National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba, Japan;
Bio-Oriented Technology Research Advancement Institution, Omiya, Japan
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Abstract |
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Introduction |
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To date, there has been no report on advantageous effects of group I introns on host organisms (see also Belfort 1990
; Edgell, Fast, and Doolittle 1996
). The superfluous insertion sequences in rDNAs, which are one of the most abundant genes in the genome, are likely to be neutral or slightly deleterious to the host's fitness. Therefore, group I introns are regarded as a selfish genetic element maintained on the basis of its capability to replicate and move within and across lineages of the host. If so, the widespread and sporadic distribution of group I introns must have been achieved through a dynamic equilibrium between gains due to their mobility and losses due to drift and/or selection. The following processes are thought to underlie the gain, maintenance, and loss of group I introns: (1) horizontal transmission, in which introns move between different species; (2) transposition, in which introns move to a different location in the host genome; (3) vertical transmission, in which introns do not move and are stably maintained through generations of the host; (4) degeneration, in which maintained introns accumulate mutations, eventually become incapable of splicing, and finally degenerate into a junk sequence; and (5) loss, in which introns are excised out and lost. To explain the sporadic distribution patterns of mobile selfish genetic elements, a hypothesis has been proposed: the cyclical model of invasion, degeneration, and loss, followed by reinvasion (Hurst and McVean 1996
; Goddard and Burt 1999
). However, there has been only one study in which the evolutionary processes were phylogenetically reconstructed in detail and the frequency of the processes was quantitatively estimated (Goddard and Burt 1999
).
In investigating the evolutionary dynamics of mobile genetic elements, the strategy of taxa sampling is very important. When comparison is made between distantly related taxa, it is expected that most evolutionary events will be untraceable and drastic horizontal transmission events will be preferentially detected. In contrast, when we analyze closely related taxa, few evolutionary events such as horizontal transmissions and losses may be detected. In this context, it would be ideal if we could examine a small and well-defined organismal group in which many group I introns exhibited a remarkable variety of insertion patterns.
An ascomycetous fungal genus, Cordyceps (family Clavicipitaceae), embraces some 300 described members which are exclusively endoparasitic to insects and other organisms (Kobayashi 1982
; Samson, Evans, and Largé 1988
; Spatafora and Blackwell 1993
; Shimizu 1994
; Nikoh and Fukatsu 2000
). In the present study, we demonstrate that nuclear rDNAs of Cordyceps fungi contain a large number of group I introns. Surprisingly, as many as 69 group I introns were identified from SSU and LSU rDNAs of 28 representatives of the genus. The evolutionary dynamics of the multiple group I introns in the genus was investigated in detail.
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Materials and Methods |
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Statistical Evaluation of Congruence Between Phylogenetic Trees
To estimate the congruence between phylogenetic trees, Brooks parsimony analysis (Brooks 1981
; Brooks and McLennan 1991
) was performed using the program package MacClade, version 3.07 (Maddison and Maddison 1992
). In this method, all terminal taxa and internal nodes of a phylogenetic tree were converted into a matrix of binary characters which represented the tree topology. Then, a test tree (in this study, an intron tree) was fitted to a binary-coded given tree (in this study, an rDNA tree) under the criteria of Wagner's parsimony. The congruency index between the trees was calculated by (number of characters in binary-coded matrix for a given tree)/(total number of characters mapped on a test tree). To statistically evaluate the level of congruence, the observed congruency index was compared with the null distribution of the index. We generated 100,000 random bifurcate trees, fitted them to the matrix of a given (rDNA) tree, calculated congruency indices for the 100,000 trees, and obtained a null distribution of the index.
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Results |
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SSU rDNA Segment
The size of the amplified fragment using primers NS1 and FS2 was expected to be approximately 1.5 kb. From 22 out of 28 species, however, longer PCR products (1.93.3 kb) were obtained.
5' Region of LSU rDNA Segment
The size of the amplified product using primers CFS2 and NLB1 was expected to be around 1.7 kb. From all 28 species, PCR products of nearly the expected size were obtained.
3' Region of LSU rDNA Segment
The size of the amplified fragment using primers ILA1 and ILB1 was expected to be approximately 0.8 kb. From 21 out of 28 species, longer PCR products (1.22.5 kb) were obtained.
Multiple Insertions Responsible for the Length Variety in the Nuclear rDNA Segments
We determined and analyzed the sequences of the SSU rDNA segment and the 5' region of the LSU rDNA segment from six species of fungi (table 1
) in addition to the sequences from 22 species described in Nikoh and Fukatsu (2000)
. The sequences of the 3' region of the LSU rDNA segment from five representative species (table 1
) in which evidently longer PCR products were identified were also determined. The lengths of the segments ranged from 1,520 to 3,300 bp in the SSU rDNA, from 1,630 to 1,781 bp in the 5' region of LSU rDNA, and from 1,215 to 2,549 bp in the 3' region of LSU rDNA.
Alignment of the sequences revealed that the remarkable length variety in the SSU rDNA and the 3' region of the LSU rDNA segments was due to multiple insertion sequences in these regions.
SSU rDNA Segment
In the SSU rDNA segment, four insertion sites were identified and named SSU516, SSU943, SSU989, and SSU1199, respectively, after the corresponding nucleotide sites in E. coli rRNA (Gutell 1993
). Of 28 sequences determined, 13 were inserted at SSU516, 13 were inserted at SSU943, 11 were inserted at SSU989, and 17 were inserted at SSU1199 (fig. 2
). When all these insertions were removed, the size of the resultant SSU rDNA segments became, as expected, around 1.5 kb. In C. kanzashiana, C. prolifica, C. inegoensis, C. cochlidiicola, and C. coccidiicola, all four sites were occupied by insertions. As a result, for example, the sizes of the SSU rDNA segment from C. kanzashiana and C. prolifica were 3,217 and 3,300 bp instead of the uninserted sizes 1,520 and 1,521 bp, respectively.
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Phylogenetic Distribution of the Multiple Insertions
In order to demonstrate the evolutionary patterns of the insertions, we conducted molecular phylogenetic analysis of the Cordyceps fungi based on their rDNA sequences. The sequences of SSU rDNA, ITS1, 5.8S rDNA, ITS2, and the 5' region of LSU rDNA, from which all of the insertion sequences were removed, were concatenated and subjected to the analysis (fig. 2
). The NJ tree and the ML-PUZZLE tree showed a good overall agreement in their topology, although minor discrepancies were found where statistical supports of the groupings were weak. As previously reported (Nikoh and Fukatsu 2000
), in the Cordyceps, several well-supported monophyletic groups were identified, such as cicada clade A (C. ramosopulvinata, C. kanzashiana, C. prolifica, Paecilomyces sp.), cicada clade B (C. sobolifera, C. yakushimensis, Cordyceps sp. 1), the truffle-cicada clade (C. japonica, C. jezoensis, C. capitata, C. ophioglossoides, C. inegoensis, C. paradoxa, T. inflatum), the moth clade (C. militaris, C. pruinosa, C. takaomontana, Cordyceps sp. 2, Cordyceps sp. 3, P. tenuipes, B. bassiana, B. brongniartii), the scale-moth clade (C. coccidiicola, C. cochlidiicola) and others. As shown in figure 2
, the insertions were generally not phylogenetically restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages. Notably, presence/absence patterns of the insertions were quite complicated, even in most of the well-supported monophyletic groups in the Cordyceps.
Characterization of the Insertion Sequences
DNA database searches revealed that all of the insertions showed significant sequence similarity to group I introns. Moreover, all of the insertions contained sequence elements P, Q, R, and S, which are thought to be needed for formation of the secondary structure of group I introns (fig. 3
; Cech 1988
).
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Phylogenetic Analysis of the Multiple Group I Introns Inserted in the rDNAs of the Cordyceps Fungi
Molecular phylogenetic analyses were performed to examine the relationships between the multiple group I introns of the Cordyceps fungi. Since the introns of the Cordyceps fungi were shown to belong to either subgroup IB3 or subgroup IC1, group I introns of these subgroups were collected from DNA databases and subjected to analysis together with the introns of the Cordyceps.
Subgroup IB3
Figure 4
is the molecular phylogenetic tree of group I introns of the subgroup IB3. Notably, the group I introns of the Cordyceps fungi inserted at the same sites constituted distinct phylogenetic groups. The introns at SSU516 and those at LSU2066 formed good monophyletic groups with high bootstrap values of 99.9% and 97.4%, respectively. The introns at SSU989 and those at LSU2449 constituted monophyletic groups with moderate bootstrap values of 83.5% and 82.9%, respectively. The introns at LSU2563 also formed a monophyletic group, although statistical support of the clade was low. The introns at SSU1199 were also grouped into a cluster, although the cluster contained an alien sequence, a group I intron of Nectria galligena. It should be noted, however, that (1) Nectria is phylogenetically closely related to the Cordyceps and placed in the same group, Hypocreales/Clavicipitales (Spatafora and Blackwell 1993
); (2) the Nectria intron is inserted at the same site, SSU1199; and (3) judging from the tree topology and statistical supports, it appears possible that the Nectria intron might be the most basal branch of the cluster in spite of the tree shape inferred.
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SSU516 Introns
Figure 6A
shows the comparison between the tree of SSU516 introns and the tree of rDNAs. The topologies of the two trees exhibited a good overall congruence. However, several discrepancies were observed in the truffle-cicada clade (C. japonica, C. capitata, C. inegoensis, C. paradoxa) and in the placement of C. heteropoda.
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SSU989 Introns
Figure 6C
represents the comparison between the tree of SSU989 introns and the tree of rDNAs. The topologies of the two trees showed very good overall congruence, with a discrepancy only in the position of Cordyceps sp. 1.
SSU1199 Introns
Figure 6D
shows the comparison between the tree of SSU1199 introns and the tree of rDNAs. The topologies of the two trees showed a good overall congruence, although a number of local discrepancies were observed.
LSU1921 Introns
Figure 6E
represents the comparison between the tree of LSU1921 introns and the tree of rDNAs. The topologies of the two trees agreed perfectly.
LSU2066 Introns
Figure 6F
shows the comparison between the tree of LSU2066 introns and the tree of rDNAs. The topologies of the two trees were perfectly congruent, although the possibility of congruence by chance is not negligible when only four taxa are analyzed.
LSU2449 Introns
Figure 6G
represents the comparison between the tree of LSU1921 introns and the tree of rDNAs. The topologies of the two trees were discrepant. Considering that C. prolifica and C. kanzashiana are closely related (fig. 2
; Nikoh and Fukatsu 2000
), the evolutionary history of LSU2449 introns might be not parallel to that of flanking rDNAs. However, because the statistical support of the grouping of C. prolifica and Cordyceps sp. 2 was very weak, it might be possible that the apparent discrepancy was due to limited resolution of the phylogenetic analysis.
LSU2563 Introns
Figure 6H
shows the comparison between the tree of LSU2563 introns and the tree of rDNAs. The topologies of the two trees were perfectly congruent, although congruence by chance is possible when only three taxa are analyzed.
Statistical Evaluation of the Phylogenetic Congruence
As shown in figure 6
, the group I intron trees and the corresponding rDNA trees appeared to be congruent in their topologies. In order to objectively evaluate the degree of congruence, SSU516, SSU943, SSU989, and SSU1199 introns were subjected to Brooks parsimony analysis (fig. 7
). In the distributions of congruency indices of 100,000 randomly generated trees, the indices of the observed intron trees exhibited very high values at a statistically significant level (P < 10-5), indicating that the congruencies between the group I intron trees and the corresponding rDNA trees could not occur by chance.
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Discussion |
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In total, 69 group I introns were identified in nuclear SSU and LSU rDNAs of 28 endoparasitic fungi representing the genus Cordyceps: 54 introns at 4 specific sites in 28 sequences of SSU rDNA, and 15 introns at 4 specific sites in 5 sequences of LSU rDNA (fig. 2
). If LSU rDNAs of the remaining 23 taxa were sequenced, the number of introns would further increase. To date, several organismal groups have been reported to contain large numbers of group I introns, such as lichen-forming fungi (Gargas, DePriest, and Taylor 1995
), ericoid mycorrhizal fungi (Perotto et al. 2000)
, chloroplasts of Chlamydomonas algae (Turmel et al. 1993
), and others. As far as we know, the Cordyceps fungi are the most prominent reservoir of group I introns so far described.
In filamentous fungi, nuclear rDNA genes are present in tandem repeats ranging from 60 copies in Coprinus (Cassidy et al. 1984
) to 220 in Neurospora crassa (Russell et al. 1984
). It should be noted, therefore, that group I introns may not necessarily be found in all rDNA repeats within the genome (Hibbett 1996
; Perotto et al. 2000
). In fact, we observed a weak intronless rDNA band coamplified with the thick intron-containing rDNA band from several samples examined in this study (data not shown). If group I introns occur at a low frequency in the rDNA repeats, we may fail to detect inserted rDNA copies. In addition, multiply inserted long rDNAs are much less efficiently amplified by PCR than intronless short rDNAs. Taking these factors into account, the number of group I introns identified in this study might still be an underestimate.
The group I introns of the Cordyceps fungi exhibited very interesting physical and phylogenetic distribution patterns. First, the introns were inserted at highly specific sites, four sites in SSU rDNA and four sites in LSU rDNA (figs. 1 and 2 ). Second, introns inserted at the same site were almost always closely related between different species, whereas introns inserted at different sites were distantly related even in the same species (figs. 4 and 5 ). Third, when the phylogenetic relationship of introns at the same site was compared with the phylogeny of their hosts, the topologies were generally highly congruent to each other (figs. 6 and 7 ). Fourth, mapped on the host phylogeny, the introns were not restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages (fig. 2 ). These patterns strongly suggest that the group I introns were present in the ancestor of the Cordyceps fungi, have seldom been mobile but were vertically transmitted through host generations, and have experienced cocladogenesis with speciation of the hosts. Therefore, in the genus Cordyceps, the group I introns appear to behave as a vertically transmitted genetic element rather than as an actively mobile genetic element. The polymorphic insertion patterns of the introns observed are best explained by stable vertical transmission of the elements occasionally interrupted by independent losses in a number of lineages.
One of the most impressive results of this study was the phylogenetic congruencies between the group I introns inserted at the same site and the hosts (fig. 6
). Although the congruencies were statistically highly significant (fig. 7
), it should be noted that the intron trees and the host trees were often not perfectly coincident, but had several local discrepancies. The rDNA trees in figure 6
, although presented as bifurcate cladograms, contained a number of clades that received weak statistical support (see fig. 2
). Because members of the genus Cordyceps are very closely related to each other, intrageneric phylogenetic relationships are sometimes not fully resolved with slow-evolving rDNA sequences (Nikoh and Fukatsu 2000)
. This is the case for the intron trees in figure 6
, because the small sequence size of the group I introns often leads to insufficient resolution and confidence in phylogenetic analysis. Therefore, most of the minor incongruencies between the intron trees and the host trees should be attributed to limited resolution of the phylogenetic analyses. In fact, when poorly supported clades were collapsed, most of the discrepancies were reconciled (data not shown). Of course, the possibility cannot be excluded that part of the discrepancies might come from local horizontal transmissions of group I introns within the genus Cordyceps.
Although vertical transmissions and losses are predominant in the evolution of group I introns in the Cordyceps, at least one or two horizontal transmission events were detected. SSU943 introns of C. prolifica and C. kanzashiana did not cluster with SSU943 introns of other Cordyceps fungi, but fell in a distinct monophyletic group that contained SSU943 introns from the distantly related ascomycetous fungi G. putredinis and R. tritirachium (fig. 5 ). This pattern strongly suggests the possibility that the common ancestor of C. prolifica and C. kanzashiana horizontally acquired the SSU943 intron from a foreign fungal donor. LSU2449 introns of C. prolifica, C. kanzashiana, and Cordyceps sp. 2 formed a clade, in which introns of C. prolifica and Cordyceps sp. 2 were related, with LSU2449 introns from the distantly related ascomycetous fungi Arxula adeninivorans and Gaeumannomyces graminis (fig. 4 ). However, molecular and morphological lines of evidence consistently supported a phylogenetic affinity between C. prolifica and C. kanzashiana, which conflicted with the intron phylogeny (Nikoh and Fukatsu 2000; fig. 6G ). This contradiction might suggest a horizontal transmission event at the LSU2449 site.
In this study, no transposition events were detected. Even in several putative horizontal transmission events described above, it was inferred that foreign group I introns were integrated into the homologous sites in rDNAs of Cordyceps members (see figs. 4 and 5
). The patterns observed in this studyno transposition, occasional horizontal transmissions into homologous sites, and frequent intron lossesmay indicate that homologous recombination is an important mechanism involved in gains and losses of group I introns in nuclear rDNAs of the Cordyceps fungi. It should be noted that reverse transcription followed by homologous recombination is believed to be involved in mobility and loss of group I introns (Dujon 1989
; Belfort and Perlman 1995
). In the group I introns of the Cordyceps fungi, we found no open reading frames that encoded proteins for mobility, such as homing endonucleases, which may also be relevant to the scarcity of horizontal transmissions, in contrast to dominating vertical transmissions and losses. Notably, our results unequivocally indicate that the common ancestor of the Cordyceps must have experienced drastic acquisitions of many group I introns, although it remains a mystery as to what happened at that time.
What processes underlie the horizontal transmission of group I introns across distinct lineages? At present, no convincing evidence is available. In some cases, spatial and ecological proximities through predation, endoparasitism, endosymbiosis, interspecific hybridization, and other processes have been hypothesized to facilitate the transfer events (Nishida and Sugiyama 1995
; Adams et al. 1998
). The horizontal transmission route of group I introns in the Cordyceps fungi may be linked to endoparasitism. Members of the genus Cordyceps are exclusively endoparasitic to various insects and other arthropods (Kobayashi 1982
; Samson, Evans, and Largé 1988
; Spatafora and Blackwell 1993
; Shimizu 1994
; Nikoh and Fukatsu 2000
). When the same insect is infected with multiple parasitic fungi including Cordyceps species, transmission of genetic materials between them might occasionally occur. It is also possible that some introns might have been transferred between the fungi and the host, although no group I introns have so far been reported from insects.
Interestingly, fungal groups that possess many group I introns tend to be highly specialized for symbiotic or parasitic lifestyles, e.g., lichen-forming fungi (Gargas, DePriest, and Taylor 1995
), ericoid mycorrhizal fungi (Perotto et al. 2000)
, yeast-like endosymbionts of anobiid beetles (Noda and Kodama 1996
), and entomoparasitic fungi of the genus Cordyceps. Are there any causal relationships between upkeep of the introns and endosymbiotic/parasitic lifestyles? Although speculative, we suggest that the slow-growing nature of symbionts/parasites might be responsible. It is expected that the more group I introns rDNAs carry, the less efficient synthesis of rRNAs becomes. Because the ribosome is a copious cellular component essential for protein synthesis, multiple group I introns in rDNAs may result in reduced cell growth and division. If so, heavily inserted rDNAs may have a negative fitness effect on most free-living organisms in which the ability to grow rapidly under favorable conditions is essential for their survival and reproduction. On the other hand, the growth rate of endosymbionts/parasites must be suppressed under a strict control to cope with limited space inside the host body, to ensure survival of the host (at least for a while), to efficiently utilize resources from the host, to synchronize their life cycle parameters with those of the host, etc. (Tanada and Kaya 1993
). Therefore, it is expected that the disadvantages due to heavily inserted rDNAs might be relaxed in slow-growing endosymbionts/parasites.
How often have group I introns been acquired and lost in the evolutionary history of the Cordyceps fungi? Figure 8 shows a phylogenetic reconstruction of gains and losses of group I introns in SSU rDNA of the Cordyceps. Based on the results presented in this study, it was assumed that the SSU516, SSU943, SSU989, and SSU1199 introns were either inherited from or acquired by the ancestor of Cordyceps and have subsequently been lost in many lineages independently. Mapped on the host phylogeny based on SSU rDNA sequences, 27 intron losses were parsimoniously estimated under this model (fig. 8 ), although the number may fluctuate due to ambiguity of the tree topology and possible homoplasy. As discussed previously, SSU943 introns of C. prolifica and C. kanzashiana are likely to be horizontally acquired from a foreign fungal donor (see fig. 5 ). In addition, four other SSU introns were identified whose phylogenetic placement significantly conflicted with that in the rDNA tree (fig. 8 , asterisks), which might suggest local horizontal transfers in the genus Cordyceps. On the assumption that these discrepancies are attributable to horizontal transfers, five independent gains of introns through horizontal transfer are parsimoniously estimated on the SSU rDNA phylogeny. If this estimate is accurate, the number of intron losses should increase to 33, because a gain of a new intron must accompany a loss of the original intron at the site in these cases.
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Acknowledgements |
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Footnotes |
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1 Keywords: Cordyceps
group I introns
nuclear ribosomal RNA genes
coevolution
mobile selfish genetic element
2 Address for correspondence and reprints: Takema Fukatsu, National Institute of Advanced Industrial Science and Technology, Agency of Industrial Science and Technology, Tsukuba Central 6, Tsukuba 305-8566, Japan. t-fukatsu{at}aist.go.jp
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