and the 322:326 base pair have no homologous position with respect to T. thermophila. These three positions are highly conserved in both introns (>95%). Figure 2 highlights the differences between the 516 and 1506 introns in the P5b and P8 domains. The P5b helix (fig. 2a ) is one of the most distinct features of the 516 intron. This bifurcated helix, present in all of the Bangiales 516 introns, replaces the typical single helix present in all IC1 introns, including the Bangialean 1506 introns (fig. 2a ). The P8 domain (fig. 2b ) is also distinct, primarily on the basis of helix length. In the 516 version of this structure, the 3-bp helix is capped by a tetraloop, while the 1506 structure has a longer helix capped by a variable-length hairpin loop.



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Fig. 1.—Phylogenetic conservation of Rhodophyta (Bangiophyceae) group I (IC1) introns (18S rRNA positions 516 and 1506) superimposed onto the Bangia sp. (IR) group I (IC1) intron secondary structure (18S rRNA position 1506). Positions with a nucleotide in >95% of sequences are shown: ACGU—95+% conserved; acgu—90%–95% conserved; •—80%–90% conserved; o—<80% conserved. Otherwise, the regions are represented by arcs. Positions shown in small square boxes and arcs with dashed lines represent features that distinguish the 516 and 1506 introns (described in table 3 ). The exon sequences that flank the intron are indicated with x's

 

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Table 3 Differences Between the 516 and 1506 Introns at Single Positions or Base Pairs Using Tetrahymena thermophila Position Numbering

 


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Fig. 2.—Gallery highlighting differences between introns 516 and 1506 using consensus diagrams and individual secondary-structure diagrams. a, P5b region of intron 516, showing bangialean consensus and differences among specific taxa. b, P5b region of intron 1506, showing a bangialean consensus and differences specific among taxa. c, Consensus of the P8 regions of introns 516 and 1506 in the Bangiales. For consensus diagrams, positions with a nucleotide in >95% of sequences are shown: ACGU—95+% conserved; acgu—90%–95% conserved; •—80%–90% conserved; o—<80% conserved. Otherwise, the regions are represented by arcs

 
Haugen et al. (1999)Citation determined that P. spiralis var. amplifolia (R) appeared to contain an open reading frame (ORF) corresponding to a 149-amino-acid His-Cys box protein within the P1 extension of the 1506 intron, as does P. tenera (KK) within the P2 extension of the 516 intron. Johansen, Embley, and Willassen (1993)Citation noted that the His-Cys box motif is a hallmark of nuclear homing endonucleases. Examination of these domains in the bangialean introns within the present study revealed that this ORF is present in those taxa containing large extensions within the P1 or P2 domain for 1506 and 516 introns, respectively. Table 2 highlights the sizes of the P2 domain (516 intron) and the P1 domain (1506 intron) for all collections examined in the study and the presence or absence of the His-Cys box motif. The size of the P2 domain in the 516 intron ranged from 62 to 552 nt, with only those >414 nt containing an ORF and His-Cys box motif. This motif has not previously been reported in the 516 intron for the following taxa: B. fuscopurpurea (Helgoland), B. fuscopurpurea (MA), P. abbottae, P. kanakaensis, and P. tenera (SK). The P1 domain in the 1506 intron ranged from 53 to 521 nt, and the His-Cys box motif was found only in those with larger insertions in this region: P. fucicola and P. umbilicalis (previously unreported).


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Table 2 Sizes (nt) of Bangialean Nuclear Group I Introns in SSU rDNA, Sizes of P1 and P2 Regions, and Presence (+) or Absence (-) of His-Cys Box Motif (HC) Within Introns Inserted at Positions 516 and 1506, Respectively

 
Haugen et al. (1999)Citation noted that the ORF within the P1 extension for intron 1506 was determined to be on the complementary strand to that encoding the SSU rRNA, and this was also the case for the ORF in the previously unreported taxa (P. fucicola and P. umbilicalis). However, this was not true for the ORF in the P2 extensions for the 516 introns, which were found on the same strand as that encoding the SSU rRNA. Figure 3 depicts an amino acid alignment of the His-Cys box motif region in the P1 domain of the 1506 intron and the P2 domain of the 516 intron. As noted by Haugen et al. (1999)Citation , P. spiralis var. amplifolia (R) and the endonuclease I-Ppo I were identical in 16 out of the 29 amino acids. The two new additional amino acid sequences, P. fucicola and P. umbilicalis (HG), were identical in 13 amino acids to the I-Ppo I endonuclease. However, the amino-acid sequence for the His-Cys box in the 516 introns was identical in only 9 or 10 amino acids (fig. 3 ). Haugen et al. (1999)Citation also noted frameshifts in the ORF of P. spiralis var. amplifolia (R) and B. fuscopurpurea (1). Frameshifts were noted in the ORF of the 516 introns in P. kanakaensis and B. fuscopurpurea (HL); however, the remaining sequences were not found to have frameshifts, although they all terminated prematurely relative to the amino acid sequence for the homing endonuclease I-Ppo I (fig. 3 ). This raises the possibility that these sequences are pseudogenes; however, endonuclease activity in the Bangiales was not tested in this study and needs to be investigated in further detail.



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Fig. 3.—Alignment of open reading frames (ORFs) coding for endonuclease-like amino acid sequences highlighting the His-Cys box motif from the 516 and 1506 introns in the Bangiales. A, 516 introns: Porphyra abbottae (P. abb.), Porphyra kanakaensis (P. kana.), Porphyra tenera (KK) (P. ten. (KK)), P. tenera (SK) (P. ten. (SK)), Bangia fuscopurpurea (Helgoland) (B. fusc. (HL)), and B. fuscopurpurea (MA) (B. fusc. (MA)). B, 1506 introns: Porphyra spiralis var. amplifolia (R), Porphyra umbilicalis (HG) (P. umb. (HG)), Porphyra fucicola, and B. fuscopurpurea (1) (B. fusc. (1)). Identical positions are indicated by dots, alignment gaps are indicated by dashes, and residues inferred from reading frameshift corrections are shown in bold lowercase letters. Conserved residues proposed to be directly involved in zinc binding (C100, C105, H110, C125, C132, H134, C138) and the active site (H98, N119) of the I-PpoI endonuclease (Flick et al. 1998Citation ) are indicated. The His-Cys box motif and frameshifts for P. tenera (KK), P. tenera (SK), B. fuscopurpurea (1), and P. spiralis var. amplifolia (R) were previously noted by Haugen et al. (1999)Citation

 
Phylogenetic Analysis of Introns
Figure 4 depicts a neighbor-joining tree derived from the analysis of the nuclear SSU rRNA genes (parsimony analyses were unobtainable due to the very long computation time required) upon which the presence (+) and absence (-) of both introns have been mapped. From these analyses, there appear to be multiple losses of both introns. For example, the first major clade of Bangia and Porphyra at the bottom of the tree contains three smaller clusters in which there have been at least two losses of the 1506 intron along each lineage (fig. 4 ). This same trend is even more evident in the larger Bangia-Porphyra clade, where taxa possessing an intron are intermingled with those that do not. There does not appear to be any consistent trend with respect to presence or absence of either intron; however, it does appear that the more derived a taxon is, the more likely the intron will be absent (e.g., P. miniata (CCAP 1379/2), P. amplissima, P. miniata, and P. miniata (NF)) (fig. 4 ). Sequences of collections of B. atropurpurea were determined to have identical nuclear SSU rRNA gene sequences, and all collections contained only intron 516.

Interestingly, some collections of B. fuscopurpurea that were identical with regard to nuclear SSU rRNA gene sequences were variable with regard to possession of either or both introns. For example, the collection from Greece contained both introns 516 and 1506; however, the other collection from Australia, with which it was identical, did not contain the 1506 intron. In addition, the collection from Ireland (IR) contained both introns 516 and 1506, whereas the other collections (WA and TX), with which it was nearly identical (differing by 8 bp) contained neither intron. A similar pattern occurred in B. fuscopurpurea from Massachussetts (MA), which did not contain the intron 1506, yet it was present in the other collections with nearly identical sequences (Helgoland and Nice, differing by 5 bp). Within Porphyra, this was only seen in one case: Porphyra yezoensis (HH) did not contain intron 1506, whereas P. yezoensis (OM) did.

Well-supported clades are along biogeographic lines with few exceptions, but the presence or absence of introns does not appear to be consistent based on geographic locations. For example, the well-supported clade (100% bootstrap) of B. fuscopurpurea containing primarily collections from the Atlantic and Mediterranean (NC, NJ, Greece, Helgoland, Nice, MA [with the exception of the Pacific samples from Australia and Mexico]) all contained intron 516, except the sample from Mexico, and the samples from Massachusetts and Australia did not contain intron 1506. Distromatic taxa (subgenus Diploderma) of Porphyra, P. miniata (CCAP 1379/2, NF) and P. amplissima (2), contained neither intron, whereas other species of Porphyra belonging to the subgenera Diplastida and Porphyra were variable in containing either intron.

Introns occur frequently within the Chlorophyta, but within the Rhodophyta, Bangia, Porphyra, and Hildenbrandia are the only genera currently known to contain introns (Ragan et al. 1993Citation ). This trend raises the question of whether the introns within Hildenbrandia and the Bangiales might be related. Hence, figure 5 also includes the two 1506 introns from H. rubra and one chlorophyte algae, C. ellipsoidea (A, B). This figure presents the one most-parsimonious tree based on analysis of 1,025 parsimony-informative characters of well-aligned positions of the group IC1 intron in positions 516 and 1506. This tree has a length of 4,589 steps and a consistency index (CI) (a measure of the amount of homoplasy exhibited by the set of characters: for no homoplasy, CI = 1) of 0.4988. This data set was also analyzed by coding the gaps as single evolutionary events (e.g., 10 consecutive gaps are treated as one event rather than 10 separate events), whereas in the previous analysis, they were treated as missing data. This analysis depicted little change in the topologies of the trees; in fact, the resolution was much lower than when the gaps were treated as missing characters.



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Fig. 5.—The one most-parsimonious tree based on analysis of 1,025 phylogenetically informative characters of well-aligned positions of the group IC1 intron in positions 516 and 1506 in the order Bangiales (length = 4,589, consistency index = 0.4988). The first number above a branch represents bootstrap support (1,000 replicates) for maximum parsimony, the second value is the percentage of times that a particular cluster was found among the 1,000 intermediate steps (QPS) using quartet puzzling, and the final value represents bootstrap resampling for distance analysis (1,000 replicates). Numbers below branches represent Bremer indices or decay values

 
Figure 5 clearly depicts the 516 and 1506 intron sequences as two separate clades that are generally well supported (516: 100% maximum-parsimony [MP] bootstrap; 50% quartet puzzling values [QPS]; 100% neighbor-joining [NJ] bootstrap; 1506: 98% MP bootstrap; <50% QPS; 99% NJ bootstrap). The two introns in H. rubra are well associated with each other (100% MP bootstrap; 88% QPS; 95% NJ bootstrap) and are well positioned within the 1506 intron sequence clade of the Bangiales (89% MP bootstrap; 89% QPS; 99% NJ bootstrap). The introns in P. spiralis var. amplifolia (position 1506) are positioned on a branch that is basal to the cluster containing the 1506 intron sequences from the Bangiales and H. rubra.

Both the nuclear SSU rRNA gene NJ tree (fig. 4 ) and the phylogenetic tree using intron sequences (fig. 5 ) show similar trends. For example, the well-supported (100% bootstrap support; fig. 5 ) cluster containing B. fuscopurpurea from New Jersey (NJ), Nice, Australia, Greece, North Carolina (NC), Helgoland, and Massachusetts (MA) is also present in the intron phylogenetic tree for both the 1506 (100% MP bootstrap; 99% QPS; 92% NJ bootstrap) and 516 introns (98% MP bootstrap; <50% QPS; 70% NJ bootstrap) (fig. 5 ). In addition, the collections from Ireland, Newfoundland (NF), and B. fuscopurpurea (2) also cluster together within the nuclear SSU rRNA gene analysis (93% bootstrap) and the intron phylogeny for both intron insertion sites. This relationship is fairly well supported for the 1506 intron sequences (72% MP bootstrap; 94% QPS; 83% NJ bootstrap) but not for the 516 intron sequences (61% MP bootstrap; <50% QPS; <50% NJ bootstrap) (fig. 5 ).

Similarly, the 516 intron sequences within the freshwater taxon B. atropurpurea from the British Isles (BI12), Netherlands (NL), Great Lakes (GL), Italy (IT), and Ireland (IR) are positioned in a well-supported clade (100% MP bootstrap; 57% QPS; 85% NJ bootstrap), as are the sequences of the nuclear SSU rRNA gene for these collections. In addition, none of the freshwater collections contained intron 1506. The relationship of the marine collection from the Virgin Islands (VIS7) was unresolved (<55% bootstrap support) in both the intron analyses, and it is only weakly associated (62% bootstrap) with one of the main clades in the NJ nuclear SSU rRNA gene tree.

The relationship among the introns in Porphyra is not as well resolved as that in Bangia. However, some groupings are evident in all three analyses. For example, Porphyra sp. (SY) and P. pseudolinearis (TT) are closely associated in the nuclear SSU rRNA gene phylogeny (75% bootstrap), and the 516 intron sequences for these two taxa are also well associated (99% MP bootstrap; <50% QPS; 99% NJ bootstrap) (fig. 5 ). In addition, the clade consisting of P. tenera (T1, TU-3), P. umbilicalis (NJ), P. yezoensis, and P. yezoensis (HH, OM) is present in the nuclear SSU rRNA gene phylogeny (although not well supported), and this cluster is also seen for the 516 introns for these taxa in figure 5 (100% MP bootstrap; 97% QPS; 76% NJ bootstrap). A close relationship among Porphyra rediviva, Porphyra sp. (Brest), and P. umbilicalis (HG) is reflected in both the nuclear SSU rRNA gene tree (100% bootstrap) and the 1506 portion of the intron phylogeny, with the relationship between P. rediviva and Porphyra sp. (Brest) being well supported (100% MP bootstrap; 100% QPS; 75% NJ bootstrap) and the 1506 intron sequence being only weakly associated with the previous clade (<50% MP bootstrap; 64% QPS; <50% NJ bootstrap). A grouping of Porphyra 1506 intron sequences consisting primarily of collections from Japan (P. yezoensis (OM, OG-1, OG-4, NA-2, NA-4), P. yezoensis narawaenis, P. tenera (KK, SK), P. pseudolinearis (TT), Porphyra sp. (SHH)) is moderately supported (82% MP bootstrap; 83% QPS; <50% NJ bootstrap). This trend is not, however, reflected in the nuclear SSU rRNA gene tree, where the relationship among many of the Japanese taxa is close but unresolved (<50% support). Many of the other relationships seen in the nuclear SSU rRNA gene NJ tree are similar to those in the intron trees, but most are not well supported by bootstrap analysis (<60%). The sequence divergence in the intron at position 516 varied considerably, ranging from 0% to as high as 31.0%. The sequence divergence for the intron in position 1506 was higher than that for the 516 intron (31.0% vs. 44.3%). Interestingly, the 1506 introns in P. spiralis var. amplifolia (Oliveira and Ragan 1994Citation ) were quite distant from all other Porphyra and Bangia taxa, ranging from 30.3% to 44.3% sequence divergence.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Introns within the nuclear SSU rRNA gene from members of the Bangiales have been reported several times within the past few years (Stiller and Waaland 1993Citation ; Oliveira and Ragan 1994Citation ; Oliveira et al. 1995Citation ; Müller et al. 1998Citation ). The presence and absence of these introns presents us with an opportunity to examine lateral intron transfer events and the evolutionary history of introns within the order Bangiales. Oliveira et al. (1995)Citation speculated that these introns could also be used to discern biogeographic or specific entities within the genus Porphyra, as they determined that variants of the group IC1 intron were present in different geographic populations of this alga. Due to the considerable number and variation (as high as 40%) in the introns in Porphyra and Bangia, this system is well suited to addressing some of these issues. In addition, group I introns that lack endonuclease coding regions appear to be nonmobile and provide a potentially valuable tool for tracing the evolutionary history of these sequences (Bhattacharya 1998Citation ).

The similarity in phylogenies between the 516 and 1506 introns and their nuclear SSU rRNA genes suggests that these introns have not been mobile during the evolution of the Bangiales. Introns have been documented in the nuclear-encoded rRNA genes of different Naegleria species. The majority of these occur at the 516 position of the nuclear SSU rRNA gene. A more extensive analysis of the Naegleria nuclear SSU rRNA genes revealed that this intron is absent in the majority of the species' nuclear SSU rRNA genes (De Jonckheere 1994Citation ). De Jonckheere (1994)Citation hypothesized that since there was probably no selective advantage arising from the presence of these introns, they were eliminated in many of the descendants. In the case of the Bangiales, it appears that after initial acquisition of the introns in the ancestor of these taxa, there was subsequent vertical inheritance and evolution within the order (and species) and in some cases loss of the introns in either insertion site, as the intron phylogenies are very similar to that of the host gene. An alternative and less favorable hypothesis is that frequent lateral transfers within the order may have taken place, but this would result in intron phylogenies that would be widely discordant with the nuclear SSU rRNA gene phylogenies (Bhattacharya et al. 1994Citation ).

The similarity among group I intron and nuclear SSU rRNA gene phylogenies has also been reported for other eukaryotic taxa. Takashima and Nakase (1997)Citation noted that introns had been transferred horizontally to distinct insertion sites in the yeast-like fungus Tilletiopsis flava and then inherited vertically. Similarly, introns 1506 and the nuclear SSU rRNA gene sequences share similar phylogenies in members of the green algal order Zygnematales (Bhattacharya et al. 1994Citation ). Wilcox et al. (1992)Citation also postulated that the distribution of introns within other green algal taxa could be explained by inheritance of these sequences along with the gene. However, VanOppen, Olsen, and Stam (1993)Citation concluded that nuclear group I introns in green algae have arisen several times and do not appear to be lineage-specific and disagreed that the distribution of introns could be explained by vertical inheritance within the green algae as postulated by Wilcox et al. (1992)Citation . In the case of the Bangiales, the latter scenario appears to be possible. For instance, the intron phylogenies appear to be in accordance with the gene phylogenies, indicating long-term immobility and vertical inheritance.

The evolutionary history of the Bangiales is quite long, as bangiophyte taxa have been reported in the fossil record from the Proterozoic (1,200 MYA) (Butterfield, Knoll, and Swett 1990Citation ), and the conchocelis stage of Porphyra has been reported in fossils up to 425 MYA (Campbell 1980Citation ). In terms of utilizing the introns within the Bangiales as specific or geographic markers as speculated by Oliveira and Ragan (1994)Citation , the present study does not appear to indicate this possibility. In fact, analyses of the group I introns appear to provide further evidence for the synonymy of these two genera, as intron phylogenies are congruent with those of the nuclear SSU rRNA gene. This is not surprising, as other molecular studies using nuclear and chloroplast genes have indicated that these taxa are not monophyletic genera (Müller et al. 1998Citation ). Thus, attempts at constructing a phylogenetic classification within the order based on morphology, life histories, and molecular analyses, and, in this case, intron sequences, have been problematic.

The observation that similar group I introns are distributed across different genomes and phylogenetically distant taxa is taken as evidence that intron transfer is relatively common (Burke 1988Citation ). The clustering of group I introns in phylogenetic trees from distinct insertion sites suggests that these introns may be traced back to one or more events in which the introns were inserted into rRNA and vertically inherited or lost (Bhattacharya et al. 1994Citation ; Bhattacharya 1998Citation ). Within the Zygnematales, bootstrap analyses yielded little support for the single origin of all group I introns at different insertion sites within rRNA (Bhattacharya et al. 1994Citation ). It is difficult to determine if this is the case with the introns within the Bangiales due to the lack of a suitable outgroup. Nonetheless, all of the 516 and 1506 introns form two separate clades distinct from each other. High sequence similarity between introns is consistent with descent from an ancestral intron that was initially acquired and vertically inherited (Schroeder-Diedrich, Fuerst, and Byers 1998Citation ). In the protist Acanthamoeba, low sequence identity (highly variable) among introns in four different positions suggests that the intron acquisition occurred independently at the four positions after divergence of the taxa within the tree (Schroeder-Diedrich, Fuerst, and Byers 1998Citation ). There is high sequence identity (0% sequence divergence in some cases) among many of the introns sequenced within the Bangiales; however, there is also considerable variability. For example, the 1506 introns in P. spiralis var. amplifolia (B, D, R) differ from all other intron sequences by 30%–44% sequence divergence. Despite this, P. spiralis var. amplifolia is well supported as grouping with the remaining 1506 introns.

Homing-Endonuclease Pseudogenes in the Bangiales
The mobility of nuclear group I introns may depend on homing through the expression of intron-encoded homing endonucleases (Belfort and Roberts 1997Citation ). The His-Cys box is one of several types of homing endonucleases that is restricted to group I introns (Johansen, Embley, and Willassen 1993Citation ). Haugen et al. (1999)Citation described unusual P1 extensions within the nuclear SSU rRNA introns of Bangia and Porphyra. Their analysis showed that the intron sequences contained His-Cys box reading frames within the P1 extension and that these may represent homing-endonuclease pseudogenes. They also demonstrated that the ORF in the 1506 intron of P. spiralis var. amplifolia occurred on the strand complementary to that encoding the SSU rRNA gene and group I intron. The present study also observed this phenomenon for the P1 domain in the introns at position 1506 for P. fucicola and P. umbilicalis (HG), both of which also contained the His-Cys box motif noted in P. spiralis var. amplifolia. The introns in the 516 position that contained an ORF as well as a His-Cys box motif were within the P2 domain and not on the complementary strand, as seen in the 1506 introns. Haugen et al. (1999)Citation also noted frameshifts in the 3' end of the P. spiralis var. amplifolia intron ORF which resulted in a truncated C-terminal end and would explain the lack of cleavage activity in the in vitro translated protein. Haugen et al. (1999)Citation suggested that the truncation and frameshifts seen in the Bangialean intron ORFs may be the result of selection against endonuclease expression which would lethally cleave chromosomal DNA. Only two of the new sequences in this study had frameshifts, P. kanakaensis and B. fuscopurpurea (Helgoland), and all sequences were found to terminate prematurely. This may indicate that these ORFs are no longer functional; however, endonuclease activity has not been studied in these sequences and needs to be investigated.

Secondary-Structure Signatures of the Group IC1 Introns in the Bangiales
In 1987, Woese rationalized that the examination of higher-order structure from a phylogenetic perspective would enable us to examine the stages of evolution of rRNA structure and aid in extrapolating the significance of changes and variation in rRNA. Subsequently, Winker and Woese (1991)Citation delineated the three primary domains using structural characteristics of 16S rRNA. They defined two types of signature characters: (1) homologous positions whose compositions are (very nearly) constant (present in at least 94% of the representative sequences) within each of the domains or kingdoms and are characteristic of at least one, and (2) nonhomologous structures that characterize and distinguish the various groupings (e.g., structures that are present in one group and absent in another and/or structural units that differ strikingly from one group to another). Within these two types there are different examples of phylogenetically constrained elements ranging from single nucleotides and base pairs to hairpin loops, noncanonical pairing, insertions, deletions, and combinations of these elements, along with coaxial stacked helices or differences in numbers between homologous structures (Gutell 1992Citation ).

In the present study, the Bangiales were observed to have sequence and structural elements, or "signatures," that differentiate these introns from each other as well as from other available intron sequences. As noted previously, the P5b domain in the 516 introns of the Bangiales contains a bifurcated helix that distinguishes these introns from all other group IC1 introns (fig. 2a ). This element is not present in the P5b domain in the 1506 bangialean introns and is an example of a nonhomologous signature. The variation in length in the P8 domain of the 1506 introns is also unique to the Bangiales and is not seen in the bangialean 516 introns. In addition to these structural elements, the introns in the Bangiales can be differentiated based on nonhomologous and homologous single-nucleotide and base pair signatures (table 3 ). For example, nucleotide 172 and base pair 322:326 have no homologous positions with respect to T. thermophila and can be considered nonhomologous signatures defining these introns. The two introns can be differentiated based on nucleotide and base pair signatures. For example, position 205 is a U in intron 516 (conserved in >95% of the sequences) and a C in intron 1506 (conserved in >95% of the sequences). These unique structural and sequence signatures provide further evidence that the introns in positions 516 and 1506 are probably the result of a single (although separate from each other) lateral transfer event and subsequent vertical inheritance. In addition, these structural signatures may provide a means for determining the possible origin of the Bangialean 516 and 1506 introns if an ancestral intron still exists. This information also yields a basis for utilizing introns to differentiate different phyla/organisms or to determine the origin of introns based on structural characteristics.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
This research was supported by NSERC grant RGP 0183503 to R.G.S., NIH grant GM 48207 to R.R.G., and an OGS Scholarship to K.M.M. Technical assistance in DNA sequencing by Angela Holliss is gratefully acknowledged. We thank John Stiller, Alison Sherwood, Ron Deckert, Blair Brace, Wilson Freshwater, Mary Koske, and John West for help in collecting or providing samples for this study.


    Footnotes
 
David Irwin, Reviewing Editor

1 Present address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada. Back

1 Keywords: Group IC1 introns Bangiales phylogeny Back

2 Address for correspondence and reprints: Robert G. Sheath, Dean's Office and Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1. rsheath{at}uoguelph.ca . Back


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 Materials and Methods
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Accepted for publication April 10, 2001.