MOLECULARPHYLOGENETICSAND

Molecular Phylogenetics and Evolution 30 (2004) 236–242

EVOLUTION

www.elsevier.com/locate/ympev

Brief communication

The advantages of the ITS2 region of the nuclear rDNAcistron for analysis of phylogenetic relationships of insects:

a Drosophila example

Irene Young and Annette W. Coleman*

Division of Biology and Medicine, Brown University, Providence, RI 02912, USA

Received 18 October 2002; revised 9 April 2003

Abstract

We examined the utility for phylogenetic reconstruction of the second internal transcribed spacer (ITS2), lying between the

nuclear 5.8S gene and the gene for large subunit ribosomal RNA, using sequences of Ceratitis, Bactrocera, Musca, and Drosophila.

We aligned and analyzed 13 sequences from GenBank and 11 new sequences from diverse species of Drosophila. Derivation of the

secondary structure of the ITS2, the RNA transcript folding pattern required for transcript processing into functional RNA units,

revealed the facets of sequence conservation common to all the sequences, that then allowed alignment of all the genera. The re-

sultant tree, though including only a sparse representation of the enormous Drosophila diversity, conforms generally with the

consensus of all prior phylogenetic reconstructions, using eight other nuclear and mitochondrial gene regions; where species rep-

resentation is greater, as in the melanogaster subgroup of the Sophophora subgenus representatives, it conforms exactly. The par-

adigm ITS2 secondary structure presented can now be used to assess the genus more thoroughly, since its base pairing pattern makes

alignment of sequences obvious. In addition, it shows that these insects share the ITS2 secondary structure characteristics of the

other major animal groups as well as the green line of eukaryote evolution. The relatively short (<400 bp) ITS2 region seems ideal

for reconstructing evolutionary relationships at the levels of species, genera, and perhaps even higher.

� 2003 Elsevier Science (USA). All rights reserved.

1. Introduction

Selection of DNA regions suitable for phylogenetic

comparisons among species and genera, even between

families, is always a challenge (Brower and DeSalle,

1994). One wants a region that faithfully reflects geneticrelationships and that can be selectively multiplied by

PCR with ease. Economic and time constraints en-

courage use of a relatively short region with high in-

formation content.

The nuclear ribosomal repeat cistrons have been

widely used for phylogenetic studies of protistan, plant,

and animal species. The nuclear RNA gene sequences

have already proven valuable for higher taxonomiclevels of insects (Pelandakis and Soliqnac, 1993). A re-

gion of these repeats more suitable for genus and species

* Corresponding author. Fax: 1-401-863-1182.

E-mail address: Annette_Coleman@brown.edu (A.W. Coleman).

1055-7903/$ - see front matter � 2003 Elsevier Science (USA). All rights res

doi:10.1016/S1055-7903(03)00178-7

comparisons is the second Internal Transcribed Spacer

region (ITS2), the one that lies between the 5.8S and

large subunit RNA genes. It is typically 200–400 bp in

length, easily amplified by PCR from even miniscule

amount of DNA, and easy to sequence.

Since the ultimate product of the ITS2 regions is anRNA, not a protein with its innate triplet punctuation,

the proper alignment of ITS2 sequences at or above the

genus level has proved challenging in the absence of

other information, for although some subregions are

very highly conserved, others seem free to vary at ran-

dom (Schl€ootterer et al., 1994). Without proper align-

ment, there is little sense in applying programs analyzing

phylogenetic relationships. Even when analysis is limitedto the subregions of unambiguous alignment, significant

information content at the species level is lost. The so-

lution to the alignment problem for ITS regions is the

recognition of the secondary structure formed by the

folding of the primary RNA transcript, a secondary

structure necessary for the ‘‘processing’’ in the nucleolus

erved.

I. Young, A.W. Coleman / Molecular Phylogenetics and Evolution 30 (2004) 236–242 237

of the long primary transcript of the ribosomal cistron(Venema and Tollervey, 1999). The products of pro-

cessing are the small and large subunits of RNA and the

5.8S RNA of the ribosome. During processing, the ITS

regions are degraded to nucleotides.

Although some of the aspects of the ITS2 secondary

structure necessary for processing are now recognized,

the actual folding pattern has largely been established by

the same procedures used to determine the secondarystructure of the RNA components of the ribosomes

themselves. This method involves comparison of po-

tential RNA folding patterns of closely related taxa to

determine which single example is common to all and

supported by compensatory nucleotide substitutions

that always preserve the pairing necessary for this sec-

ondary structure.

One of the first phylogenetic explorations of ITS2using RNA secondary structure as a guide to alignment

was the study of eight Drosophila species by Schl€oottereret al. (1994). Their proposed ITS2 secondary structure

was both incomplete and inapplicable, except for one

hairpin loop, to their outgroup, Musca. Since 1994, the

ITS2 secondary structure has been determined for a

wide variety of eukaryote groups, both plant and ani-

mal, and most recently, with the refolding of the originalyeast model (Joseph et al., 1999), it has become clear

that all these eukaryote groups share the same overall

secondary structure (Coleman and Vacquier, 2002; Mai

and Coleman, 1997; Michot et al., 1999). These devel-

opments open a much broader vista for phylogenetic

application of ITS2.

Since the universal ITS2 secondary structure now

recognized (Coleman, 2003) differs from that originallyproposed by Schl€ootterer et al. (1994), we decided to re-

visit the drosophilid case. Specifically, we wished to

determine if there is indeed a single secondary structure

that characterizes all drosophilids, and if it conforms

with the general eukaryote model. If so, it would make

ITS2 valuable once again for phylogenetic analyses in

this group, and if alignment guided by secondary

structure led to success in aligning even more distantlyrelated insects, a much wider applicability in insect

evolutionary studies.

2. Materials and methods

Data on the species of Drosophila sequenced for

this study are listed in Fig. 2, along with ITS2 se-quences available from GenBank that were also used.

To obtain template DNA, flies were squashed and the

DNA extracted using the protocol for single fly DNA

extraction given as ‘‘1995’’ on the FlyBase website

(http://flybase.bio.indiana.edu). Two microliters of this

DNA extract was added to a 50 ll mixture of buffer,

primers, deoxynucleotides, and MgCl2, as described in

the Taq polymerase protocol of Promega (Madison,WI). After the mixture had first reached 95 �C, 1–

1.5U of Taq polymerase were added. The thermocy-

cler profile was that of Schl€ootterer et al. (1994), and

the forward and reverse primers we used initially are

given in that paper. Subsequently, an internal forward

primer representing a sequence in the 5.8S region

specific to drosophilids (‘‘D5.8S for’’¼ 50-AGAACG

AGCAAACTGTGC-30) was substituted for the origi-nal forward primer, which is near the 30 end of the

SSU RNA gene, and a shorter PCR program was

used (see Coleman et al., 2001).

PCR products were purified from agarose gels by use

of the Qiaquick Gel Extraction kit (Qiagen, Valencia,

CA) and sequenced in both directions, using the ABI

Prism 377 DNA sequencer and Big Dye Chemistry

protocol (Applied Biosystems, Foster City, CA) and thesame primers as for PCR. In two cases, PCR products

were also subcloned into pGem T-vector (Promega,

Madison, WI) for comparison with sequence generated

from direct PCR sequencing.

Sequences were edited and aligned using MacVector

software (Kodak, International Biotechnologies, New

Haven, CT), and the termini of ITS2 conform to those

used in Schl€ootterer et al. (1994). Optimal alignment wasgreatly aided by knowledge of the secondary structure of

the ITS2 region of the primary transcript. This was es-

tablished by submission of the primary sequence of all

the species to the RNA folding website supporting mfold

version 3.1 (available from http://bioinfo.math.rpi.edu/

~mfold/rna/form1.cgi) using the default parameters for

folding (Zuker et al., 1999). Comparisons among the

results for the various species revealed the folding pat-tern common to them all, which in turn established the

regions of relatively conserved primary structure and

hence homology for alignment. Thus, pairing positions

on the 50 side of a hairpin were aligned, and their cor-

responding pairing partners on the 30 side were likewise

aligned for each sequence.

Aligned sequences were subjected to phylogenetic

analysis using PAUP* version 4.0b10 (Swofford, 2002).All nucleotides were weighted equally, and gaps were

treated as missing data. Both parsimony (branch and

bound analysis for tree construction) and distance

(matrix generated by the Kimura two parameter algo-

rithm and tree building by neighbor joining) methods,

with other parameters left at the default position, were

used to derive trees, and variations were tried that in-

cluded all nucleotides, only those present in regions I, II,and III, and only those in regions I and II. Coding gaps

as ‘‘fifth nucleotide’’ did not add any detail to the trees.

The sequence of Musca served as outgroup to the dro-

sophilids. Bootstrap support was evaluated with 100

(parsimony) or 500 (neighbor joining) iterations. New

sequences have been deposited in GenBank and the

alignment is available upon request.

238 I. Young, A.W. Coleman / Molecular Phylogenetics and Evolution 30 (2004) 236–242

3. Results

With either the Schl€ootterer et al. (1994) primer pair or

our ‘‘D5.8S for’’ plus the Schl€ootterer reverse primer,

typically only a single band was found when the PCR

products were run on an agarose gel. Its identity as the

nuclear ribosomal region was confirmed by comparison

of the very conservative 5.8S sequence with that of

previously sequenced insect examples. The length of theDrosophila ITS2, as determined by sequencing, was 320–

429 bp and the percent GC content ranged from 18 to

26% for the full-length sequences.

3.1. RNA transcript secondary structure

The putative secondary structure of the ITS2 RNA

transcript region of Drosophila melanogaster is shown inFig. 1, and serves for all the organisms sequenced, in-

cludingMusca. It is fundamentally similar to that of other

eukaryotes in having: (a) four helix loop regions, of which

(b) the helix in region II is highly conserved and bears a

pyrimidine mismatch within the basal seven nucleotide

pairings, and (c) helix III displays on its 50 side, near theterminus, the single most conserved region of primary

sequence among all the species.A stretch of 10 nucleotidesthere (marked in Fig. 1) is identical among all the Dro-

sophila sequences, as well asMusca,Bactrocera,Ceratitis,

and even Glossina, the tse-tse fly (Chen et al., 1999). This

region is part of a 25 nucleotide sequence essentially

identical among all Drosophila species (also marked in

Fig. 1). There are two sites of variation: Drosophila cru-

cigera, the Hawaiian species, has UC rather than CU in

the nucleotide bulge on the 50 side; and for the A–Upairingmarked by the arrow, all the subgenusSophophora

have the A–U pairing illustrated except Drosophila fi-

cusphila (G–U), Drosophila sturtevant and Drosophila

willistoni (G–C), and Drosophila pseudoobscura (A–C);

the remaining Drosophila species have G–C, except D.

crucigera (A–U). In each case, the nucleotide substitution

is such that pairing potential is maintained.

The helix in region I is also remarkably similaramong the species. In region IV, traditionally the most

variable region of ITS2, D. melanogaster and its five

closest relatives clearly have the potential for two loops

(IVA and IVB in Fig. 1), while the other species and

genera probably have but one, perhaps Y-shaped, the

more standard situation in other organisms. The proof

of secondary structure in region IV would require se-

quencing additional related species for comparisons.One characteristic of the drosophilid secondary struc-

ture that is not often found among other eukaryotes is

the short helix labeled IIa that appears between helix II

and helix III. This is totally absent in all the non-dro-

sophilids examined.

The ‘‘proof’’ of putative secondary structure in RNA

has been the presence of CBCs (Compensatory Base

Changes) as defined in Gutell and Larsen (1994). A CBCis a pairing position in a helix where the sequences of

two related organisms differ at both positions yet retain

the pairing potential (for example, see Coleman, 2003).

Among just the drosophilids used here, there are six

CBCs among the basal nine pairings of helix I; the basal

10 pairings of helix II are identical and the 11th pairing

has a CBC, as does the 12th; and in the relatively con-

served region of helix III marked in Fig. 1 with an ar-row, there is the one position where CBCs occur, as

already noted by Schl€ootterer et al. (1994). Additional

CBCs and numerous hemi-CBCs, pairings where only

one the of the nucleotides is altered appropriately (e.g.,

G–C becomes G–U), are found in the less conserved

regions of helix.

3.2. Phylogenetic analysis

The total ITS2 alignment of Musca and 18 species of

Drosophila evaluated with PAUP* had 597 positions of

which 195 were parsimony informative. The single most

parsimonious tree (Fig. 2) has superimposed on it

bootstrap values obtained from both parsimony and

distance analyses. There were no conflicts among all

methods of tree building as to which clades emerged,only in the level of support for various of these clades.

The tree topology was unchanged, whether using the

entire ITS2 length or only selected regions encompassing

the more conserved helices.

Fig. 2 (inset A) presents a distance tree including

Ceratitis, Bactrocera, Musca, and all the Drosophila

species used. It reaffirms the monophyletic nature of

these drosophilids with respect to the outgroups, but forfurther phylogenetic analysis, the Ceratitis and Bactro-

cera sequences were omitted because they added no

clarity to the trees, only homoplasy. The Drosophila

serrata sequence was also omitted because the sequence

was incomplete (only regions I, II, and 50 of III), but it isclearly most similar to Drosophila takahashi.

4. Discussion

The selection of Drosophila species used here was

chosen both to overlap with previous studies and to

encompass a sufficient range of the diversity within this

large genus to derive and analyze the common ITS2

transcript folding. Both in our experience, and that of

Schl€ootterer, polymorphisms for the entire ITS1-5.8S-ITS2 are minimal (0–0.05%). We failed to find poly-

morphism within an individual, and our sequences of

Drosophila virilis and D. pseudoobscura were identical to

those already in GenBank, suggesting that concerted

evolution (Dover, 1982) has homogenized ITS2 of the

numerous ribosomal repeats sufficiently for its use in

phylogenetic analysis.

Fig. 1. Secondary structure diagram of the primary RNA transcript of Drosophila melanogaster ITS2. The four major folding domains of ITS2 are

designated with roman numerals. The characteristic pyrimidine mismatch of helix II is indicated by an arrowhead. In the region of highest primary

sequence conservation, on the 50 side of helix III, the 10-nucleotide primary sequence common to Drosophila, Musca, Bactrocera, Ceratitis, and

Glossina is circled, while the 25-nucleotides very highly conserved among the drosophilids are bracketed. Arrows indicate the nucleotide pairing in

this conserved portion of the helix that shows CBCs within the genus Drosophila.

I. Young, A.W. Coleman / Molecular Phylogenetics and Evolution 30 (2004) 236–242 239

Approximately 115 nucleotide positions in ITS2 are

relatively conserved (Mai and Coleman, 1997). In

agreement with this, Schl€ootterer et al. (1994) estimated

that 40% of the total ITS is constrained by selection in

Drosophila, and the remainder they found to be un-

constrained positions evolving with a rate that is close

to the neutral rate in this species group. Thus ITS2

combines information most germane to species and

subspecies levels with that useful for comparing even

genera and higher levels.

240 I. Young, A.W. Coleman / Molecular Phylogenetics and Evolution 30 (2004) 236–242

b

Fig. 2. Single most parsimonious tree found by branch and bound analysis of Drosophila species, using all nucleotide positions of ITS2 and Musca as

outgroup. Subgenera designations of drosophilids are shown. Tree length¼ 726 steps, consistency index¼ 0.705, and homoplasy index¼ 0.299. Step

length is shown below each line, and bootstrap values greater than 50% above each line, the first from parsimony and the second from distance

analysis (Kimura two parameter, neighbor joining) of the same data. Inset A: Distance tree, with bootstrap values, using the Drosophila and Musca

sequences, plus Ceratitis and Bactrocera, rooted with the Musca sequence. GenBank sequences used are D. virilis Z28415, D. pseudoobscura Z28460,

Drosophila yakuba Z28416, Drosophila orena Z28549, Drosophila simulans Z28413, Drosophila sechellia Z28412, Drosophila mauritiana Z28538, D.

melanogaster M21017, Musca domistica Z28417, Bactrocera dorsalis AF276516, Bactrocera pyrifoliae AF332590, Bactrocera cucumis AF276515,

Ceratitis capitata AF189691. New sequences, with their Tucson Stockcenter Nos. (//stockcenter.arl.arizona.edu), are D. busckii (#13000-0081.0)

AF551741, Drosophila flavomontana (#15010-0981.0) AF551742, D. crucigera (#15287-2531.0) AF551743, D. takahasii (#14022-0311.0) AF551744,

D. ananassae (#14024-0371.0) AF551750, D. ficusphila (#14025-0441.1) AF551747, Drosophila eugracilis (#14026-0451.1) AF551745, Drosophila

elegans (#14027-0461.0) AF551746, D. serrata (#14028-0681.0) AY175379, D. sturtevanti (#14043-0871.1) AF551748, D. willistoni (#14030-0811.0)

AF551749.

I. Young, A.W. Coleman / Molecular Phylogenetics and Evolution 30 (2004) 236–242 241

Whereas the earlier study that included ITS2(Schl€ootterer et al., 1994) failed to obtain a secondary

structure applicable to all the species used, and hence

missed such aspects as recognition of helix I and its

homologous helix in Musca, our secondary structure

reveals all four major regions and their parallels with

ITS2 of other eukaryotes. This permits alignment of not

only the drosophilids, but also Musca, Bactrocera,

Ceratitis, and Glossina, at least for regions I, II, and III,with great confidence. A cursory examination of the

ITS2 sequences of Chironomus and Glyptotendipes

available from GenBank shows that the first three heli-

ces are easily recognizable, as is the most conserved re-

gion of primary structure, that on the 50 side of helix III.

Thus ITS2 alignments might be made of all the Diptera

for regions I, II, and III.

4.1. Phylogenetic comparisons with other data sets

It was not our purpose to produce a detailed phylo-

genetic analysis of the drosophilids, a project that could

now be carried out using many of the species we have

omitted. The only previous evaluation using ITS was

that of Schl€ootterer et al. (1994) for eight Drosophila

species. In their ITS tree, the arrangement is identical tothat in Fig. 2.

Previous analyses of subsets of Drosophila species

overlapping with those included here made use of se-

quences from the large subunit nuclear ribosomal DNA

gene (Pelandakis and Soliqnac, 1993), mitochondrial

DNA (e.g., DeSalle, 1992), and nuclear genes Adh, Sod,

Gpdh (Katoh et al., 2000; Kwiatowsky and Ayala, 1999;

Russo et al., 1995) and Ddc and amd (Tatarenkov et al.,2001). Further analyses combining data from these

sources and additional species include O�Grady and

Kidwell (2002) and Remsen and O�Grady (2002). Within

the Sophophora subgenus the only notable difference

from these previous studies is the position of D. pseud-

oobscura basal to the Drosophila sturtevanti–Drosophila

willistoni–Drosophila ananassae clade. For the moment,

we attribute this to the absence in our study of the ad-ditional species expected to clarify the basal regions of

the tree. Bettencourt and Feder (2001) summarize the

distribution of species having duplicated heat shockprotein sequences (hsp70), representing a major event in

the Sophophora phylogenetic history. It is particularly

interesting to note that exactly that branch, the clade of

D. ficusphila and more terminal species, is supported by

very high bootstrap values in Fig. 2.

The tree in Fig. 2 (inset A), as in previous studies

limited to these representatives, finds the genus Dro-

sophila monophyletic. The subgenus Drosophila includesthe Hawaiian species, as also found with previous mo-

lecular studies, and the subgenus Sophophora includes

the D. willistoni subspecies clade. Not all of the previous

analyses included the interesting species, Drosophila

busckii, generally treated as belonging to a third sub-

genus, Dorsilopha. A trichotomy directly into the three

subgenera is found in Fig. 2 (inset A).

In summary, the ITS2 evaluation alone, guided bytranscript secondary structure for alignment, does not

conflict with significant support from expectations

from previous studies, and reproduces in remarkable

detail, considering the paucity of included species, the

consensus tree supported by eight other nuclear and

mitochondrial DNA regions. What our complement of

Drosophila ITS2 sequences has shown is that phylog-

eny as analyzed by ITS2 is at least as informative asthat obtained by using any other DNA locus. Where

sufficient species representation is present, the highly

supported clades are the same. The insects, ecdysids,

appear to share the same ITS2 characteristics as do

the other protostome and the deuterostome animal

groups, and higher fungi and plants as well. In 2001,

Tatarenkov et al. suggested in their review of molec-

ular data that ‘‘no single gene has yet produced anunequivocal phylogeny of the Drosophilidae’’; perhaps

the ITS2 region of DNA will contribute very signifi-

cantly to that goal.

Acknowledgments

The authors gratefully acknowledge the kind gift ofdrosophilids from Dr. Kristi Wharton and Dr. David

Rand, Brown University.

242 I. Young, A.W. Coleman / Molecular Phylogenetics and Evolution 30 (2004) 236–242

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