Insect Molecular Biology (2005) 14(6), 625–643© 2005 The Royal Entomological Society 625Blackwell Publishing, Ltd.Assessin...
626 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Kellogg’s whitefly, ...
Bizarre insertions in strepsipteran 18S rRNA 627© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
628 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Figure 1. The secon...
Bizarre insertions in strepsipteran 18S rRNA 629© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
630 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Psocoptera 0.43, 61...
Bizarre insertions in strepsipteran 18S rRNA 631© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
632 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643is similar in all s...
Bizarre insertions in strepsipteran 18S rRNA 633© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
634 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643
Bizarre insertions in strepsipteran 18S rRNA 635© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
636 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Figure 4. (Continued)
Bizarre insertions in strepsipteran 18S rRNA 637© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
638 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643structural characte...
Bizarre insertions in strepsipteran 18S rRNA 639© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
640 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643female (Johnston et...
Bizarre insertions in strepsipteran 18S rRNA 641© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
642 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643loops in ribosomal ...
Bizarre insertions in strepsipteran 18S rRNA 643© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–...
Upcoming SlideShare
Loading in …5
×

Gutell 095.imb.2005.14.625

398 views

Published on

Gillespie J.J., McKenna C.H., Yoder M.J., Gutell R.R., Johnston J.S., Kathirithamby J., and Cognato A.I. (2005).
Assessing the Odd Secondary Structural Properties of Nuclear Small Subunit Ribosomal RNA Sequences (18S) of the Twisted-Wing Parasites (Insecta: Strepsiptera).
Insect Molecular Biology, 14(6):625-643.

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
398
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
0
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Gutell 095.imb.2005.14.625

  1. 1. Insect Molecular Biology (2005) 14(6), 625–643© 2005 The Royal Entomological Society 625Blackwell Publishing, Ltd.Assessing the odd secondary structural properties ofnuclear small subunit ribosomal RNA sequences (18S) ofthe twisted-wing parasites (Insecta: Strepsiptera)J. J. Gillespie*, C. H. McKenna*, M. J. Yoder*,R. R. Gutell†, J. S. Johnston*, J. Kathirithamby‡ andA. I. Cognato**Department of Entomology, Texas A & M University,College Station, TX, USA; †Institute for Cellular andMolecular Biology, University of Texas, Austin, TX, USA;and ‡Department of Zoology, University of Oxford,Oxford, UKAbstractWe report the entire sequence (2864 nts) and second-ary structure of the nuclear small subunit ribosomalRNA (SSU rRNA) gene (18S) from the twisted-wingparasite Caenocholax fenyesi texensis Kathirithamby& Johnston (Strepsiptera: Myrmecolacidae). The majorityof the base pairings in this structural model map on tothe SSU rRNA secondary and tertiary helices that werepreviously predicted with comparative analysis.Theseregions of the core rRNA were unambiguously alignedacross all Arthropoda. In contrast, many of the variableregions, as previously characterized in other insecttaxa, had very large insertions in C. f. texensis. Thehelical base pairs in these regions were predicted witha comparative analysis of a multiple sequence alignment(that contains C. f. texensis and 174 published arthropod18S rRNA sequences, including eleven strepsipterans)and thermodynamic-based algorithms. Analysis of ourstructural alignment revealed four unusual insertionsin the core rRNA structure that are unique to animal18S rRNA and in general agreement with previouslyproposed insertion sites for strepsipterans. One curi-ous result is the presence of a large insertion within ahairpin loop of a highly conserved pseudoknot helix invariable region 4. Despite the extraordinary variabilityin sequence length and composition, this insertioncontains the conserved sequences 5′′′′-AUUGGCUUAAA-3′′′′ and 5′′′′-GAC-3′′′′ that immediately flank a putativehelix at the 5′′′′- and 3′′′′-ends, respectively. The longersequence has the potential to form a nine base pairhelix with a sequence in the variable region 2, consistentwith a recent study proposing this tertiary interaction.Our analysis of a larger set of arthropod 18S rRNAsequences has revealed possible errors in some of thepreviously published strepsipteran 18S rRNA sequences.Thus we find no support for the previously recoveredheterogeneity in the 18S molecules of strepsipterans.Our findings lend insight to the evolution of RNA struc-ture and function and the impact large insertions poseon genome size. We also provide a novel alignmenttemplate that will improve the phylogenetic placementof the Strepsiptera among other insect taxa.Keywords: rRNA, ribosome, Strepsiptera, secondarystructure, insertion, homology, variable regions, 18S.IntroductionFor nearly a decade it has been known that the ribosomalRNA (rRNA) genes of strepsipteran insects possessextraordinarily expanded sequences in less conservedregions of the rRNA molecules when compared to otherarthropods (Chalwatzis et al., 1995; Whiting et al., 1997;Hwang et al., 1998; Choe et al., 1999b). This peculiarcharacteristic of strepsipteran 18S rRNA has been hypoth-esized to correlate with the unusual biology exhibited bythese bizarre insects (Chalwatzis et al., 1995). However, ithas been shown that other organisms with less unusualbiologies also have greatly expanded rRNA genes, espe-cially in expansion segments and variable regions (e.g.Schnare et al., 1996;Wuyts et al., 2000;Alvares et al., 2004).In particular, complete sequences of the 18S rRNA gene ofseveral arthropods (two crustaceans and three hemipterans)are exceptionally larger than average (1800–1900 bp):3214 bp in the soil bug, Armadillidium vulgare (Choe et al.,1999a); 2293 bp in the water flea, Daphnia pulex (Crease &Colbourne, 1998); 2469 bp in the pea aphid, Acyrthosiphonpisum (Kwon et al., 1991);2373 bp in the California red scale,Aonidiella aurantii (Campbell et al., 1994); and 2496 bp indoi: 10.1111/j.1365-2583.2005.00591.xReceived 6 March 2005; accepted after revision 11 June 2005.Correspondence: Joseph J. Gillespie, Department of Entomology, Texas A& M University, College Station, TX 77843, USA. Tel.: +1 979 458 0579;fax: +1 979 845 6305; e-mail: pvittata@hotmail.com (for correspondence),pvittata@gmail.edu (for large attachments)
  2. 2. 626 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Kellogg’s whitefly, Pealius kelloggii (Campbell et al., 1995).Alist of other unusually long metazoan 18S rDNA sequencesis provided by Giribet & Wheeler (2001). Given these data,we question whether or not the extremely expanded rRNAsequences of strepsipterans can actually be associated withthe highly unusual biology exhibited by this bizarre insect taxon.Indeed, strepsipterans are odd in that the larvae are freeliving in the first instar, later developing into apodousendoparasites of other insect species (Kathirithamby, 1989).Females (except Mengenillidae) reside inside their hostsfor the remainder of their life. The majority of the male lifecycle is spent as a larval endoparasite, with short-livedwinged adults seeking females for reproduction. Particu-larly strange are the Myrmecolacidae, in which males andfemales parasitize hosts in different insect orders, a form ofparasitism referred to as heterotrophic heteronomy (Walter,1983). Myrmecolacid males exploit Hymenoptera (ants) ashosts, and the females parasitize a range of species inseveral orthopteroid orders (Ogloblin, 1939; Kathirithamby,1991a; Kathirithamby & Hamilton, 1992). While it is gener-ally accepted that koinobiont endoparasites (those livingwithin a mobile and defensive host) have a narrower hostrange than ectoparasitic ones (Askew & Shaw, 1986;Strand& Peach, 1995), a phenomenon likely due to the constraintsof the host immune system on endoparasites (Strand,1986), strepsipterans defy this rule by having an extremelyvast host range relative to species richness. Only 596species of Strepsiptera have been described as of 2004,yet there have been reports of species parasitizing sevenorders and thirty-four families of Insecta (Kathirithamby,1989).This wide host range is likely greater than that of anygroup of parasitoid insects (Kathirithamby et al., 2003) andit is hypothesized that this biology promotes the extremesexual dimorphism (females are highly reduced morpho-logically) observed in this insect taxon (Kathirithamby, 1989).Few entomologists would argue against a correlationbetween the unusual life history of strepsipterans and weirdmorphological characteristics. However, evidence fortantamount odd molecular differences is not well known.Perhaps if rampant host switching is associated with anincrease in the rate of molecular evolution, then strepsipteranDNA sequences may have undergone an accelerated rateof nucleotide substitution, much like that reported for thestem lineage of Diptera (flies) (Friedrich & Tautz, 1997a,b).However, as no comprehensive molecular phylogeny existsfor the order Strepsiptera, as does for the Diptera (Yeates& Wiegmann, 1999; Wiegmann et al., 2003), an acceler-ated rate of nucleotide evolution in strepsipterans remains aspeculation, particularly due to the paucity of existing rRNAsequences for the order and the difficulty in objectively aligningthem with often much shorter sequences from other insects.Earlier secondary structure models for the strepsipteranrRNAs were predicted from a comparative analysis of a limitednumber of taxa (Hwang et al., 1998; Choe et al., 1999b).Nevertheless, using three 18S rRNA sequences Choe et al.(1999b) identified the locations that contain the majority of theextra length present in the strepsipterans and absent in theother arthropods.We have re-evaluated the atypical structureof strepsipteran 18S rRNA with a larger number of availablesequences and the prediction of a refined double pseudoknotstructure in variable region 4 of 18S rRNA that was publishedafter these earlier predicted models (Wuyts et al., 2000).In this paper, we report the sequence and secondarystructure of the entire 18S rDNA gene region from the strep-sipteran Caenocholax fenyesi texensis Kathirithamby &Johnston, 2004 (Myrmecolacidae).Based on our analyses,we: (1) provide a secondary structure model for the entirestrepsipteran 18S rRNA that includes the variable regionswithin the conserved core structure; (2) characterize thehigher order ribosome structure in these unique regions ofstrepsipteran rRNA; (3) offer an alignment with strong cov-ariation support of 175 arthropod sequences that will proveuseful for future investigations on arthropod phylogenetics;and (4) discuss how our findings are related to the evolutionof genome size in organisms with high occurrences ofnucleotide insertions.Results and DiscussionPredicted secondary structureOur predicted 18S rRNA secondary structure for C. f.texensis is shown in Fig. 1.The diagram follows the secondarystructural model of Drosophila melanogaster (Gutell, 1993,1994; Cannone et al., 2002), with refinement to the variableregion 4 (Van de Peer et al., 1999) made from the modelof Wuyts et al. (2000). The length of the 18S rRNA inC. f. texensis is 2864 nts, which is currently the fourth largestarthropod 18S rRNA known (only the strepsipteransXenos vesparum and X. pecki of the family Stylopidae andthe soil bug Armadillidium vulgare (Crustacea) are larger).While the core rRNA sequences of strepsipterans superim-pose on to the other arthropod sequences and predictedstructure with little or no ambiguity, most of the variableregions previously characterized for SSU rRNA are greatlyexpanded (Table 1). Additionally, several regions of strep-sipteran 18S rRNA, localized within universally conservedcore elements, contain unique features of animal SSU rRNA(Choe et al., 1999b).These characteristics of strepsipterannuclear SSU rRNA are discussed below.18S rRNA features unique to StrepsipteraH143: This putative helix is present in some arthropods asa small helix (2–4 bp) except for the psocopteranLiposcelissp. that has a putative 12 bp helix that is not energeticallystable. In contrast, this helix in strepsipterans is highlyexpanded, ranging from 15 to 19 bp. Comparative supportfor this helix is minimal across closely related taxa; however,the helix predicted with a thermodynamic-based algorithm
  3. 3. Bizarre insertions in strepsipteran 18S rRNA 627© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643
  4. 4. 628 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Figure 1. The secondary structure model of the nuclear SSU rRNA (18S) from the strepsipteran Caenocholax fenyesi texensis (accession number DQ026302).(A) Domains I–II. (B) Domain III. Helix numbering follows the system of Cannone et al. (2002), except for variable region 4 (V4) for which the notation of Wuytset al.(2000) is used.Variable regions are coloured light blue and the naming follows Van de Peeret al.(1999).Regions coloured red depict both highly expandedvariable regions and insertions in the core rRNA specific to Strepsiptera. Differences between our sequence and previously published C. fenyesi sequences(U65190 and U65191) are coloured purple, with insertions (dark arrows), deletions (open arrows) and substitutions (parentheses) shown. Sequences colouredgreen depict conserved motifs within the pseudoknot 13/14 insertion.Helices aligned across all sampled panarthropods are boxed in grey.Regions of ambiguousalignment (RAA) are boxed in green and characterized following the method of Gillespie (2004). A single ambiguity in helix H829a is boxed in red. Base pairing(where there is strong comparative support) and base triples are shown connected by continuous lines. Base-pairing is indicated as follows: standard canonicalpairs by lines (C–G, G–C, A–U, U–A); wobble G·U pairs by dots (G·U); A·G pairs by open circles (A°G); other non-canonical pairs by filled circles (e.g. C•A).Universal primers, as well as primers designed in this study, are mapped on the structure in orange with the first primer position circled. A primer table is postedat the jRNA website (http://hymenoptera.tamu.edu/rna).The diagram was generated using the program XRNA (Weiser, B. & Noller, H., University of Californiaat Santa Cruz) with manual adjustment.
  5. 5. Bizarre insertions in strepsipteran 18S rRNA 629© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Table 1. Summary statistics on the variable regions of the 18S rRNA in the major arthropod classes and hexapod orders. Numbers have been adjusted toexclude taxa wherein sequences are partial for a given region. Values in bold depict uniquely long sequence lengths in Strepsiptera. The mean range ofstrepsipterans is significantly greater than that of all other orders (PROC GLM Sheffe P < 0.01; SAS 8.22; SAS Inst., Inc. Cary, NC). For each region the rangesobserved for strepsipterans were compared to that of all other orders using orthogonal contrasts in PROC GLM of SAS (***P < 0.0001, **P < 0.01, *P < 0.05).All pairwise mean AU% differences were highly significant (Sheffe P < 0.01) except for comparisons between Strepsiptera and Diptera, and Strepsiptera andTardigrada. Within columns, A/U composition is listed followed by ranges of nucleotide lengths within defined variable regions. See Figure 1 for the location ofvariable regionsV1 H143 V2 H184c H198b V3Tardigradaa0.55, 33 0.73, 11 0.61, 100 0, 0 0.40, 5 0.44, 34Onychophora 0.52, 37–38 0.40, 12–13 0.41, 97–118 0, 0 0.71, 3–4 0.46, 35Chelicerata 0.53, 33–35 0.66, 10–12 0.48, 95–128 0, 0 0.42, 5–6 0.48, 34Myriapoda 0.51, 33–42 0.62, 10 0.45, 99–101 0, 0 0.52, 5–19 0.44. 34–36Crustacea 0.45, 33 0.63, 9–13 0.50, 90–116 0, 0 0.27, 4–5 0.40, 34Collembola 0.61, 33–34 0.69, 9 0.53, 92–94 0, 0 0.32, 4–5 0.48, 34Protura 0.42, 34–35 0.70, 10 0.44, 130–132 0, 0 0.56, 5–6 0.29, 31Diplura 0.32, 37 0.42, 10 0.38, 99–165 0, 0 0.43, 8–10 0.16, 31–35Archaeognatha 0.49, 33–34 0.67, 10–12 0.49, 95–114 0, 0 0.31, 5–6 0.43, 34Thysanura 0.56, 33–36 0.74, 11 0.58, 102–117 0, 0 0.56, 6 0.38, 34Odonata 0.49, 32–33 0.61, 11–12 0.50, 94–103 0, 0 0.25, 5–6 0.38, 34Ephemeroptera 0.52, 29–33 0.70, 10–11 0.46, 102–105 0, 0 0.34, 5–6 0.38, 34Dermaptera 0.53, 33 0.83, 12 0.68, 103 0, 0 0.54, 6 0.38, 34Plecoptera 0.54, 30–33 0.66, 11–12 0.53, 90–123 0, 0 0.33, 5–6 0.38, 34Grylloblattodea 0.48, 33 0.67, 12 0.51, 128–130 0, 0 0.40, 5 0.38, 34Embioptera 0.51, 30–33 0.58, 12 0.43, 102 0, 0 0.33, 6 0.38, 34Blattaria 0.47, 33 0.64, 10 0.48, 98–133 0, 0 0.38, 5–6 0.38, 34Isoptera 0.48, 33 0.61, 9–10 0.48, 101–133 0, 0 0.33, 6 0.38, 34Mantodea 0.48, 33 0.60, 10 0.49, 97 0, 0 0.17, 6 0.38, 34Phasmatodea 0.53, 33 0.65, 10 0.53, 104–105 0, 0 0.25, 6 0.40, 34Orthoptera 0.51, 33 0.58, 10–11 0.51, 104–105 0, 0 0.44, 6 0.38, 34Hemiptera 0.53, 33 0.57, 10–12 0.49, 99–107 0, 0 0.47, 6 0.39, 34Psocoptera 0.53, 33–48 0.49, 10–39 0.55, 102–146 0, 0 0.50, 5–6 0.46, 35Phthiraptera 0.52, 33 0.64, 12–16 0.53, 224–244 0, 0 0.30, 5–7 0.48, 35–36Thysanopteraa0.55, 33 0.73, 11 0.51, 102 0, 0 0.33, 6 0.38, 34Hymenoptera 0.47, 33 0.67, 12 0.56, 110–117 0, 0 0.42, 6 0.38, 34Mecoptera 0.49, 33 0.64, 12 0.58, 109–110 0, 0 0.47, 5 0.38, 34Siphonapteraa0.48, 33 0.64, 11 0.55, 110 0, 0 0.40, 5 0.38, 34Lepidoptera 0.43, 33 0.65, 10 0.55, 99–110 0, 0 0.30, 5 0.44, 34Trichoptera 0.51, 33 0.65, 10 0.46, 114 0, 0 0.40, 5 0.35, 34Neuroptera 0.47, 33 0.61, 11–12 0.50, 119–124 0, 0 0.40, 5 0.38, 34Coleoptera 0.49, 33 0.67, 12 0.52, 103–119 0, 0 0.29, 4–5 0.38, 34Diptera 0.61, 33 0.82, 10–12 0.65, 88–110 0, 0 0.50, 5 0.57, 35–36Strepsiptera 0.64, 38–69* 0.72, 49–57*** 0.66, 149–225*** 0.77, 54–194*** 0.58, 32–71** 0.53, 34V4E23-1, 2bE23-5cE23-6 E23-7dE23-8-14eTotalTardigradaa0.52, 56 0, 0 0, 0 0.49, 41 0.61, 126 0.56, 242Onychophora 0.17, 63–64 0, 0 0.25, 8 0.30, 38–43 0.51, 132–147 0.39, 255–274Chelicerata 0.38, 51–55 0.41, 2–30 0.43, 7 0.40, 33–36 0.61, 125–126 0.50, 231–365Myriapoda 0.33, 54–61 1, 1 0.36, 11 0.35, 35–40 0.54, 127–137 0.49, 236–265Crustacea 0.36, 55–74 0.40, 5 0.25, 8 0.39, 27–40 0.59, 125–133 0.48, 233–278Collembola 0.46, 54–55 0, 0 0, 0 0.54, 33–35 0.60, 125 0.55, 231–234Protura 0.28, 73 0.27, 61–64 0, 0 0.27, 11 0.50, 130 0.38, 292–295Diplura 0.28, 58–65 –f–f–f0.46, 123–131 0.37, 235–524Archaeognatha 0.33, 54–66 0, 0 0, 0 0.41, 35–36 0.57, 124–130 0.47, 234–252Thysanura 0.37, 55–59 0, 0 0, 0 0.52, 35–38 0.61, 127–131 0.52, 201–245Odonata 0.32, 57–58 0.31, 16–19 0.73, 5 0.46, 35 0.59, 129 0.47, 261–265Ephemeroptera 0.33, 56–58 0.21, 21–43 0.62, 5–9 0.33, 33–40 0.60, 129–131 0.45, 265–289Dermaptera 0.52, 58–60 0.59, 68–85 0.70, 5 0.46, 28–41 0.64, 126 0.58, 310–331Plecoptera 0.34, 64–68 0.30, 54–237g0.58, 5–7 0.45, 34–39 0.63, 126–129 0.44, 303–488Grylloblattodea 0.43, 63 0.50, 246–250 0.86, 7 0.43, 36 0.60, 128 0.52, 499–503Embioptera 0.35, 62 0.30, 37 0.89, 9 0.43, 34 0.59, 128 0.47, 289Blattaria 0.32, 59–62 0.23, 20–115 0.81, 8 0.37, 32–37 0.58, 127–130 0.42, 265–359Isoptera 0.32, 61–62 0.22, 70–73 0.74, 7 0.36, 35–39 0.57, 130–132 0.41, 251–326Mantodea 0.35, 62 0.19, 20–45 0.86, 7 0.38, 35 0.58, 131 0.44, 273–298Phasmatodea 0.39, 62 0.24, 44 0.56, 9 0.37, 34 0.61, 129 0.46, 297Orthoptera 0.38, 62–64 0.24, 60–87 0.71, 7–9 0.37, 34–35 0.60, 127–129 0.44, 312–342Hemiptera 0.34, 58–60 0.27, 66–79 0.53, 7–9 0.40, 34–40 0.61, 129–132 0.44, 314–329
  6. 6. 630 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Psocoptera 0.43, 61 0.37, 34–53 0.58, 9–10 0.36, 34–35 0.63, 128–131 0.50, 285–309Phthiraptera 0.35, 60–62 0.32, 56h0.61, 6–12 0.32, 34–39 0.57, 128–134 0.44, 309–464Thysanopteraa0.33, 60 0.27, 41 0.56, 18 0.38, 34 0.61, 126 0.46, 298Hymenoptera 0.42, 58–60 0.30, 25 0.88, 8 0.41, 35 0.62, 129 0.52, 274–276Mecoptera 0.41, 59–62 0.36, 26–27 0.81, 9 0.44, 35 0.65, 130 0.54, 278–282Siphonapteraa0.38, 60 0.33, 27 0.78, 9 0.37, 35 0.63, 128 0.51, 278Lepidoptera 0.34, 60 0.35, 25–26 0.84, 9–10 0.37, 35 0.66, 121–132 0.51, 270–281Trichoptera 0.38, 33–58 0.25, 18–26 0.41, 9–18 0.24, 25–29 0.61, 132–134 0.47, 253–267Neuroptera 0.38, 60–61 0.25, 119–161 0.81, 8–13 0.36, 34–35 0.61, 131 0.42, 377–414Coleoptera 0.29, 58–60 0.27, 25–104 0.64, 7–13 0.32, 34–35 0.62, 131 0.45, 275–360Diptera 0.55, 62–78 0.69, 19–54 0.83, 9–33 0.54, 33–38 0.67, 129–131 0.63, 264–336Strepsiptera 0.67, 67–74 0.70, 40–140 0.75, 90–217*** 0.60, 32–146* 0.71, 259–505*** 0.70, 599–859***V4E23-1, 2bE23-5cE23-6 E23-7dE23-8-14eTotalV7V5 V6 H1118b-2 H1118c Total V8 V9Tardigradaa0.50, 44 0.57, 44 0, 0 0.43, 21 0.56, 59 0.55, 71 0.44, 41Onychophora 0.27, 44–46 0.47, 44 0, 0 0.53, 21–26 0.47, 62–65 0.35, 64–66 0.28, 41–67Chelicerata 0.37, 44 0.58, 44 0, 0 0.44, 17–26 0.52, 49–66 0.43, 61–65 0.22, 41–43Myriapoda 0.36, 44–45 0.53, 44 0, 0 0.40, 18–32 0.46, 52–64 0.42, 62–67 0.24, 28–49Crustacea 0.38, 44–45 0.54, 44 0, 0 0.46, 17–24 0.51, 49–62 0.40, 58–63 0.30, 38–67Collembola 0.46, 44–45 0.52, 44 0, 0 0.57, 17–20 0.55, 51–53 0.52, 58–59 0.39, 41Protura 0.26, 45 0.54, 52 0, 0 0.47, 25–26 0.45, 58–60 0.35, 112 0.25, 56–59Diplura 0.28, 44–51 0.46, 44–51 0, 0 0.16, 20–22 0.28, 52–54 0.33, 62–64 0.32, 38–49Archaeognatha 0.37, 43–44 0.56, 44 0, 0 0.37, 22–27 0.46, 52–58 0.38, 61 0.20, 50–59Thysanura 0.37, 44 0.59, 44 0, 0 0.72, 15–22 0.61, 57–60 0.43, 59–63 0.42, 35–47Odonata 0.40, 43–44 0.54, 44 0, 0 0.61, 22 0.53, 56–57 0.39, 59 0.26, 47–50Ephemeroptera 0.34, 44 0.57, 44 0, 0 0.37, 21–22 0.46, 54–56 0.39, 59–62 0.25, 39–44Dermaptera 0.40, 44 0.54, 44 0, 0 0.43, 20 0.51, 61 0.42, 59 0.52, 44–45Plecoptera 0.33, 43–44 0.59, 44 1, 5 0.42, 25 0.52, 69–74 0.43, 61–63 0.30, 39–44Grylloblattodea 0.32, 44 0.52, 44 0, 0 0.35, 23 0.53, 64 0.44, 59 0.43, 40Embioptera 0.34, 44 0.52, 44 0, 0 0.39, 22 0.49, 61 0.39, 59 0.38, 41Blattaria 0.34, 44 0.51, 42–44 0, 0 0.37, 22–23 0.48, 50–63 0.39, 59 0.29, 38–42Isoptera 0.34, 44 0.50, 43–44 0, 0 0.35, 22 0.49, 50–62 0.40, 59 0.18, 38Mantodea 0.34, 44 0.50, 44 0, 0 0.32, 22 0.44, 62 0.39, 59 0.21, 41Phasmatodea 0.34, 44 0.52, 44 0, 0 0.35, 23 0.49, 63 0.41, 59 0.35, 41Orthoptera 0.31, 44 0.54, 44 0, 0 0.41, 21–23 0.50, 60–63 0.42, 59 0.34, 41–44Hemiptera 0.34, 44–48 0.56, 44 0, 0 0.41, 19–25 0.53, 60–67 0.39, 59–62 0.30, 34–44Psocoptera 0.38, 44–46 0.63, 43–44 0.41, 9–32 0.38, 19–21 0.49, 66–95 0.46, 63–64 0.43, 45–46Phthiraptera 0.42, 44–45 0.54, 44 0.69, 9–28 0.42, 18–104 0.53, 78–137 0.47, 63–65 0.34, 45–73Thysanopteraa0.43, 44 0.57, 44 0, 0 0.57, 23 0.51, 63 0.41, 59 0.30, 40Hymenoptera 0.35, 44 0.53, 44 0.90, 5 0.43, 49 0.52, 94 0.41, 59 0.28, 40Mecoptera 0.37, 44 0.57, 44 0.80, 5 0.45, 62–67 0.45, 105–112 0.45, 59 0.43, 40–41Siphonapteraa0,34, 44 0.52, 44 0.80, 5 0.41, 63 0.49, 108 0.44, 59 0.35, 40Lepidoptera 0.39, 43–44 0.55, 44 0, 0 0.38, 47 0.48, 85 0.42, 59 0.26, 38Trichoptera 0.47, 44–45 0.52, 44 0, 0 0.34, 80 0.45, 125 0.39, 59 0.30, 40Neuroptera 0.41, 44 0.52, 44 0.78, 9 0.30, 155–215 0.37, 205–264 0.43, 59 0.41, 41Coleoptera 0.32, 44 0.52, 44 0.75, 8 0.29, 27–136 0.40, 62–184 0.37, 59 0.34, 40–45Diptera 0.54, 43–45 0.66, 44 0.42, 8–11 0.59, 21–103 0.63, 68–164 0.52, 57–60 0.55, 41–52Strepsiptera 0.48, 45–46 0.60, 44 0.63, 143–307*** 0.59, 128–300*** 0.61, 432–569*** 0.60, 60–72 0.54, 41–43aOnly one taxon sampled.bIncludes RAA (14). (See Experimental procedures for further information)cIncludes RAA (15) and RAA (17).dIncludes RAA (19) and RAA (21).eIncludes RAA (22).fStructures predicted in this region (37–310 nts) did not conform to the alignment model.gTwo sequences, Isoperla obscura (196 nts) and Megarcys stigmata (231 nts) have sequences with odd, unalignable structures.hThree taxa, Pediculus humanus, Haematomyzus elephantis and Heterodoxus calabyi, range from 128–211 nts and form odd structures.Table 1. (Continued)
  7. 7. Bizarre insertions in strepsipteran 18S rRNA 631© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Figure 2. A gallery of diverse secondary structure diagrams of the variable region 2 (V2) and related core elements from selected strepsipterans.Helices H143,H184c and H198b are specific to Strepsiptera.(A) Mengenilla chobauti, AF423800.(B) Xenos vesparum, X77784.(C) Mengenilla chobauti, X89441.(D) Stylopsmelittae, X89440. The explanations of base pair symbols, helix numbering and reference for software used to construct structure diagrams are in Fig. 1.
  8. 8. 632 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643is similar in all strepsipterans (Fig. 2). This insertion sitewas proposed by Choe et al. (1999b) to foster a helix rang-ing from 15 to 17 bp, yet their proposed structures lackfrom 1–4 bp in the basal region of the helix, with threealternate and less stable base pairs in one taxon (Xenosvesparum, X77784). These inconsistencies resulted fromour model being based on the boundaries of core helicesH122 and H144 of the Escherichia coli 16S-like model(Cannone et al., 2002), which has been verified by the recentcrystalline structures of the ribosome (Ban et al., 2000;Wimberly et al., 2000;Schluenzen et al., 2000; Yusupov et al.,2001; Gutell et al., 2002). Interestingly, the pogonophoranworm Siboglinum fiordicum also contains a helical insertionin this region of the 18S rRNA (Winnepenninckx et al., 1995).H184c: This helix occurs strictly in Strepsiptera and rangesfrom 21 to 73 bp (Table 1). The majority of length variationin this region of the 18S in non-strepsipteran insects occursto the 5′-side of helix H184c [RAA (8)], with the 3′-sequencejust before helix H198 unambiguously aligned acrossArthropoda. Adjacent to the 5′-end of this conserved sequ-ence (flanking the 3′-end of helix H184c) a 5′-AA-3′sequence occurs that is found only in strepsipterans (Fig. 2).The structure predicted for X. vesparum by Choe et al.(1999b) for H184c differs from our model and is likely basedon discrepancies in algorithms used to predict both struc-tures. However, the predictions by Choe et al. (1999b)for Stylops melittae (X89440, Stylopidae) and Mengenillachobauti (X89441, Mengenillidae) of H184c are inaccuratedue to the inclusion of some paired nucleotides in H198bwithin their structures (Fig. 3).H198b: The extension of helix H198 (8 bp) occurs in sev-eral arthropod groups and is usually no greater than 2 bp.However, this helix is greatly expanded in Strepsipteraand ranges from 21 to 34 bp (Fig. 2). In contrast to variableregions (Gerbi, 1985), it is unclear how the expansion andcontraction of an otherwise highly conserved core helixeffects ribosome assembly and function. Similar evidencefor the expansion and contraction of a conserved core helixhas recently been detected in helix H604 in domain II of28S rRNA in Hymenoptera (Gillespie et al., 2005a,b).Interestingly, unpublished data from our labs suggests helixH604 is extraordinarily hypervariable in sequence lengthand base composition in strepsipterans.E23-13/14: A large and unusual insertion occurs exclu-sively in Strepsiptera in the hairpin-stem loop of the pseu-doknot 13/14 in the V4 region (Choe et al., 1999b). Insertsvary from 118 to 366 nts, with putative helical regionssupported by thermodynamic algorithms and comparativeevidence across closely related taxa. A gallery of diversesecondary structure predictions illustrates the lack ofsequence conservation and structure in this insertion site(Fig. 4). Despite the lack of significant conservation in thisregion across the strepsipterans, the conserved sequence5′-AUUGGCUUAAA-3′ always occurs immediately 5′ to ahelical structure that is flanked on its 3′-end by a 5′-GAC-3′sequence. However, the precise location of this conservedsequence does vary within this insertion (Fig. 4). Interest-ingly, these two highly conserved sequences only differ inthe two individuals of Mengenillidae, the proposed sistertaxon to the rest of the families of Strepsiptera (Kinzelbach,1971; Kathirithamby, 1989).The distribution of these hyper-variable sequences within the two conserved sequencesin the hairpin loop of pseudoknot 13/14 is summarized inTable 2. Because the highly conserved boundaries ofpseudoknot 13/14 were not used as anchors by Choeet al. (1999b) for structure prediction, the structures theyproposed are very different to our predictions, with theTable 2. Distribution of sequence and structure within the strepsipteran insertion in the second hairpin loop of pseudoknot 2 within the variable region 4 (V4)of the 18S rRNA molecule. Nucleotides in bold show conserved sequences across Strepsiptera; underlined nucleotides show conserved sequences across allfamilies apart from MengenillidaeAccessionnumber Taxon5′ Helix (?)/structureb5′ Conservedsequencec3′ Sequence/structured3′ ConservedsequenceU65159 Triozocera mexicana No UUUGAAAUUGGCUUAAA 238 nts; 87 bp GACU65160 Caenocholax fenyesi No UUUGAAAUUGGCUUAAA 235 nts; 87 bp GACU65161 Caenocholax fenyesi (2); 12, 18 bp UUUGAAAUUGGCUUAAA 43 nts; 17 bp GACDQ026302aCaenocholax fenyesi (2); 12, 18 bp UUUGAAAUUGGCUUAAA 43 nts; 17 bp GACU65163 Crawfordia sp. (1); 12 bp ---AUUAUUGGCUUAAA 173 nts; 69 bp GACX89440 Stylops melittae (1); 22 bp --AGAAAUUGGCUUAAA 163 nts; 63 bp GACX74763 Xenos vesparum (1); 17 bp -AUAAAAUUGGCUUAAA 284 nts; 109 bp GACX77784 Xenos vesparum (1); 17 bp -AUAAAAUUGGCUUAAA 283 nts; 109 bp GACU65164 Xenos pecki (1); 12 bp --UAAAAUUGGCUUAAA 329 nts; 118 bp GACX89441 Mengenilla chobauti No --------AGGCUUUU- 173 nts; 63 bp G-CAF423800 Mengenilla chobauti No --------AGGCUUUU- 140 nts; 51 bp G-CaSequenced in this study.bNumber of base pairs within putative helices.cIncludes all unpaired nucletides flanking the 5′-end of conserved sequence.dTotal base pairs in all putative helices.
  9. 9. Bizarre insertions in strepsipteran 18S rRNA 633© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643conservation of the above-mentioned unpaired sequencesinvolved in nonhomologous base pairings (data not shown,Choe et al., 1999b; their fig. 3).Given the lack of conservation in sequence and struc-ture, it is likely that the insertion in pseudoknot 13/14 is partof the mature SSU rRNA and is probably not an intron.Themajor insertion points for introns in SSU rRNA have beenwell characterized (Wuyts et al., 2001; Jackson et al., 2002)and usually occur at the subunit interface or in conservedsites with known tRNA–rRNA interaction (Jackson et al.,Figure 3. A comparison between our predicted structures for helix H184c and those of Choe et al. (1999b). Shaded regions in our diagrams (left) depictnucleotides found within their models.The explanations of base pair symbols, helix numbering and reference for software used to construct structure diagramsare in Fig. 1. Models from Choe et al. (1999b) were reproduced manually.
  10. 10. 634 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643
  11. 11. Bizarre insertions in strepsipteran 18S rRNA 635© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–6432002). Additionally, both the 18S rDNA and rRNA of Xenosvesparum were sequenced by Chalwatzis et al. (1995) andshowed no differences in length. Given this, the functionalsignificance of a peculiar insertion specific to Strepsipterawithin a conserved pseudoknot across all Eukaryotaremains unknown. However, recent evidence for a con-served sequence of the V4 forming a putative helix with aregion in the V2 (our helix H184b-1) suggests a tertiaryinteraction between the two expansion segments is prob-able (Alkemar & Nygård, 2003), given their close proximity inthe three-dimensional structure of the ribosome (Spahnet al., 2001). Within our conserved sequence 5′-AUUG-GCUUAAA-3′, isolated by a variety of secondary structures(Fig. 4), the sequence 5′-AUUGGCUUA-3′ can form a helixwith the 5′-strand of helix H184-1 and flanking nucleotides(Fig. 1). An analysis of base pair frequencies and degree ofcovariation for both helix H184-1 and this putative tertiaryhelix, named here helix HV2/V4, reveals stronger supportfor the tertiary interaction (Table 3).However, because bothstructures can form across all Arthropoda, we cannot ruleout the possibility that they both occur at different stages ofribosome assembly and function. It should be noted thatin all non-strepsipteran arthropods, the formation of helixHV2/V4 entails the dissolution of the first base pair in helixE23-13, thus providing evidence against the proposedbase triple this base pair forms with the unpaired positionimmediately flanking the 5′-end of helix E23-8 (Wuyts et al.,2000).E23-13/14 in published sequencesPreviously, sequence heterogeneity was found in the 18SrRNA genes within single strepsipteran individuals (Whitinget al., 1997).In that study, an automated alignment program(Malign; Wheeler & Gladstein, 1994) was used to alignthese strepsipteran sequences with seventy-nine othersequences from the major lineages of insects for the pur-pose of estimating a phylogeny. Interestingly, the alignmentof the majority of the insertion within hairpin 13/14 acrossthese seven strepsipterans (Fig. 5) included two divergentsequences each from individuals of C. fenyesi Pierce, 1909and Xenos pecki (Whiting et al., 1997).The authors explainedthat there were sequencing problems for the V4 for thesetaxa due to the presence of multiple amplicons (Whitinget al.,1997). Thus, the amplicons were cloned and sequenced,which resulted in two different sequences for both species.This result is in conflict with our analysis of seven strep-sipteran taxa and our identification of two highly conservedshort sequences within the variable secondary structuresin the V4 pseudoknot 13/14. Thus we conclude that thisheterogeneity of the strepsipteran rDNA sequences islikely erroneous for the following reasons. First, the ‘long’sequence of C. fenyesi is probably Triozocera mexicanaPierce, 1909 (Corioxenidae) (Fig. 5) because of high simi-larity in primary sequence (only one substitution and fourindels out of 238 nts) and secondary structure (Table 2,Fig.4A,C).Confusion of these sequences could have occurredFigure 4. A gallery of diverse secondary structure diagrams of the insertion within the hairpin loop of pseudoknot 13/14 within variable region 4 (V4) fromselected strepsipterans. The conserved sequences described in the text are bold-italicized and shaded. (A) Caenocholax fenyesi, U65160. (B) Caenocholaxfenyesi, U65161. (C) Triozocera mexicana, U65159. (D) Crawfordia sp., U65163. (E) Stylops melittae, X89440. (F) Xenos pecki, U65164. (G) Xenos vesparum,X77784. (H) Xenos vesparum, X74763. (I) Mengenilla chobauti, X89441. (J) Mengenilla chobauti, AF423800. Differences between C. fenyesi (U65190) andC. fenyesi (U65191), and between C. fenyesi (U65190) and Triozocera mexicana (U65159) are depicted using the same symbols described in Fig. 1, with µrepresenting a substitution.F.The‘short’unpublished sequence of X.pecki from Whiting et al.(1997) is shown under X. pecki (U65164) with an asterisk depictingthe missing sequence and potential inclusion of a putative cloning vector. The explanations of base pair symbols, helix numbering and reference for softwareused to construct structure diagrams are in Fig. 1.
  12. 12. 636 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Figure 4. (Continued)
  13. 13. Bizarre insertions in strepsipteran 18S rRNA 637© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643through several means, nonetheless, our sequence of thisregion of the 18S rRNA from C. f. texensis (Fig. 1A) favoursthe probability that the ‘short’ sequence of C. fenyesi(Fig. 4B) from Whiting et al. (1997) is correct.Second, individuals of X. pecki do not contain ‘short’ and‘long’inserts in the loop of hairpin 13/14 because the ‘short’sequence appears to be an artefact of cloning. X. pecki‘short’(Fig. 5) comprises three different sequences, of whichonly one should be aligned with the other strepsipteransequences in the ‘insert 23’alignment of Whiting et al. (1997:56). The sequence in the dashed box (Fig. 5) depicts aregion that is identical to X. pecki ‘long’, except that this ispart of the conserved alignment of Whiting et al.(1997: 47).The regions that are boxed (Fig. 5) depict identicalsequences that are misaligned due to the inclusion of thestretch of nucleotides from the unambiguous alignmentmisplaced in ‘insert 23’. Finally, the remaining sequence(139 nts) of X. pecki ‘short’, boxed and shaded dark (Fig. 5),is likely the 5′-end of the cloning vector. A sequencesimilarity search with the program BLAST (Altschul et al.,1990) revealed four separate cloning vectors, all with 93%sequence identity (AF335420, AF335419,Y10545, U14118).Although a X. pecki ‘short’ sequence (minus the cloningvector) is still possible, verification awaits sequences of the3′-strands of helices E23-14a and E23-14b, as well as theassociated unpaired flanking regions (Fig. 4F).The importance of using structure to align rRNA sequencesThe elucidation of secondary structure of rRNA moleculesguides the assignment of positional nucleotide alignment(Gutell et al., 1985, 1992, 1994; Kjer, 1995) and has beenshown to improve phylogeny estimation (Dixon & Hillis,1993; Kjer, 1995; Titus & Frost, 1996; Morrison & Ellis,1997; Uchida et al., 1998;Mugridge et al., 1999; Cunninghamet al., 2000; Gonzalez & Labarere, 2000; Hwang & Kim,2000; Lydeard et al., 2000; Morin, 2000; Xia, 2000; Xiaet al., 2003; Kjer, 2004).The regions of length heterogene-ous sequence alignments that are the most difficult toestablish homology across often contain valuable phyloge-netic signal (Lee, 2001), and secondary structure providesan objective means for retrieving this information (Gillespie,2004). While often uninformative at the nucleotide leveldue to the rapidly evolving nature of rRNA variable regionswhen compared across highly divergent taxa, secondaryFigure 4. (Continued)
  14. 14. 638 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643structural characters can provide morphological evidencefor similarity that is not immediately apparent with primarysequence data (reviewed in Gillespie et al., 2004). Ithas been stated (e.g. Woese et al., 1980; De Rijk et al.,1994; Gutell & Damberger, 1996; Kjer, 1997) and demon-strated (Xia et al., 2003; Kjer, 2004) that structuralalignment also provides a means to ‘proofread’ rRNAsequences for their accuracy, which is analogous to convert-ing protein-encoding DNA sequences to their respectiveamino acid sequences to check for shifts in the readingframe and unexpected stop codons. Our study also exem-plifies the benefit of structure for the quality assessment ofnew and published data.Implications for the systematic position of StrepsipteraGiven their peculiar biology and morphology, it is not sur-prising that the Strepsiptera are difficult to place phyloge-netically within the Insecta. Some workers have allied theStrepsiptera with Coleoptera (beetles) based on the simi-larity of hind-wing-based flight (Kinzelbach, 1990; Kathirith-amby, 1991b). However, several molecular phylogeneticestimations (all based on rDNA sequences) recover thestrepsipterans as a sister taxon to the flies (Chalwatziset al., 1995, 1996; Whiting et al., 1997).This is not surpris-ing, as the exceedingly autapomorphic rDNA sequences ofStrepsiptera are highly unlike those of other holometabo-lous insects (Gillespie, unpubl. data), and possibly groupwith dipteran rDNA sequences based on nucleotideconvergence due to similar rapid rates of evolution (Hwanget al., 1998) and similar AU base composition biases(Table 1). In fact, when convergence in nucleotide rates ofsubstitution is accommodated for via character weightingor maximum likelihood modelling, the strepsipteransand dipterans are not recovered as a monophyletic group(Carmean & Crespi, 1995; Huelsenbeck, 1997; Hwanget al., 1998). Interestingly, the majority of these phylogenyestimations have excluded the variable regions of insectrRNA molecules due to the difficulty in establishing align-ment across heterogeneous sequences (but see Hwanget al., 1998). The putative strepsipteran/dipteran synapo-morphy of a doubly branched V7 region by Choe et al.(1999b) is not supported in our study, as the V7 containstwo helices (H1118b and H1118c; Fig. 1B) in many taxawithin the Holometabola (Table 1).Our predicted 18S rRNA structure model divides theV4, one of the most commonly sequenced molecules forarthropod phylogeny reconstruction (Cannone et al., 2002),into 80 discrete regions defined by secondary structure(Fig. 1 and alignment at http://hymenoptera.tamu.edu/rna).These partitions will be of use to a wide range of phylogeneticTable 3. Composition and degree of compensation for the base pairs of putative helices H184-2 and HV2-V4 of the 18S rRNA across 175 arthropods. For basecomposition percentages, bold values represent any base pair present at 2% or greater in the alignment. Asterisks denote positions that strictly covary for agiven base pair (representing 3% or more of total base pair types), with the summed numbers providing a percentage of covariation. Note: percentages ignorephylogenetic correlationHelixabpb# SeqcBase pair composition, %dGape(–)Canonical Non-canonicalGC CG UA AU GU UG AA AC AG CA CC CU GA GG UC UUH184b1 176 – 0.6 89.7 – – – – – – – – – – – – – 9.72 176 – 0.6 89.1 – – 0.6 – – – – – – – – – – 9.73 176 – 0.6 – 86.3 – – – 0.6 2.9 – – – – – – – 9.74 176 0.6 – 86.9 – – 0.6 – – – – – – – – 2.3 – 9.75 176 – 0.6 69.1 – – 19.4 – – – – 0.6 – – – – 0.6 9.76 176 1.1 – – 75.4 10.9 0.6 – 2.3 – – – – – – – – 9.77 176 – 77.1* 1.7 – – 4.0* – 0.6 – 6.3* – 0.6 – – – – 9.78 176 – 87.4 – – – – – – 1.1 0.6 – – – 0.6 – 0.6 9.79 176 1.1 0.6 1.1 79.4 4.6 – 1.7 0.6 0.6 – – 0.6 – – – – 9.710 176 0.6 0.6 1.1 71.4 0.6 – 4.0 1.7 5.1 – – – – 1.1 – 0.6 11.4HV2-V41 176 1.7 – – 78.9 0.6 – 2.3 4.6 – – – – – – – – 0.62 176 0.6 – – 84.0 – – – 2.3 – – – – – – – 0.6 0.63 176 1.1 – – 79.4 6.3 – – – – – – – 0.6 – – – 0.64 176 10.9* – – 68.6* 0.6 – – 7.4* 0.6 – – – – – – 0.6 0.65 176 – 82.9 – – – 5.1 – – 0.6 – – – – – – – 0.66 176 – 86.9 – – – 0.6 – – 1.1 – – – – – – – 0.67 176 3.4* – 1.1 81.1* 2.3 – – – – – – 0.6 – – – – 0.68 176 0.6 – 1.1 82.3 1.1 – – – – – 0.6 – – – – 0.6 0.69 176 – 5.1* 3.4* – – 77.1* – – 1.1 – – – 0.6 – – – 0.6aHelix numbering refers the nucleotide positions shown in Figure 1.bBase pairs are numbered from 5′-end of 5′-strand of each helix.cNumbers vary at eachposition due to missing data (?), deletions (–) and possible presence of IUPAC-IUB ambiguity codes.dThe first nucleotide is that in the 5′-strand.eGaps representsingle insertion or deletion events, not indels.
  15. 15. Bizarre insertions in strepsipteran 18S rRNA 639© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643analysis programs which incorporate mixed evolutionarymodels. Additionally, we characterize the remainingregions of the 18S rRNA in concordance with publishedrRNA models and provide an alignment template that iscustom for the Arthropoda.This should contribute greatly tofuture studies that employ structure into the process ofhomology assignment for the estimation of hexapodrelationships.The evolution of bizarre insertions in strepsipteran rDNAgenesThe arthropod nucleotide insertions relative to other arthro-pod 18S rRNAs are usually confined to a few variableregions (usually V4 and V7; e.g. Crease & Taylor, 1998).However, strepsipterans have insertions in nearly everydefined variable region of SSU rRNA, often with differentbase compositions in these regions when compared tomost other arthropod groups (Table 1). In addition, thestrepsipterans have unique insertions in the highly con-served core rRNA structure (Choe et al., 1999b).This sug-gests that the strepsipteran 18S rRNA genes are moretolerant of certain mutations rather than selected against orpruned by processes of gene conversion and/or unequalcrossing over (Arnheim et al., 1980; Ohta, 1980; Dover,1982; Arnheim, 1983; Flavell, 1986; Nagylaki, 1988). Highrates of nucleotide substitution occur in strepsipterans(Hwang et al., 1998), and perhaps the rate of mutationexceeds the rate at which gene conversion and/or unequalcrossing over can remove novel insertion events, leading totheir eventual fixation (Ohta, 1982).The fixation of insertionevents relative to their removal by purifying selection wouldbe directly affected by the size of the strepsipteran genomeand the number of copies of rDNA genes.In organisms with rapid rates of nucleotide evolution, alow rDNA copy number would allow gene conversion and/or unequal crossing over to keep the highly evolving rDNAcopies concerted. Unfortunately, the rDNA copy number forany strepsipteran genome has yet to be established. How-ever, a recent study determined that the genome sizeof C. f. texensis has one of the smallest C-values of anyanimal (Johnston et al., 2004). As rDNA copy number isusually positively correlated with genome size (Prokopowichet al., 2003), it is likely that C. f. texensis has a low rDNAcopy number. Further support for this comes from theevidence that C. f. texensis undergoes endoreduplication, andcan have up to sixteen copies of its genome in male flightmuscle and in the tissues that support the hundreds ofthousands of rapidly developing embryos in the matureFigure 5. Recreated MALIGN alignment of Whiting et al. (1997: p56). Sequences include the majority of the insertion depicted in Fig. 3, plus the conserved3′-strand of helix E23-14b. The conserved sequences described in the text are bolded. The dashed box in Xenos pecki (short) depicts a misaligned region ofthe 18S that should be in the unambiguously aligned data of Whiting et al. (1997: p47). The boxes without shading show homologous (identical) sequencesshared between X. pecki (long) and (short) as depicted with the grey arrow.The darkly shaded box depicts a sequence with 93% similarity to published cloningvectors. The lightly shaded boxes illustrate the near identity of Caenocholax fenyesi (long) with Triozocera mexicana.
  16. 16. 640 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643female (Johnston et al., 2004). It has been shown that,despite having only one rDNA gene per genome, the ciliatedprotozoan Tetrahymena thermophila endoreduplicates itsgenome approximately 200-fold (Gall, 1974; Yao et al.,1974). Thus, the ability to endoreduplicate certain regionsof the genome would allow for organisms with low copies ofrDNA arrays to still produce enough rRNAs to meet thedemands of the cell.In the case of strepsipterans, low rDNAcopy number and endoreduplication could possibly combatthe rapid rates in nucleotide evolution by accommodatingthe homogenization process of the rDNA copies.We present this hypothesis for a low rDNA copy numberin strepsipterans in light of recent controversy regardingthe evolution of genome size. It has been proposed thatvariation in genome size can be indicative of biases insmall insertions and deletions (indels) (Petrov et al., 1996,2000, 2001, 2002; Bensasson et al., 2001). In general,these studies ascribe to the logic that larger genomes tendto accumulate more insertions over time, with smallergenomes likely riddled with more deletion events. However,these generalizations of genome size are probably morecomplicated (Gregory, 2003, 2004), and there are likelyother selective factors, such as cell volume (Bennett, 1972;Cavalier-Smith, 1985; Gregory, 2001) and endoreduplication(Nagl, 1978), responsible for shaping genome size. Thestrepsipterans certainly pose a paradox to the C-value enigma(Gregory, 2003), having one of the smallest animal genomesand possessing some of the largest insertions in all of thedocumented SSU rRNA sequences.Further study is neededto understand the tolerance strepsipterans have for accu-mulating large insertions within an otherwise tiny genome.Experimental proceduresTaxa examinedThe majority of the published 18S rRNA sequences used in thisstudy were compiled from two recent phylogenetic studies oninsects (Kjer, 2004) and arthropods (Mallatt et al., 2004).A taxon listwith respective GENBANK accession numbers and other informationcan be found at the jRNA website (http://hymenoptera.tamu.edu/rna). The accession number for C. f. texensis is DQ026302. Thevoucher specimens were deposited in either the Texas A & Minsect collection or the Museum of Natural History, Oxford.Genome isolation, PCR, and sequencingFor the sequence generated in this study, total genomic DNA wasisolated using DNeasy™ Tissue Kits (Qiagen, Valencia, CA). PCRconditions followed those of Cognato & Vogler (2001). A completelist of previously published primers, as well as newly designedprimers specific to C. f. texensis, is posted at the jRNA website.Double-stranded DNA amplification products were sequenceddirectly with ABI PRISM™ (PerkinElmer, Boston, MA) Big DyeTerminator Cycle Sequencing Kits and analysed on an AppliedBiosystems (PerkinElmer) 377 automated DNA sequencer. Bothantisense and sense strands were sequenced for all taxa, andedited manually with the aid of Sequence Navigator™ (AppliedBiosystems, Foster City, CA). During editing of each strand, nucle-otides that were readable, but showed either irregular spacingbetween peaks, or had some significant competing backgroundpeak, were coded with lower case letters or IUPAC-IUB ambiguitycodes. Consensus sequences were exported into MicrosoftWord™ for manual alignment.Multiple sequence alignmentOur C. f. texensis 18S rDNA sequence, as well as eleven otherpublished strepsipteran sequences and the panarthropod taxafrom Mallatt et al. (2004), were aligned to the recent structuralalignment of Kjer (2004). Adjustments to Kjer’s alignment weremade either in strict adherence to the 16S-like models on thecomparative RNA website http://www.rna.icmb.utexas.edu (Gutellet al., 1994; Cannone et al., 2002) or from information provided bycovariation analysis (see below). Additionally, the V4 region wasrealigned according to the model of Wuyts et al. (2000), with thetertiary interaction between the V2 and V4 included (Alkemar& Nygård, 2003).The structural notation of the alignment followedGillespie et al. (2004). Length variable sequences, especially hair-pin-stem loops, were evaluated in the program mfold (version 3.1;http://bioinfo.math.rpi.edu/∼zukerm/), which folds rRNA based onfree energy minimizations (Mathews et al., 1999; Zuker et al.,1999).These free energy-based predictions were used to facilitatethe search for potential base-pairing stems, which were con-firmed only by the presence of compensatory base changesacross a majority of taxa. Thermodynamic-based folding algo-rithms usually predict several plausible suboptimal structure mod-els in addition to the optimal one, and in many situations thedifference in energy value between the optimal and suboptimalis very small. Thus, all of these predicted structures should beconsidered, not just the optimal one. Consequently, we are moreconfident of a predicted structure that contains certain sequence/structure motifs characteristic of rRNA, such as hairpin loops withthree U nucleotides,YUCG and GNRA tetra-hairpin loops (Woeseet al., 1990) and AA and AG juxtapositions at the ends of helices(Elgavish et al., 2001). Regions of the alignment wherein homol-ogy assignments could not be made with a high level of confidencewere treated following the methodology of Gillespie (2004).Alignment-based statistics and structure diagramsOur alignment was modified into a Nexus file for further manipula-tion using scripts available at the jRNA website. Scripts were usedto calculate base pair frequency tables (providing a percentage ofcovariation for each base pair within a putative helix), nucleotidecomposition in non-pairing regions of the alignment, and meanlength ranges for the variable regions throughout the 18S rRNA.Secondary structure diagrams were generated with the computerprogram XRNA (developed by B. Weiser and H. Noller, Universityof Santa Cruz, CA) and adjusted manually for production of the fig-ures. All alignment formats, alignment-based statistics, structurediagrams (including a panarthropod consensus model), andscripts used to parse and analyse the data are available at thejRNA website following the links to ‘arthropoda’.AcknowledgementsWe are especially grateful to Karl Kjer for providing us withhis 18S rRNA alignment prior to publishing it.The comments
  17. 17. Bizarre insertions in strepsipteran 18S rRNA 641© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643of Karl Kjer and one anonymous reviewer greatly improvedthis manuscript. We thank Meagan Wheeler for helprecreating Fig. 5. J.J.G. greatly appreciates invigoratingdiscussion with Jamie Cannone regarding the structuralfindings presented here. This study was supported inpart by NSF grants DEB-0328920 to A.I.C., DEB-0328922to Robert Wharton (TAMU), and NIH grant GM067317 toR.R.G.ReferencesAlkemar, G. and Nygård, O. (2003) A possible tertiary rRNAinteraction between expansion segments ES3 and ES6 ineukaryotic 40S ribosomal subunits. RNA 9: 20–24.Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.(1990) Basic local alignment search tool. J Mol Biol 215: 403–410.Alvares, L.E., Wuyts, J., Van de Peer, Y., Silva, E.P., Coutinho, L.L.,Brison, O. and Ruiz, I.G.R. (2004) The 18S rRNA from Odon-tophrynus americanus 2n and 4n (Amphibia: Anura) revealsunusual extra sequences in the variable region V2. Genome47: 421–428.Arnheim, N. (1983) Concerted evolution of multigene families. In:Evolution of Genes and Proteins (Nei, M. and Koehn, R.K.,eds), pp. 38–61. Sinauer, Sunderland, Massachusettes.Arnheim, N., Krystal, M., Shmickel, R., Wilson, G., Ryder, O. andZimmer, E. (1980) Molecular evidence for genetic exchangesamong ribosomal genes on nonhomologous chromosomes inman and apes. Proc Natl Acad Sci USA 77: 7323–7327.Askew, R.R. and Shaw, M.R. (1986) Parasitoid communities:Their size, structure and development. In: Insect Parasitoids(Waage, J. and Greathead, D., eds), pp. 225–264. 13th Sym-posium of the Royal Entomological Society of London, 18–19September 1985.Ban, N., Nissen, P., Hansen, J., Moore, P.B. and Steitz, T.A. (2000)The complete atomic structure of the large ribosomal subunitat 2.4 Å resolution. Science 289: 905–920.Bennett, M.D. (1972) Nuclear DNA content and minimum genera-tion time in herbaceous plants. Proc Roy Soc Lond B Biol Sci181: 109–135.Bensasson, D., Petrov, D.A., Zhang, D.-X., Hartl, D.L. and Hewitt, G.M.(2001) Genomic gigantism: DNA loss is slow in mountaingrasshoppers. Mol Biol Evol 18: 246–253.Campbell, B.C., Steffen-Campbell, J.D. and Gill, R.J. (1994)Evolutionary origin of whiteflies (Hemiptera: Sternorrhyncha:Aleyrodidae) inferred from 18S rDNA sequences. Insect MolBiol 3: 175–194.Campbell, B.C., Steffen-Campbell, J.D., Sorensen, J.T. and Gill, R.J.(1995) Paraphyly of Homoptera and Auchenorrhyncha inferredfrom 18S rDNA nucleotide sequences. Syst Entomol 20: 175–194.Cannone, J.J., Subramanian, S., Schnare, M.N., Collett, J.R., Du, Y.,D’Souza, L.M., Feng, B., Lin, N., Madabusi, L.V., Muller, K.M.,Pande, N. and Shang, Z., Yu, N. and Gutell, R.R. (2002) Thecomparative RNA web (CRW) site: an online database of com-parative sequence and structure information for ribosomal,intron, and other RNAs. BMC Bioinformatics 3: 2. [Correction.BMC Bioinformatics 3: 15.].Carmean, D. and Crespi, J.B. (1995) Do long branches attractflies? Nature 373: 666.Cavalier-Smith, T. (1985) Cell Volume and the evolution of eukary-otic genome size. In: The Evolution of Genome Size (Cavalier-Smith, T., ed.), pp. 104–184. John Wiley & Sons, Chichester.Chalwatzis, N., Baur, A., Stetzer, E., Kinzelbach, R. and Zimmer-mann, R.K. (1995) Strongly expanded 18S ribosomal-RNAgenes correlated with a peculiar morphology in the insect orderof Strepsiptera. Zool Anal Complex Systems 98: 115–126.Chalwatzis, N., Hauf, J., Van de Peer, Y., Kinzelbach, R. andZimmermann, R.K. (1996) 18S ribosomal-RNA genes of insects:Primary structure of the genes and molecular phylogeny of theHolometabola. Ann Entomol Soc Am 89: 788–803.Choe, C.P., Hancock, J.M., Hwang, U.W. and Kim, W. (1999a)Analysis of the primary sequence and secondary structure ofthe unusually long SSU rRNA of the soil bug, Armadillidiumvulgare. J Mol Evol 49: 798–805.Choe, C.P., Hwang, U.W. and Kim, W. (1999b) Putative secondarystructures of unusually long strepsipteran SSU rRNAs and itsphylogenetics implications. Mol Cells 9: 191–199.Cognato, A.I. and Vogler, A.P. (2001) Exploring data interactionand nucleotide alignment in a multiple gene analysis of Ips(Scolytinae). Syst Biol 50: 758–780.Crease, T.J. and Colbourne, J.K. (1998) The unusually long small-subunit ribosomal RNA of the crustacean, Daphnia pulex:Sequence and predicted secondary structure. J Mol Evol 46:307–313.Crease, T.J. and Taylor, D.J. (1998) The origin and evolution ofvariable-region helices in V4 and V7 of the small-subunit ribos-omal RNA of branchiopod crustaceans. Mol Biol Evol 15:1430–1446.Cunningham, C.O., Aliesky, H. and Collins, C.M. (2000) Sequenceand secondary structure variation in the Gyrodactylus(Platyhelminthes: Monogenea) ribosomal RNA gene array. JParasitol 86: 567–576.De Rijk, P., Van de Peer, Y., Chapelle, S. and De Wachter, R.(1994) Database on the structure of large ribosomal subunitRNA. Nucleic Acids Res 22: 3495–3501.Dixon, M.T. and Hillis, D.M. (1993) Ribosomal secondary structure:Compensatory mutations and implications for phylogeneticanalysis. Mol Biol Evol 10: 256–267.Dover, G.A. (1982) Molecular drive: a cohesive mode of speciesevolution. Nature 299: 111–117.Elgavish, T., Cannone, J.J., Lee, J.C., Harvey, S.C. and Gutell, R.R.(2001) AA.AG@Helix.Ends: A:A and A:G base-pairs at theends of 16S and 23S rRNA helices. J Mol Biol 310: 735–753.Flavell, R.B. (1986) Structure and control of expression ofribosomal RNA genes. Oxf Surv Plant Mol Cell Biol 3: 252–274.Friedrich, M. and Tautz, D. (1997a) An episodic change of rDNAnucleotide substitution rate has occurred at the time of theemergence of the insect order Diptera. Mol Biol Evol 14: 644–653.Friedrich, M. and Tautz, D. (1997b) Evolution and phylogeny of theDiptera: a molecular phylogenetic analysis using 28S rDNAsequences. Syst Biol 46: 674–698.Gall, J.G. (1974) Free ribosomal RNA genes in the macronucleusof Tetrahymena. Proc Natl Acad Sci USA 71: 3078–3081.Gerbi, S.A. (1985) Evolution of ribosomal DNA. In: Molecular Evo-lutionary Genetics (MacIntyre, R.J., ed.), pp.419–517.PlenumPress, New York.Gillespie, J.J. (2004) Characterizing regions of ambiguous align-ment caused by the expansion and contraction of hairpin-stem
  18. 18. 642 J. J. Gillespie et al.© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643loops in ribosomal RNA molecules. Mol Phylogenet Evol 33:936–943.Gillespie, J.J., Cannone, J.J., Gutell, R.R. and Cognato, A.I. (2004)A secondary structural model of the 28S rRNA expansion seg-ments D2 and D3 from rootworms and related leaf beetles(Coleoptera: Chrysomelidae; Galerucinae). Insect Mol Biol 13:495–518.Gillespie, J.J., Yoder, M.J. and Wharton, R.A. (2005a) Predictedsecondary structures for 28S and 18S rRNA from Ichneumo-noidea (Insecta: Hymenoptera: Apocrita): Impact on sequencealignment and phylogeny estimation. J Mol Evol 61: 114–137.Gillespie, J.J., Munro, J.B., Heraty, J.M., Yoder, M.J., Owen, A.K.and Carmichael, A.E. (2005b) A secondary structural model of the28S rRNA expansion segments D2 and D3 for chalcidoid wasps(Hymenoptera: Chalcidoidea). Mol Biol Evol 22: 1593–1608.Giribet, G. and Wheeler, W.C. (2001) Some unusual small-subunitribosomal RNA sequences of metazoans. Am Mus Novitates3337: 1–14.Gonzalez, P. and Labarere, J. (2000) Phylogenetic relationships ofPleurotus species according to the sequence and secondarystructure of the mitochondrial small-subunit rRNA V4, V6 andV9 domains. Microbiology 146: 209–221.Gregory, T.R. (2001) Coincidence, coevolution, or causation?DNA content, cell size, and the C-value enigma. Biol Rev 76:65–101.Gregory, T.R. (2003) Is small indel bias a determinant of genomesize? Trends Genet 19: 485–488.Gregory, T.R. (2004) Insertion-deletion bias and the evolution ofgenome size. Gene 324: 15–34.Gutell, R.R. (1993) Collection of small subunit (16S- and 16S-like)ribosomal RNA structures. Nucleic Acids Res 21: 3051–3054.Gutell, R.R. (1994) Collection of small subunit (16S- and 16S-like)ribosomal RNA structures: 1994. Nucleic Acids Res 22: 3502–3507.Gutell, R.R. and Damberger, S.H. (1996) Comparative sequenceanalysis of experiments performed during evolution. In: Ribos-omal RNA and Group I Introns (Green, R. and Schroeder, R.,eds), pp. 15–33. R.G. Landes Company, Austin, Texas.Gutell, R.R., Weiser, B., Woese, C.R. and Noller, H.F. (1985) Com-parative anatomy of 16S-like ribosomal RNA. Prog NucleicAcid Res Mol Biol 32: 155–216.Gutell, R.R., Power, A., Hertz, G., Putz, E. and Stormo, G. (1992)Identifying constraints on the higher-order structure of RNA:continued development of comparative sequence analysismethods. Nucleic Acids Res 20: 5785–5795.Gutell, R.R., Larsen, N. and Woese, C.R. (1994) Lessons from anevolving rRNA: 16S and 23S rRNA structures from a compar-ative perspective. Microbiol Rev 58: 10–26.Gutell, R.R., Lee, J.C. and Cannone, J.J. (2002) The accuracy ofribosomal RNA comparative structure models. Curr OpinStruct Biol 12: 301–310.Huelsenbeck, J.P. (1997) Is the Felsenstein zone a fly trap? SystBiol 46: 69–74.Hwang, S.K. and Kim, J.G. (2000) Secondary structure andphylogenetic implications of nuclear large subunit ribosomalRNA in the ectomycorrhizal fungus Tricholoma matsutake.Curr Microbiol 40: 250–256.Hwang, U.I., Kim, W., Tautz, D. and Friedrich, M. (1998) Molecularphylogenetics at the Felsenstein zone: Approaching theStrepsiptera problem using 5.8S and 28S rDNA sequences.Mol Phylogenet Evol 9: 470–480.Jackson, S.A., Cannone, J.J., Lee, J.C., Gutell, R.R. andWoodson, S.A. (2002) Distribution of rRNA introns in the three-dimensional structure of the ribosome. J Mol Biol 323: 35–52.Johnston, J.S., Ross, L.D., Bean, L., Hughes, D.P. and Kathirithamby,J. (2004) Tiny genomes and endoreduplication in Strepsiptera.Insect Mol Biol 13: 581–585.Kathirithamby, J. (1989) Review of the order Strepsiptera. SystEntomol 14: 41–92.Kathirithamby, J. (1991a) Stichotrema robertsoni spec. n. (Strep-siptera: Myrmecolacidae): The first report of stylopization inminor workers of an ant (Pheidole sp. Hymenoptera: Formicidae).J Entomol Soc South Afr 54: 9–15.Kathirithamby, J. (1991b) Strepsiptera.In: The Insects of Australia:a Textbook for Students and Research Workers, Vol. 1(Naumann, I.D., Carne, P.B., Lawrence, J.F., Nielsen, E.S., Sprad-berry, J.P., Taylor, R.W., Whitten, M.J. and Littlejohn, M.J., eds),pp. 684–695. CSIRO, Melbourne University Press, Melbourne.Kathirithamby, J. and Hamilton, W.D. (1992) More covert sex: Theelusive females of Myrmecolacidae.Trends Ecol Evol 7:349–351.Kathirithamby, J., Ross, L.D. and Johnston, J.S. (2003) Masquer-ading as self?: Endoparasitic Strepsiptera (Insecta) enclosethemselves in host-derived epidermal bag. Proc Natl Acad SciUSA 100: 7655–7659.Kinzelbach, R.K. (1971) Morphologische Befunde anFächerflüglern und Ihre Phylogenetische Bedeutung (Insecta:Strepsiptera). Schweizerbart’sche-Verlagsbuchhandlung,Stuttgart, Germany.Kinzelbach, R. (1990) The systematic position of Strepsiptera(Insecta). Am Entomol 36: 292–303.Kjer, K.M. (1995) Use of rRNA secondary structure in phylogeneticstudies to identify homologous positions: an example of align-ment and data presentation from the frogs. Mol PhylogenetEvol 4: 314–330.Kjer, K.M. (1997) An alignment template for amphibian 12S rRNA,domain III: conserved primary and secondary structural motifs.J Herpetol 31: 599–604.Kjer, K.M. (2004) Aligned 18S and insect phylogeny. Syst Biol 53:506–514.Kwon, O.Y., Ogino, K. and Ishikawa, H. (1991) The longest 18Sribosomal RNA ever known. Eur J Biochem 202: 827–833.Lee, M.S.Y. (2001) Unalignable sequences and molecular evolu-tion. Trends Ecol Evol 16: 681–685.Lydeard, C., Holznagel, W.E., Schnare, M.N. and Gutell, R.R.(2000) Phylogenetic analysis of molluscan mitochondrial LSUrDNA sequences and secondary structures. Mol PhylogenetEvol 15: 83–102.Mallatt, J., Garey, J.R. and Shultz, J.W. (2004) Ecdysozoan phyl-ogeny and Bayesian inference: First use of nearly complete28S and 18S rRNA gene sequences to classify the arthropodsand their kin. Mol Phylogenet Evol 31: 178–191.Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. (1999)Expanded sequence dependence of thermodynamic parame-ters improves prediction of RNA secondary structure. J MolBiol 288: 911–940.Morin, L. (2000) Long branch attraction effects and the status of‘basal eukaryotes’: Phylogeny and structural analysis of theribosomal RNA gene cluster of the free-living diplomonadTrepomonas agilis. J Eukaryot Microbiol 47: 167–177.Morrison, D.A. and Ellis, J.T. (1997) Effects of nucleotide sequencealignment on phylogeny estimation: a case study of 18S rDNAsof Apicomplexa. Mol Biol Evol 14: 428–441.
  19. 19. Bizarre insertions in strepsipteran 18S rRNA 643© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 625–643Mugridge, N.B., Morrison, D.A., Johnson, A.M., Luton, K., Dubey, J.,Votypka, J. and Tenter, A.M. (1999) Phylogenetic relationshipsof the genus Frenkelia: a review of its history and new know-ledge gained from comparison of large subunit ribosomalribonucleic acid gene sequences. Int J Parasitol 29: 957–972.Nagl, W. (1978) Endopolyploidy, and Polyteny in Differentiationand Evolution: Towards an Understanding of Quantitative andQualitative Variation of Nuclear DNA in Ontogeny and Phylog-eny. Elsevier/North-Holland Biomed, New York.Nagylaki, T. (1988) Gene conversion, linkage, and the evolution ofmultigene families. Genetics 120: 291–301.Ogloblin, A.A. (1939) The Strepsiptera parasites of ants. Int CongrEntomol Berlin 1938(2): 1277–1284.Ohta, T. (1980) Evolution and Variation of Multigene Families.Springer-Verlag, Berlin.Ohta, T. (1982) Allelic and nonallelic homology of a supergenefamily. Proc Natl Acad Sci USA 79: 3251–3254.Petrov, D.A. (2001) Evolution of genome size: new approaches toan old problem. Trends Genet 17: 23–28.Petrov, D.A. (2002) Mutational equilibrium model of genome sizeevolution. Theor Pop Biol 61: 533–546.Petrov, D.A., Lozovskaya, E.R. and Hartl, D.L. (1996) High intrinsicrate of DNA loss in Drosophila. Nature 384: 346–349.Petrov, D.A., Sangster, T.A., Johnston, J.S., Hartl, D.L. and Shaw, K.L.(2000) Evidence for DNA loss as a determinant of of genomesize. Science 287: 1060–1062.Prokopowich, C.D., Gregory, T.R. and Crease, T.J. (2003) The cor-relation between rDNA copy number and genome size ineukaryotes. Genome 46: 48–50.Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J., Gluehmann, M.,Janell, D. et al. (2000) Structure of functionally activated smallribosomal subunit at 3.3 Å resolution. Cell 102: 615–623.Schnare, M.N., Damberger, S.H., Gray, M.W. and Gutell, R.R.(1996) Comprehensive comparison of structural characteristicsin eukaryotic cytoplasmic large subunit (23S-like) ribosomalRNA. J Mol Biol 256: 701–719.Spahn, C.M., Beckmann, R., Eswar, N., Penczek, P.A., Sali, A.,Blobel, G. and Frank, J. (2001) Structure of the 80S ribosomefrom Saccharomyces cerevisiae-tRNA-ribosome and subunit–subunit interactions. Cell 107: 373–386.Strand, M.R. (1986) The physiological interactions of parasitoidswith their hosts and their influence on reproductive strategies.In: Insect Parasitoids (Waage, J. and Greathead, D., eds),pp. 97–136. 13th Symposium of the Royal Entomological Soci-ety of London, 18–19 September 1985.Strand, M.R. and Peach, L.L. (1995) Immunological basis for com-patibility in parasitoid-host relationships. Annu Rev Entomol40: 31–56.Titus, T.A. and Frost, D.R. (1996) Molecular homology assessmentand phylogeny in the lizard family Opluridae (Squamata:Iguania). Mol Phylogenet Evol 6: 49–62.Uchida, H., Kitae, K., Tomizawa, K.I. and Yokota, A. (1998) Com-parison of the nucleotide sequence and secondary structure ofthe 5.8S ribosomal RNA gene of Chlamydomonas tetragamawith those of green algae. DNA Seq 8: 403–408.Van de Peer, Y., Robbrecht, E., De Hoog, S., Caers, A., De Rijk, P.and De Wachter, R. (1999) Database on the structure of smallsubunit ribosomal RNA. Nucleic Acids Res 27: 179–183.Walter, G.H. (1983) ‘Divergent male ontogenies’ in Aphelinidae(Hymenoptera: Chalcidoidea): a simplified classificationand a suggested evolutionary sequence. Biol J Linn Soc 19:63–82.Wheeler, W.C. and Gladstein, D.L. (1994) MALIGN, Version 1.93.American Museum of Natural History, New York.Whiting, M.F., Carpenter, J.C., Wheeler, Q.D. and Wheeler, W.C.(1997) The Strepsiptera problem: phylogeny of the holometab-olous insect orders inferred from 18S and 28S ribosomal DNAsequences and morphology. Syst Biol 46: 1–68.Wiegmann, B.M., Yeates, D.K., Thorne, J.L. and Kishino, H. (2003)Time flies, a new molecular time-scale for brachyceran flyevolution without a clock. Syst Biol 52: 745–756.Wimberly, B.T., Brodersen, D.E., Clemons, W.M. Jr, Morgan-Warren,R.J., Carter, A.P. et al. (2000) Structure of the 30S ribosomalsubunit. Nature 407: 327–339.Winnepenninckx, B., Backeljau, T. and De Wachter, R. (1995)Phylogeny of protostome worms derived from 18S rRNAsequences. Mol Biol Evol 12: 641–649.Woese, C.R., Magrum, L.J., Gupta, R., Siegel, R.B., Stahl, D.A.,Kop, J., Crawford, N., Brosius, J., Gutell, R., Hogan, J.J. andNoller, H.F. (1980) Secondary structure model for bacterial 16Sribosomal RNA:Phylogenetic, enzymatic and chemical evidence.Nucleic Acids Res 8: 2275–2293.Woese, C.R., Winker, S. and Gutell, R.R. (1990) Architecture ofribosomal RNA: constraints on the sequence of Tetra-loops.Proc Natl Acad Sci USA 87: 8467–8471.Wuyts, J., De Rijk, P., Van de Peer, Y., Pison, G., Rousseeuw, P.and De Wachter, R. (2000) Comparative analysis of more than3000 sequences reveals the existence of two pseudoknots inarea V4 of eukaryotic small subunit ribosomal RNA. NucleicAcids Res 28: 4698–4708.Wuyts, J., Van de Peer, Y. and De Wachter, R. (2001) Distributionof substitution rates and location of insertion sites in the tertiarystructure of ribosomal RNA. Nucleic Acids Res 29: 5017–5028.Xia, X. (2000) Phylogenetic relationship among horseshoe crabspecies: The effect of substitution models on phylogeneticanalyses. Syst Biol 49: 87–100.Xia, X., Xie, Z. and Kjer, K.M. (2003) 18S ribosomal RNA andtetrapod phylogeny. Syst Biol 52: 283–295.Yao, M.-C., Kimmel, A.R. and Gorovsky, M.A. (1974) A smallnumber of cistrons for ribosomal RNA in the germinal nucleusof a eukaryote, Tetrahymena pyriformis. Proc Natl Acad SciUSA 71: 3082–3086.Yeates, D.K. and Wiegmann, B.M. (1999) Congruence and contro-versy: Toward a higher-level phylogeny of the Diptera. AnnuRev Entomol 44: 397–428.Yusupov, M.M., Yusupova, G.Z., Baucom, A., Lieberman, K.,Earnest, T.N., Cate, J.H. and Noller, H.F. (2001) Crystal struc-ture of the ribosome at 5.5 Å resolution. Science 292: 883–896.Zuker, M., Mathews, D.H. and Turner, D.H. (1999) Algorithms andthermodynamics for RNA secondary structure prediction: apractical guide. In: RNA Biochemistry and Biotechnology,NATO ASI Series (Barciszewski, J. and Clark, B.F.C., eds),pp. 11–43. Kluwer Academic Publishers, Boston, MA.

×