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Opinion
TRENDS in Genetics Vol.19 No.7 July 2003
ITS2 is a double-edged tool for
eukaryote evolutionary comparisons
Annette W. Coleman
BioMed, Brown University, Providence, RI 02912, USA
The internal transcribed spacer (ITS) region of the
nuclear rDNA cistrons is one of the more frequently
utilized regions for phylogenetic analyses at the genus
and species levels. It has proven valuable for phylogenetic reconstruction of species and genus relationships, using comparisons of primary sequence. Although
potential transcript secondary structure homology is
often utilized to aid alignment in comparisons of ribosomal gene sequences, such consideration has rarely
been applied to ITS primarily because secondary structures for its transcript were not available. Here, we
describe the value of applying ITS2 RNA transcript secondary structure information to improve alignments,
which then allows comparisons at even deeper taxonomic levels. The evolutionarily conserved subportions
of ITS2, apparently necessary for positioning of the
multimolecular transcript processing machinery, also
provide material for distinguishing evolutionarily rare
events, ‘Compensatory Base Changes’ in the relatively
conserved regions, that might be useful in recognizing
how arbitrary are the assignments of classical taxonomic ranks in disparate groups of eukaryotes. This
relatively short and easily sequenced region of DNA –
ITS2 – has yet to be fully exploited in phylogenetics.
The internal transcribed spacer (ITS; see Glossary)
conventionally includes the entire ITS1, 5.8S gene and
ITS2 portion of the nuclear rDNA cistron (Fig. 1). Initial
misgivings about the suitability of the ITS for phylogenetic
comparisons arose from the observation that there are
hundreds of tandem copies of the rRNA cistrons at the
nucleolar organizer locus in a typical eukaryote genome –
that is, it is a multigene family with the potential for
variation among repeats. Accumulating evidence now
suggests that significant variation among ITS sequences
of an organism is found only within organisms that are
hybrids, either diploid or polyploids, of disparate
parents [1]. Except in these, a process called concerted
evolution rapidly homogenizes the many copies of this
multigene family, such that the ITS can be treated as a
single gene [2].
At the genus and species level, a majority of the
numerous studies that have utilized the ITS for phylogenetic reconstruction in diverse eukaryotes report the ITS
to be more informative than gene sequences of the same
organisms (Fig. 2). The ITS-derived relationships conflict
in a few cases with analyses based on morphology, but not
with relationships as recognized from mating studies.
Where such data are available, this is universally true,
down to the finest level of detection [3,4]. As a result, the
ITS has now become the single most frequently utilized
DNA region, at least for plant studies [2], yet its deeper
application has been limited to the level of comparisons of
genera in a family owing to uncertainties in alignment
at higher taxonomic levels. Secondary structure of the
ITS2 transcript provides the key to this problem. With
the insight provided by RNA transcript folding, this
same region is now revealing relationships at order and
higher levels.
Properties of ITS2
Within the ITS, the 5.8S gene sequence is highly conserved
and is useful for verifying the identity of a sequence – that
is, recognizing possible contaminant DNA – and for
designing primers. ITS1 and ITS2 are much more variable
in primary sequence. Initially, ITS sequences appeared to
differ so much between major groups of organisms that it
seemed useless for phylogenetic studies above the species/genus level [5]. However, ITS2, as compared with
ITS1, has subregions of fairly high conservation, sufficient
that Hershkovitz and Lewis [6] and Hershkovitz and
Zimmer [7] attempted an alignment of ITS2 across
angiosperms and beyond. Nevertheless, satisfactory alignment above the genus level remained a problem in the
absence of the innate punctuation provided by the triplet
code for regions producing a protein product. Although
ITS2 seemed to contain information potentially useful
for comparisons at family, order and even higher levels,
these have not generally been attempted, and even the
Glossary
CBC (and hemi-CBC) : compensatory base change involving change of the
nucleotide at both of the two positions that pair with each other in a doublestranded helix. Hemi-CBC is where only one of the two changes, but still
preserves the pairing.
ITS, ETS, SSU, 5.8S, LSU : subportions of each nuclear ribosomal DNA cistron;
respectively, internal transcribed spacer, external transcribed spacer, small
subunit ribosomal RNA gene, 5.8S ribosomal RNA gene, large subunit
ribosomal RNA gene. Cistrons are separated by a region of nontranscribed
spacer.
NOR : Nucleolar organizer region, where ribosomal RNA is transcribed from
the multiple cistrons of an rDNA locus and processed. The result is the
nucleolus, a recognizable region within the nucleus dense with nucleic acids
and proteins.
Taxonomic rank : from lowest to highest category used here: subspecies,
species, genus, family, order, class.
Corresponding author: Annette W. Coleman ([email protected]).
http://tigs.trends.com 0168-9525/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0168-9525(03)00118-5
Opinion
18S (SSU)
5′ ETS
TRENDS in Genetics Vol.19 No.7 July 2003
ITS1 5.8S ITS2
28S (LSU)
3′ ETS
ITS
rRNA transcript
TRENDS in Genetics
Fig. 1. Diagram illustrating the organization of the nuclear ribosomal cistrons (gray
boxes) of a typical eukaryote and their primary RNA transcript.
variable regions most useful at species and population
levels are often omitted owing to lack of guidance for
appropriate alignment.
ITS2 transcript folding – the common secondary
structure model
The ITS2 represents an untranslated insertion near the
50 end of Domain 1 of the region of eukaryotic rDNA
encoding the large ribosomal subunit. The initial transcript of a nuclear ribosomal cistron is a long RNA that
includes all three RNA genes plus the ITS1 and ITS2
spacers. It promptly folds, forming helices that provide
the necessary recognition and docking signals for the
molecular complex that processes this primary transcript
in the nucleolar organizing region into the rRNAs,
reducing ITS1 and ITS2 to nucleotides [8– 10]. As first
analyzed by Schlötterer et al. [11], the more variable
portions of ITS2 appear to evolve at a rate close to neutral,
implying no selection, while the relatively conserved
regions (, 40% of the ITS length in his Drosophila species)
are clearly stabilized by selection. The selective pressure
arises from the need for correct rRNA processing.
If RNA transcript folding structure – secondary
structure – is crucial to the function of ITS2, how does
Order
LSU and
SSU
rDNA
Family
rbcL
ITS2
Genus
mtDNA
ITS
Species
Subspecies
TRENDS in Genetics
Fig. 2. Diagram of the rank order of taxonomic categories. Brackets indicate the
approximate range for which some DNA sequences commonly utilized for phylogenetic comparisons apply. Abbreviations: ITS, internal transcribed spacer;
mtDNA, non-coding regions of animal mitochondrial DNA; rbcL, the large subunit
of the chloroplast gene encoding ribulose bisphosphate carboxylase. The ITS2
bracket represents the range possible by use of transcript secondary structure.
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371
one determine that structure? Although chemical determination of which nucleotide positions are nearest
neighbors in the secondary structure has been utilized,
the major approach to solving the problem is use of
comparative biology, the same method used for rRNAs
[12,13], but at a lower taxonomic level. The potential
folding patterns [14] of the ITS2 transcripts of species
and genera of a family are cross-compared, to discover
the single secondary structure common to them all [15].
The proof of the common secondary structure, as
opposed to other possible folding patterns, lies primarily in the predominance of compensatory basepair
changes (CBCs; see Glossary) [13] that preserve the
pairings essential to the secondary structure helices,
even across an order or higher. Essentially all
nucleotide variation in regions paired in the secondary
structure preserves the pairing potential with CBCs
(meaning, for example, A-U replaced by G-C) or hemiCBCs. The exceptions, where we have reinvestigated,
prove to be sequencing errors. Apparently, other variants
do not survive.
This procedure has so far been applied to ITS2
sequences of green and brown algae, terrestrial plants
and the major animal lineages represented by flatworms, mollusks, insects and vertebrates [4,15– 22]. The
surprising result is that, among the examined eukaryote
groups, all share the same ITS2 secondary structure
model – a four-fingered hand; and so also do the higher
fungi, now that the original yeast model has been
reexamined [23]. In the four-helix model, certain constant
aspects remain recognizable (Fig. 3). Among plants and
green algae, nucleotide sequence evolves most rapidly in
region IV, and next most rapidly in helix I. Helix II is more
stable and characteristically has a pyrimidine – pyrimidine
bulge. Helix III contains on the 50 side the single most
conserved primary sequence, a region of , 20 nucleotides
encompassing the TGGT (or its variants in algae). In the
animal examples, helix II maintains its hallmark pyrimidine bulge. The helix that maintains the longest region
of primary sequence identity is labeled, in some cases
[18,20,23], helix IV, thanks to a short, variable helix
intervening following helix II. For both plants and
animals, the single-stranded regions of the palm of the
four-fingered hand lying between helices I and IV are
also very conserved and are purine rich. In mammals,
this region contains a site just 30 of helix II that is
cleaved early in RNA processing [18]. The net result of
these constraints on ITS2 evolution is that , 110 – 118
nucleotide positions are relatively conserved, whatever
the total length of the ITS2 among organisms of the same
family or order.
Once secondary structure has been established for a
group of related organisms, it can serve a much broader
function than might have been anticipated. It can be used
as a guide to alignment of all the nucleotide positions of the
ITS2 sequences of families and orders, and even higher
taxonomic levels. The pairing pattern in the helices
dictates which nucleotides line up. This has been applied
above the family level only for green algae and flat worms
[19,23], but should be applicable more widely. Hence, we
explore one example here, the angiosperm order Fagales,
Opinion
372
TRENDS in Genetics Vol.19 No.7 July 2003
III
Fagus grandifolia
II
Fagales
The Fagales include familiar trees such as the beeches and
oaks. There are hundreds of Fagales ITS2 sequences in
GenBank, thanks to more than a dozen studies at the
genus and species level. Figure 3 illustrates the secondary
structure of the Fagus grandifolia ITS2 RNA transcript,
established by the standard folding methods and crosscomparison of related species and genera. As expected, it
displays the universal features, four helices, the pyrimidine– pyrimidine bulge on helix II and the TGGT
preserved on the 50 side of helix III. Little alteration is
required to accommodate any other genus in the order; for
example the elbow in helix III shifts over one nucleotide in
the genus Quercus, as compared with Fagus.
After checking for any unexpected ITS2 diversity
among the multiple available sequences of widespread
species, and comparing sequences of species in each genus,
we selected one sequence of each genus for an alignment,
guided by secondary-structure-defined homologous positions. Two sequences from different families of Cucurbitales, a sister order, were added as outgroups. A simple
distance-based analysis with 1000 bootstrap iterations
produced the tree in Fig. 4.
80
92
59
91
59
IV
90
93
100
50
64
54
I
98
TRENDS in Genetics
Fig. 3. Diagram of secondary structure of ITS2 transcript of Fagus grandifolia
(GenBank AF457015) derived by comparisons among all Fagales genera, supported by CBCs and hemiCBCs (see Glossary) that preserve the helix pairings. The
four domains, each with a stem –loop, are labeled I –IV. In helix II, the characteristic
pyrimidine–pyrimidine bulge is obvious. In helix III, the TGGT common to essentially all Angiosperms is indicated by a bracket. In domains II and III and in the
‘palm’ of the four-fingered hand, nucleotide positions that show a conservation
level among Fagales of $1.3 (here, essentially the same nucleotide in .83% of
sequences) are circled. These represent the relatively conserved positions. The conservation value (C) at each position was calculated using the formula in Gutell [12].
Four nucleotide pairs are boxed in helix III; these show CBCs in Cucumis and/or
Corynocarpus, by comparison with the Fagales sequences. In the proximal box,
A –U is G– C in Corynocarpus, the next G –C is A–U in Cucumis, and, in the next
two boxes, C– G and A –U are, respectively, U– A and G –C in both outgroup taxa.
to see whether this group of flowering plants is similar to
groups previously studied and to illustrate both the
application of secondary structure to alignment and the
remarkable degree of ITS2 conservation.
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67
86
90
Corynocarpus
Cucumis
Betula
Corylus
Carpinus
Betulaceae
Ostrya
Ostryopsis
Alnus
Rhoipteleaceae
Rhoiptelea
Carya
Pteryocarya
Juglandaceae
Juglans
Engelhardia
Nothofagus
Nothofagaceae
Fagus
Lithocarpus
Castanea
Castanopsis
Chrysolepis
Q. ilex
Fagaceae
Q. engelmannii
Q. virginiana
Q. rubra
Trigonobalanus
Colombobalanus
TRENDS in Genetics
Fig. 4. Dendrogram of 20 genera of Fagales, plus Cucurbitales genera Corynocarpus similis (AF149009) and Cucumis melo (M36377) as an outgroup. Brackets link
genera in each of five families [38]; GenBank entries are Betula papyrifera
(AF432067), Corylus heterophylla (AY006355), Carpinus betulus (AF432027), Ostrya
carpinifolia (AF432059), Ostreopsis davidiana (AF081527), Alnus accuminata
(AJ251673), Rhoiptelea chilianthus (AF303800), Carya ovata (AF303822), Pteryocarya stenoptera (AF179587), Juglans ailanthifolia (AF179567), Engelhardia spicata
(AF303802), Nothofagus grandis (U96859), Fagus grandifolia (AF457015) Lithocarpinus echinifer (AY040399), Castanea seguinii (AY040395), Castanopsis echinocarpa (AY040375), Chrysolepis chrysophylla (AF389087), Quercus ilex (AF098432),
Quercus engelmannii (AF098420), Quercus virginiana (AF098427), Quercus rubra
(AF0098418), Trigonobalanus verticillata (AF098413) and Colombobalanus excelsa
(AF098412). PAUP* version 4.0b10 [39] was used to derive the tree from a Kimura
two-parameter distance matrix analyzed by the neighbor joining algorithm, with
all sites weighted equally and ignoring gaps in the alignment. Tree length ¼ 540;
CI ¼ 0.55; HI ¼ 0.45. Values at nodes ¼ bootstrap support expressed as percentage
of 1000 repetitions. There are 114 parsimony informative positions in the total
alignment of 263 characters.
Opinion
TRENDS in Genetics Vol.19 No.7 July 2003
In the Fagales tree, derived solely from ITS2 data, each
family is indeed a clade. Branching topography does not
conflict with any prior analyses of these families, using 18S
rDNA or the chloroplast genes atp B, mat K and rbc L,
although further resolution in terminal branches would
require addition of more sequences. The branching pattern
within the family Fagaceae is the same as that obtained by
Manos [24], using the entire ITS region. A fundamental
dichotomy at the base of the tree, separating the family
Fagaceae from the remainder, is characteristic of all
previous trees where authors have sampled the entire
order using plastid genes rbc L and atp B of five genera [25],
or 18S plus rbc L and atp B of nine genera [26]. The only
prior trees that included the interesting genus Nothofagus
are those in Manos and Steele [27], where 16 genera of
Fagales were compared using mat K and rbc L. The trees
differ from Fig. 4 only in that they found support for
Nothofagus as being basal to the remainder of the genera,
and we do not find support for this with ITS2. With the
exception of this moot point, ITS2 analysis alone reproduces the phylogenetic pattern found using all these
other loci, from genus through to ordinal level.
Exceptional ITS2 sequences among Fagales
Many examples in the literature of multiple cloned
sequences of an organism suggest that, at most, one or a
very few nucleotides differ among the ITS copies of an
organism, or even among multiple specimens of the same
species; and the variant nucleotides are not distributed
randomly, but almost always in regions of the secondary
structure that remain unpaired. Almost all sequences of
Fagales in GenBank conform to these prior expectations.
However, the analysis of Fagales ITS2 turned up exceptions, some of which are illustrative of experience with
other eukaryote groups as well. One set of exceptional
sequences from various oak species are all seriously
variant, for they show deletions of portions crucial to the
ITS2 secondary structure. They all stem from a single
study [28] where there was some problem in methodology
or DNA preparation (discussed in [29]).
The second area of exception involves hybrids. Among
known species hybrids of Fagales genera, the parents in
most cases are near identical in ITS2 sequence. However,
one case stands out, that of two oak species, Quercus robur
and Quercus petraea, known to be interfertile. In an
extensive study, Muir et al. [30] found that individual trees
of either species could yield the same three types of ITS2
sequence per individual tree sampled. The implication is
that these two taxa derive from an ancient cross, and ITS
cistron homogenization has not yet gone to completion. To
see which cistron type was active, Muir et al. determined
sequences of transcribed RNA in one such tree and found
that only one of the three cistrons seemed to be expressed.
In some other eukaryote groups, variant ITS sequences,
typically with significant deletions affecting secondary
structure, are known to be pseudogenes, for example in
Drosophila melanogaster (GenBank X15707). However,
analysis of the transcript secondary structure of all three
of the Q. petraea/robur ITS2 types mentioned above
suggests that they maintain folding perfectly, thanks to
several compensating nucleotide changes. Hence, there is
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no evidence that they are pseudogenes from their
sequence. Interestingly, these and other species of oaks
have nucleolar organizer region (NOR; see Glosssary) sites
on more than one pair of homologs [31], probably an
indication of past polyploidy and/or hybridization. Where
there are multiple NOR regions in a genome, homogenization might not be as rapid [1]. Alternatively, or in
addition, the other two ITS2 types might be expressed in
another of the specimen trees, if examined. In plants
particularly, methylation often leads to silencing (repression of transcription) of some of the multiple copies
of a gene family [1]. Further analyses of the origin of
Q. petraea/robur might give insight into the role of
polyploidy, which is so common in plants.
Methods for comparing higher taxonomic levels
Knowledge of transcript secondary structure might help
clarify how arbitrary are assignments of traditional
taxonomic ranks (see Glosssary) among major eukaryote
groups [32]. Taxonomic designations clearly do not
represent comparable levels of evolutionary divergence
across eukaryotes. Several authors have tried deriving an
ITS or ITS2 mutation rate to compare evolutionary
divergence among organisms. In addition to the universal
problem of rate heterogeneity, there is an innate problem
with ITS2, thanks to its length variation among different
compared taxa. Because the overall length of ITS2 varies,
the longer the ITS2, the greater the proportion of
nucleotide sites free to vary, and hence the greater the
percentage pairwise difference between related organisms. This makes calculation of a single mutation rate for
ITS2, based on the entire sequence, not broadly applicable
and helps to explain why there is no consensus on the
percentage pairwise difference that indicates species level
or genus level.
A totally different approach to measuring the passage of
evolutionary time is to ascertain where in a lineage the
first CBC appears that involves a pairing of two relatively
conserved positions (red circled in Fig. 3) that participate
in transcript helix pairings, and compare this with the
taxonomic level to see whether there is some broad
consistency between eukaryote groups [33]. This is
particularly intriguing because essentially all eukaryotes
have an ITS2, and the transcript secondary structures are
now seen to be directly comparable. This level of
comparison arises just at the point where homoplasy
from single nucleotide variation begins to overwhelm the
value of direct sequence comparison.
There are no CBCs involving ITS2 conserved nucleotide
positions in the order Fagales (for the five of eight families
examined). However, there are four pairing positions
where CBCs differentiate all Fagales ITS2 sequences
from those of the Cucurbitales used for comparison (Fig. 3).
Two of them are found in both outgroup sequences, one
characterizes Corynocarpus alone, and one is found in
Cucumis only. Thus, although the Fagales conserved
sequence pairings exhibit no CBCs, the Curcurbitales as
an order clearly do. The only other group of Angiosperms
analyzed similarly is the subfamily Maloideae of the
Rosales, where no CBCs involving the conserved nucleotide positions of ITS2 were found among 20 genera of the
374
Opinion
TRENDS in Genetics Vol.19 No.7 July 2003
subfamily, but CBCs appear above the family level [15].
These observations provide a satisfying confirmation of
what most botanists recognize but rarely discuss.
Among other eukaryotes analyzed, the abalone genus
(Haliotis) has no CBCs [16], and the enormous genus
Drosophila has three CBCs [4]. The only order of green
algae analyzed so far, the Volvocales, which includes
hundreds of both unicellular and colonial flagellate
genera, encompasses at least 29 CBCs, each unique to
one or more genera [33]. Whether the level of the CBC
clade [33], derived from analysis of the ITS2 conserved nucleotide positions, can contribute significantly
to our broader biological understanding of the taxonomic
hierarchy remains to be seen.
Concluding remarks
In sum, once secondary structure has been established for
the ITS2 transcript of a group of organisms, supported by
CBC analyses across multiple species and genera, one can
align ITS2 sequences across even broader taxonomic
levels, certainly for orders and perhaps higher. Thus,
with a single locus of a few hundred nucleotides, relatively
easy to sequence, one can analyze relationships from
the subspecies through to the ordinal level, meanwhile
benefiting from the double-check on possible sequence
errors, which become obvious in alignments guided by
secondary structure, and the ready recognition of pseudogenes and hints of past hybridization events. The
advantages are both convenience and a broader taxonomic
range of comparison, when based on a total alignment. A
detailed example of the methodology of using secondary
structure to aid alignment is given by Kjer [34], using LSU,
where the practice has become common; and Mai and
Coleman [23] describe the reiterative process of deriving
ITS2 secondary structure.
Paradigm structures of completely folded ITS2, supported by CBC analyses utilizing comparisons of related
species and genera, are available for plants, fungi, animals
and green and brown algae, but none has yet been
elaborated for such diverse protistan groups as diatoms,
dinoflagellates and red algae. The very abbreviated ITS2
(55 nucleotides) reported for the protozoan Giardia [35,36]
remains a puzzle, as does the even smaller ITS2 reported
for Variomorpha [37]; interestingly, both these organisms
are parasites. As ITS2 secondary structures accumulate,
their variant conformations should contribute information
useful to students of the evolution of RNA processing in
eukaryotes [18,23]. Eventually, we will understand what
underlies the remarkably conserved secondary structure
of this genetic region with a transcript half-life probably
measured in seconds to minutes, but absolutely necessary
for the formation of ribosomes and hence for life.
Acknowledgements
The criticisms and suggestions of three good reviewers are very much
appreciated.
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References
1 Buckler, E.S. et al. (1997) The evolution of ribosomal DNA: divergent
paralogues and phylogenetic implications. Genetics 145, 821 – 832
2 Hershkovitz, M.A. et al. (1999) Ribosomal DNA sequences and
http://tigs.trends.com
27
angiosperm systematics. In Molecular Systematics and Plant Evolution (Hollingsworth, P.M. et al., eds), pp. 268 – 326, Talor & Francis
Fabry, S. et al. (1999) Intraspecies analysis: comparison of ITS
sequence data and gene intron sequence data with breeding data for
a world-wide collection of Gonium pectorale. J. Mol. Evol. 48, 94 – 101
Young, I. and Coleman, A.W. The advantages of the ITS2 region of the
nuclear rDNA cistron for analysis of phylogenetic relationships of
insects: a Drosophila example. J. Mol. Evol. (in press)
Baldwin, B.G. (1995) The ITS region of nuclear ribosomal DNA: a
valuable source of evidence on angiosperm phylogeny. Ann. Missouri
Bot. Gard. 82, 247– 277
Hershkovitz, M.A. and Lewis, L.A. (1996) Deep-level diagnostic value
of the rDNA-ITS region. Mol. Biol. Evol. 13, 1276 – 1295
Hershkovitz, M.A. and Zimmer, E.A. (1996) Conservation patterns in
angiosperm ITS2 sequences. Nucleic Acids Res. 24, 2857– 2867
Hadjiolova, K.V. et al. (1994) Processing of truncated mouse or human
rRNA transcribed from ribosomal minigenes transfected into mouse
cells. Mol. Cell. Biol. 14, 4044 – 4056
Lalev, A.I. and Nazar, R.N. (1999) Structural equivalence in the
transcribed spacer of pre-rRNA transcripts in Schizosaccharomyces
pombe. Nucleic Acids Res. 27, 3071– 3078
Venema, J. and Tollervey, D. (1999) Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet. 33, 261 – 311
Schlötterer, C. et al. (1994) Comparative evolutionary analysis of
rDNA ITS regions in Drosophila. Mol. Biol. Evol. 11, 513 – 522
Gutell, R.R. et al. (1985) Comparative anatomy of 16-S-like ribosomal
RNA. Prog. Nucleic Acid Res. Mol. Biol. 32, 155 – 215
Gutell, R.R. et al. (1994) Lessons from an evolving rRNA: 16S and 23S
rRNA structures from a comparative perspective. Microbiol. Rev. 58,
10 – 26
Zuker, M. et al. (1999) Algorithms and thermodynamics for RNA
secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology In NATO ASI Series (Barciszewski, J. and
Clark, B.F.C., eds) pp. 11 – 43, Klewer Academic Publishers
Mai, J.C. and Coleman, A.W. (1997) The internal transcribed spacer 2
exhibits a common secondary structure in green algae and flowering
plants. J. Mol. Evol. 44, 258 – 271
Coleman, A.W. and Vacquier, V.D. (2002) Exploring the phylogenetic
utility of ITS sequences for animals: a test case for abalone (Haliotis).
J. Mol. Evol. 54, 246 – 257
Michot, B. et al. (1993) Conserved secondary structures in the ITS2 of
trematode pre-rRNA. FEBS Lett. 316, 247– 252
Michot, B. et al. (1999) Evolutionarily conserved structural features in
the ITS2 of mammalian pre-rRNAs and potential interactions with the
snoRNA U8 detected by comparative analysis of new mouse sequences.
Nucleic Acids Res. 27, 2271– 2282
Morgan, J. and Blair, D. (1998) Trematode and Monogenean rRNA
ITS2 secondary structures support a four-domain model. J. Mol. Evol.
47, 406 – 419
Oliverio, M. et al. (2002) ITS2 rRNA evolution and its congruence with
the phylogeny of muricid neogastropods (Caenogastropoda, Muricoidea). Mol. Phylogenet. Evol. 25, 63 – 69
Wesson, D.M. et al. (1992) Sequence and secondary structure
comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae). Mol.
Phylogenet. Evol. 1, 253 – 269
Yatsukura, N. et al. (1999) Little divergence in ribosomal DNA internal
transcribed spacer-1 and -2 sequences among non-digitate species of
Laminaria (Phaeophyceae) from Hokkaido, Japan. Phycol. Res. 47,
71 – 80
Joseph, N. et al. (1999) Ribosomal internal transcribed spacer 2 (ITS2)
exhibits a common core of secondary structure in vertebrates and
yeast. Nucleic Acids Res. 27, 4533– 4540
Manos, P.S. and Steele, K.P. (1997) Phylogenetic analyses of ‘higher’
Hamamelididae based on plastid sequence data. Am. J. Bot. 84,
1407– 1419
Savolainen, V. et al. (2000) Phylogenetics of flowering plants based on
combined analysis of plastid atpB and rbcL gene sequences. Syst. Biol.
49, 306 – 362
Soltis, D.E. et al. (2000) Angiosperm phylogeny inferred from
18S rDNA, rbc L, and atp B sequences. Bot. J. Linnean Soc. 133,
381– 461
Manos, P.S. et al. (2001) Systematics of Fagaceae: phylogenetic tests of
reproductive trait evolution. Int. J. Plant Sci. 162, 1361 – 1379
Opinion
TRENDS in Genetics Vol.19 No.7 July 2003
28 Samuel, R. et al. (1998) ITS sequences from nuclear rDNA suggest
unexpected phylogenetic relationships between Euro-Mediterranean,
East Asiatic and North American taxa of Quercus (Fagaceae). Plant
Syst. Evol. 211, 129– 139
29 Mayol, M. and Rossello, J.A. (2001) Why nuclear ribosomal DNA
spacers (ITS) tell different stories in Quercus. Mol. Phylogenet. Evol.
19, 167 – 176
30 Muir, G. et al. (2001) Three divergent rDNA clusters predate the
species divergence in Quercus petraea (Matt.) Liebl. and Quercus robur
L. Mol. Biol. Evol. 18, 112 – 119
31 Zoldos, V. et al. (1999) Molecular-cytogenetic studies of ribosomal
genes and heterochromatin reveal conserved genome organization
among 11 Quercus species. Theor. Appl. Genet. 99, 969 – 977
32 Ereshevsky, M. (2001) The Poverty of the Linnaean Hierarchy, pp. 326,
Cambridge University Press
33 Coleman, A.W. (2000) The significance of a coincidence between
evolutionary landmarks found in mating affinity and a DNA sequence.
Protist 151, 1 – 9
34 Boothroyd, J.C. et al. (1987) An unusually compact ribosomal DNA
repeat in the protozoan Giardia lamblia. Nucleic Acids Res. 15,
4065– 4083
35 Kjer, K.M. (1995) Use of rRNA secondary structure in phylogenetic
studies to identify homologous positions: and example of alignment
and data presentation from the frogs. Mol. Phylogenet. Evol. 4,
314– 330
36 Healey, A. et al. (1990) Complete nucleotide sequence of the ribosomal
RNA tandem repeat unit from Giardia intestinalix. Nucleic Acids Res.
18, 4006
37 Vossbrinck, C.R. et al. (1993) Ribosomal DNA sequences of
Encephalitozoon hellem and Encephalitozoon cuniculi; species identification and phylogenetic construction. J. Eukaryot. Microbiol. 40,
354– 362
38 The Angiosperm Phylogeny Group, (1998) An ordinal classification for
the families of flowering plants. Ann. Missouri Bot. Gard. 85, 531 – 537
39 Swofford, D.L. (2002) PAUP*. Phylogenetic Analysis Using Parsimony
(* and Other Methods). Version 4. Sinauer Associates
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