*Department of Genetics, University of Cambridge, Cambridge, England;
Abteilung für Evolutionsbiologie, Institut für Spezielle Zoologie, Westfälische Wilhelms-Universität Münster, Münster, Germany;
Comparative Sequence Analysis Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, England; and
§Department of Biology, University College London, London, England
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Abstract |
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Introduction |
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In the majority of metazoans, ITS1 elements generally provide sufficient levels of nucleotide variation for phylogenetic inferences below the species or genus level (e.g., Schlötterer et al. 1994
; Bowles, Blair, and McManus 1995
; McLain et al. 1995
; Miller, Crabtree, and Savage 1996
; Blair et al. 1997
; Hugall, Stanton, and Moritz 1999
; Schilthuizen et al. 1999
; Schizas et al. 1999
; Harris and Crandall 2000
). However, the extents of variation often differ between closely related taxa (e.g., Miller, Crabtree, and Savage 1996
; Blair et al. 1997
; Harris and Crandall 2000
). Moreover, in some groups, this spacer seems to be too conserved for resolving phylogenetic relationships within genera or even subfamilies (e.g., Kuperus and Chapco 1994
; Booton et al. 1999
). Although the observed differences in the extent of sequence variation between taxonomic groups may have simply resulted from differences in their evolutionary histories (e.g., speciation rates), it is worth noting that they may also reflect different constraints at the molecular level.
ITS1 sequence variation also seems to differ across the spacer region. Throughout the whole subclass Digenea (Platyhelminthes), ITS1 elements have been found to be conserved at the 3' end, whereas complete spacer sequences could only be aligned with confidence for closely related taxa of the same genus or family (von der Schulenburg, Englisch, and Wägele 1999
). Similar findings have also been made for drosophilid dipterans (Insecta) (Schlötterer et al. 1994
). In both cases, 3'-end sequence conservation indicated the presence of functional constraints, supported by studies of yeast for which the 3' end of ITS1 contains recognition sites and conserved secondary structure motifs which have all been found to be essential for rRNA maturation (e.g., Henry et al. 1994
; van Nues et al. 1994
; Weaver, Sun, and Chang 1997
).
In addition, ITS1 evolution seems to be shaped by internal repetition, leading to ITS1 size variation. This repetition includes repetitive elements with comparatively long repeat units, e.g., in trematodes (Platyhelminthes) and dipterans (Paskewitz, Wesson, and Collins 1993
; Kane et al. 1996
; Tang et al. 1996
; van Herwerden, Blair, and Agatsuma 1998, 1999
), or, more commonly, "simple" repetitive sequence motifs, as in various arthropods and in humans (Gonzalez et al. 1990
; Kwon and Ishikawa 1992
; Wesson, Porter, and Collins 1992
; Wesson et al. 1993
; Kuperus and Chapco 1994
; Vogler and DeSalle 1994
; McLain et al. 1995
; Miller, Crabtree, and Savage 1996
; Fenton, Malloch, and Moxey 1997
; Remigio and Blair 1997
; Kumar, Fenton, and Jones 1999
; Harris and Crandall 2000
). In these cases, evolution of repetitive sequences is subject to molecular processes such as replication slippage, unequal crossing over, and biased gene conversion, which are involved in the generation of length variation and/or lead to concerted evolution (e.g., Dover 1982
; Levinson and Gutman 1987
; Elder and Turner 1995
).
ITS1 evolution may also be affected by the extent of homogenization between repeat units of the ribosomal cistron. Inefficient homogenization between repeats, particularly between those on different chromosomes, may then result in intraindividual ITS1 heteroplasmy, as hypothesized for some insects, crustaceans, and trematodes (Wesson, Porter, and Collins 1992
; Vogler and DeSalle 1994
; van Herwerden, Blair, and Agatsuma 1998
; Harris and Crandall 2000
; see also Schlötterer and Tautz 1994
).
Consequently, the evolution of these spacers seems to be characterized by a variety of different factors. The respective underlying evolutionary forces and their relationship to each other are as yet poorly understood. Such information should be of value for our general understanding of the evolution of transcribed spacer DNA and is additionally expected to be of importance in employment of ITS1 as a reliable marker in phylogenetic studies.
The discovery of extreme length and length variation of ITS1 in ladybird beetles (Coleoptera: Coccinellidae) gave us an ideal opportunity to extend our current knowledge on the evolutionary dynamics of these spacers. Using a comparative analysis of the complete range of ITS1 size classes from taxa with different degrees of relatedness, we specifically addressed the following questions: What is the origin of extreme length and length variation? Is it associated with previously identified sources of length variation such as "simple" repetition or long repeat elements? To what extent is a high degree of length variation reflected by a high rate of sequence evolution? Is there evidence for functional constraints on ITS1 evolution? How do such constraints relate to the factors creating length variation?
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Materials and Methods |
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Putatively homologous sequence similarities between and within coccinellid ITS1 sequences were analyzed using three approaches: (1) Dotplot analysis was employed to visualize sequence similarities. We used the program DOTTYPLOT (Gilbert 1990
) with a window size of 35 and a stringency of 19, in accordance with previous studies of length-variable sequences (e.g., Hancock and Dover 1988
). (2) Sequence similarities were assessed using progressive sequence alignment algorithms. For this purpose, alignments were generated with the program CLUSTAL W (Thompson, Higgins, and Gibson 1994
), using a range of gap open and gap extension penalties. (3) Previous studies highlighted that ITS1 elements contain conserved secondary structure motifs, some of which are likely to be of functional importance (cf. von der Schulenburg, Englisch, and Wä gele [1999
] and references therein). Therefore, further support for putative homologies in primary nucleotide sequence was sought via identification of such conserved secondary structures. For this purpose, secondary structures of complete ITS1 elements were calculated using the program MFOLD (Zuker 1989
; see also discussion on relevance of inferred secondary structures as an indication of sequence homologies in Kjer [1995
] Hancock and Vogler [1998
]). Final alignments were then produced taking into consideration the results obtained in the above analyses.
Phylogenetic analysis was performed on putatively homologous ITS1 sequence regions using maximum likelihood as implemented in the program PAUP*, version 4.0b4a (Swofford 1999
). Phylogenetic trees were reconstructed with the heuristic search modus (branch swapping by tree bisection and reconnection) and random addition of sequences based on 10 repetitions. Tree estimation was repeated using a variety of different substitution models. These all yielded the same tree topology. The utility of different substitution models was subsequently assessed with the program MODELTEST (Posada and Crandall 1998
), following the strategy outlined by Huelsenbeck and Crandall (1997)
. The general time- reversible model including rate heterogeneity across sites (Rodríguez et al. 1990
; Yang 1996
) was identified as the best-fitting model. It was therefore employed for estimation of branch lengths and assessment of the robustness of the inferred topology. The latter was performed via nonparametric bootstrapping (Felsenstein 1985
) using the heuristic search modus (branch swapping by tree bisection and reconnection), random addition of sequences with 10 repetitions, and 100 bootstrapped data sets. All alignment illustrations were created with the program GENEDOC, version 2.6.001 (Nicholas and Nicholas 1997
), and phylogenetic trees were created with the program TREEVIEW, version 1.5.2 (Page 1996
).
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Results |
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There was no unambiguous indication of intraindividual ITS1 sequence variation, as the extent of nucleotide differences between the three clones, sequenced for each specimen, was consistent with the error rate of the Taq polymerase used (see Materials and Methods). Base compositions of coccinellid ITS1 sequences were highly similar, ranging from 47% A+T in both Coccidula rufa and Scymnus suturalis to 56% A+T in Psyllobora vigintiduopunctata (21%26% A, 23%29% G, 25%31% T, 19%25% C). However, differences were generally larger between coccinellids and the chrysomelid beetle, D. virgifera, which has a base composition of 70% A+T (33% A, 15% G, 37% T, 15% C).
Finally, analysis based on the BLAST algorithm revealed that nucleotide similarities between the isolated sequences and published data are present only in the included part of the 5.8S rRNA gene. Further similarities to any other previously published sequences could not be identified.
DNA Sequence Similarity Between ITS1 Elements
ITS1 sequence regions from different taxa were considered putatively homologous only if they were (1) consistently identified by progressive sequence alignment programs under a range of gap open and extension penalties and (2) indicated by dotplot analysis. Using this strategy, putatively homologous sequence similarities both between all taxa considered and between the different coccinellid species (excluding D. virgifera) could be inferred for only three regions (figs. 1 and 2
): (1) the 8 bp of the adjacent 18S rRNA gene and 4 bp at the 5' end of ITS1, (2) about 20 bp in the middle of the spacer (block D in fig. 2
), and (3) 87 bp of the adjacent 5.8S rRNA gene and 8 bp at the 3' end of ITS1. With one exception, further interspecific ITS1 sequence homologies could not be ascertained in any subsets of the data, e.g., closely related taxa such as the members of the subfamily Chilocorinae, or those with similar ITS1 sizes. The only exception was for the subfamily Coccinellinae, which showed a high degree of sequence similarity at about 550 nucleotide positions in six regions within the ITS1 element (figs. 1 and 2
): one at the 5' end (190 bp, block A), two at the 3' end (
150 bp and
70 bp for blocks E and F, respectively), and three small dispersed regions of 3050 bp in the middle of the spacer (blocks BD). An additional conserved sequence region corresponds to a repetitive element and will be dealt with below. Secondary-structure calculations, based on complete ITS1 and the adjacent partial 18S and 5.8S rRNA genes, indicated the presence of conserved structural elements in four of these regions (blocks A, C, and EF), further supporting homology of the observed sequence similarities in these regions.
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The results thus obtained revealed sequence divergence between repeats to be low for C. renipustulatus (<5% nucleotide differences), and, to a lesser extent, for H. axyridis (half of the comparisons with <25% and all of them with 40% nucleotide differences). For E. quadripustulatus, the repeats showed considerable sequence variation (26%63%). However, variation was consistently lower within the tandem repeats (26% 45%) than between these and the dispersed copies or between the two dispersed copies (52%63%). The repeats of both A. bipunctata and A. decempunctata yielded about 34%65% differences. For these two species, similarity between repeat units was generally higher between than within species, particularly for those copies present in the same order (fig. 5
).
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Consequently, size increases due to repetitive elements (excluding one copy of the respective repeat unit; simple repetitive elements with copy numbers of 5 were only considered for A. bipunctata) consisted of 13.4%38.5% of the spacer length. Without such repetition, ITS1 would show lengths of 2,162 in E. quadripustulatus and 6761,022 in the other species (table 2
).
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Discussion |
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Sequence analysis on the data from coccinellids indicated a number of factors which characterize ITS1 evolution in this group. In particular, size variation between coccinellid species was associated with a high degree of sequence divergence. For all taxa and almost all subsets of the data, putatively homologous sequence similarities were identified only for three small regions: one in the middle of the spacer and one each at the 3' and 5' ends of ITS1. Hence, sequence similarities are present only in restricted areas, suggesting that they are due to functional constraints. This seems likely for the small region in the middle of the spacer, although such a singular conserved fragment has not yet been reported for any other taxon and its functional importance remains to be determined. Functional constraints are known from the 3' and 5' end of ITS1 (see below). However, only <10 bp are conserved at both the very end and the beginning of ITS1. Therefore, in these cases, sequence conservation could also have been caused indirectly by selection on the adjacent rRNA genes.
In addition, our analysis included data for more than two taxa from two subfamilies of ladybird beetles, the Coccinellinae and the Chilocorinae. Apparent sequence similarities were observed for almost half of the spacer in the Coccinellinae. These were associated with conserved secondary-structure motifs and were primarily found at the 3' and 5' ends. These ITS1 regions have been shown in yeast to contain various elements required for ribosome biogenesis (Henry et al. 1994
; van Nues et al. 1994
; Weaver, Sun, and Chang 1997
). For metazoans, functional importance has been implicated at least for the 3' end (Schlötterer et al. 1994
; von der Schulenburg, Englisch, and Wägele 1999
). Moreover, sequence similarities seemed to be correlated with the degree of relatedness of the taxa included (cf. Ková
1996
; see fig. 1
), and conserved sequence regions were shown by phylogenetic tree reconstruction to contain a highly informative and consistent phylogenetic signal. These observations, taken together, suggest that sequence similarities in the Coccinellinae are due to identity by descent and are most likely the result of direct functional constraints. In contrast, putatively homologous sequence similarities of such an extent could not be identified for the Chilocorinae. This may reflect a recent origin of the subfamily Coccinellinae, whereas the Chilocorinae represent a phylogenetically older taxon. Interestingly, the latter subfamily includes the longest spacer and also shows a much higher degree of size variation than the Coccinellinae. It is therefore also possible that ITS1 evolutionary rates differ between taxonomic groups due to factors associated with the generation of extreme size variation. However, on the basis of the current knowledge of the phylogeny of ladybird beetles (e.g., Ková
1996
), it is not possible to differentiate between these hypotheses.
Extreme size and size variation seem to be associated with "long" repetitive elements. Such a finding is paralleled by previous studies of trematodes and flies. However, in these cases, size variation resulted from copy number differences of homologous repetitive elements (Paskewitz, Wesson, and Collins 1993
; Kane et al. 1996
; Tang et al. 1996
; van Herwerden, Blair, and Agatsuma 1998, 1999
), whereas in coccinellids, it is to a large extent determined by the presence or absence of nonhomologous repetition. In addition, coccinellid ITS1 size variation does not seem to be produced by simple repetition, which has previously been recorded for a variety of organisms to generate size increases in different parts of the genome, including ribosomal DNA (e.g., Hancock and Dover 1988
; Hancock 1995a, 1995b
). In coccinellids, simple repetition was found to be significant only for A. bipunctata, for which it only accounted for minor size differences.
Long repetitive elements, identified for 6 of the 10 coccinellid species studied, were always confined to the middle of the spacer. As functional importance has previously been indicated for the 3'- and 5'-end regions of ITS1, lack of such constraints in the middle of the spacer may have favored the rise of repetition in this region. This is also supported by the fact that repetition has arisen at least four times independently. In particular, three of the six repetitive elements do not show any interspecific similarities, thus implying their independent origins. The remaining elements, all from taxa of the tribe Coccinellini, bear sequence similarities. In this case, the inferred phylogeny supports two equally parsimonious alternatives for their origin: either repetition arose twice independently in the lineages leading to H. axyridis and the genus Adalia, or it arose only once at the base of the tribe, followed by its loss in the lineage leading to C. septempunctata.
These repetitive elements can occupy up to 40% of the spacer length. Without repetition, five of the six species with repetition (only excluding the exceptional case of E. quadripustulatus) would produce a maximum ITS1 length of about 1,000 bp, much more similar in size to those taxa that lack internal repetition. However, extreme size and size variation cannot always be attributed directly to internal repetition. In particular, it is insufficient to explain the size of ITS1 of E. quadripustulatus, where absence of repetition would only reduce ITS1 to a length of 2,162 bp. Furthermore, the ITS1s of Platynaspis luteorubra and S. suturalis are comparatively long (>1,000 bp) but do not bear internal repeats. Nevertheless, the following characteristics suggest that long repetitive elements have generally played a major role in coccinellid ITS1 evolution and may also represent the original source of size increases in the above cases:
Taken together, these observations suggest that internal repetition has occurred in the past and led to current length variation but is no longer detectable due to the obscuring effects of high substitution rates and inefficient homogenization across repeats. The alternative hypothesis, that ITS1 size increases were the result of insertion events, was not confirmed by the results of the BLAST analysis. There were no sequence similarities indicated between coccinellid ITS1 sequences and those of known insertion elements. An analysis of ITS1s of additional taxa, particularly those which are closely related to E. quadripustulatus, P. luteorubra, and S. suturalis, may help us in the future to understand the origin of ITS1 size increases in these cases if they allow detection either of relics of internal repetition or of insertions.
In conclusion, our results indicate that a variety of factors play a role in the evolution of ITS1 elements. ITS1 data from additional coccinellid species may in the future permit a more detailed assessment of the exact relevance of such factors (see above). Comparative analyses of other taxonomic groups, in each case including taxa with different degrees of relatedness, should also be of great value. In particular, such studies may allow identification of a general pattern in the distribution of conserved and thus putatively functionally important ITS1 regions in relation to those that are highly variable. Similarly, they may aid in establishing whether differences in concerted evolution across repetitive elements and repeats of the ribosomal cistron represent a common feature of rDNA evolution, thus favoring the disruption of internal repetition, which may give rise to the presence of hypervariable regions.
Finally, we note that extreme ITS1 size variation should represent a suitable marker for species diagnostics. This may prove useful in ecological studies which involve analysis of early developmental stages of different coccinellid species, such as eggs and first-instar larvae, which cannot be easily identified using morphological traits.
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Acknowledgements |
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Footnotes |
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1 Keywords: Coccinellidae
concerted evolution
internal transcribed spacer
repetitive DNA
ribosomal DNA
2 Address for correspondence and reprints: J. Hinrich Graf von der Schulenburg, Abteilung für Evolutionsbiologie, Institut für Spezielle Zoologie, Westfälische Wilhelms-Universität Münster, Hüfferstr. 1, 48149 Münster, Germany. hschulen{at}uni-muenster.de
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