A Secondary Structural Model of the 28S rRNA Expansion Segments D2 and D3 for Chalcidoid Wasps (Hymenoptera: Chalcidoidea)

http://www.100md.com 分子生物学进展 2005年第7期

     * Department of Entomology, Texas A&M University; and Department of Entomology, University of California, Riverside

    Correspondence: E-mail: pvittata@hotmail.com.

    Abstract

    We analyze the secondary structure of two expansion segments (D2, D3) of the 28S ribosomal (rRNA)-encoding gene region from 527 chalcidoid wasp taxa (Hymenoptera: Chalcidoidea) representing 18 of the 19 extant families. The sequences are compared in a multiple sequence alignment, with secondary structure inferred primarily from the evidence of compensatory base changes in conserved helices of the rRNA molecules. This covariation analysis yielded 36 helices that are composed of base pairs exhibiting positional covariation. Several additional regions are also involved in hydrogen bonding, and they form highly variable base-pairing patterns across the alignment. These are identified as regions of expansion and contraction or regions of slipped-strand compensation. Additionally, 31 single-stranded locales are characterized as regions of ambiguous alignment based on the difficulty in assigning positional homology in the presence of multiple adjacent indels. Based on comparative analysis of these sequences, the largest genetic study on any hymenopteran group to date, we report an annotated secondary structural model for the D2, D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related apocritan taxa.

    Key Words: rRNA ? ribosome ? chalcidoid ? secondary structure ? expansion segment ? homology ? multiple sequence alignment ? 28S

    Introduction

    With an estimated 400,000 species, of which less than 6% have been described, the superfamily Chalcidoidea is one of the largest and least-understood groups of Hymenoptera (Noyes 2000; Heraty and Gates 2003). Adults are usually 2–5 mm in size but can range from 0.13 mm to more than 25 mm in total body length (Mockford 1997; Gibson, Heraty, and Woolley 1999). They are extremely diverse morphologically, and yet commonly display trends involving the reduction or convergence of morphological traits (Heraty and Schauff 1998; Gibson, Heraty, and Woolley 1999). Larvae develop as parasitoids of 13 insect orders, spider eggs, ticks, pseudoscorpions, and nematodes, or as primary or secondary feeders on plant tissue (Grissell and Schauff 1997; Gibson, Heraty, and Woolley 1999). Their behavior is just as diverse as their morphology and host range. Parasitic Chalcidoidea are very important for the control of phytophagous insects in both natural and agricultural insect populations (Noyes 1978; Greathead 1986; La Salle 1993).

    The superfamily currently comprises 19 families, approximately 89 subfamilies, and about 21,000 described species (Noyes 1978, 1990, 2002; Gibson, Heraty, and Woolley 1999). Much of the diversification of Chalcidoidea had occurred by the late Cretaceous based on the presence of fairly modern representatives of Mymaridae, Trichogrammatidae, and Tetracampidae in Canadian amber (Yoshimoto 1975). Because of their small size, convergence of morphological traits, and the frequent reduction or loss of features, it can be difficult to assess the relationships of species or even the relationships between genera with any degree of confidence. The taxonomy of the group is not well resolved, with many family-group taxa considered paraphyletic or polyphyletic, and several subfamilies are unplaced at the family level (Gibson, Heraty, and Woolley 1999). Morphological features have been proposed that support the monophyly of the superfamily (Gibson 1986). However, a phylogeny based on morphological characters within the superfamily has not been proposed, although "intuitive" phylogenies have been postulated (Gibson 1990; Noyes 1990; cf., Heraty, Woolley, and Darling 1997). The lack of available hypotheses of relationships prevents the application of the predictive power of phylogenetic systematics to practical and theoretical aspects of Chalcidoidea classification and biology.

    Several recent studies have focused on the higher level relationships of parasitic Hymenoptera using molecular data (Dowton and Austin 1994, 1998; Belshaw and Quicke 1997, 2002; Gimeno, Belshaw, and Quicke 1997; Belshaw et al. 1998, 2001; Dowton, Austin, and Antolin 1998; Whitfield and Cameron 1998; Mardulyn and Whitfield 1999; Kambhampati, Volkl, and Mackauer 2000; Dowton et al. 2002). A few chalcidoid taxa have been included in studies of relationships among Hymenoptera (Derr et al. 1992a, 1992b; Dowton and Austin 1994, 1995, 1998, 2001; Schulmeister 2003), but so far only one admittedly preliminary study was carried out for 109 species of Chalcidoidea (Campbell et al. 2000). The mitochondrial 16S ribosomal RNA (rRNA) transcript was the focus of earlier studies that included Chalcidoidea (Derr et al. 1992a, 1992b; Dowton and Austin 1994, 1995, 1997). More recently, there has been a shift toward including various regions of the nuclear rRNA large subunit (LSU) 28S rRNA for higher level relationships of Chalcidoidea within Hymenoptera (Dowton and Austin 2001; Schulmeister 2003), across Chalcidoidea (Campbell et al. 2000), within families of Chalcidoidea (Rasplus et al. 1998; Gauthier et al. 2000; Lopez-Vaamonde et al. 2001; Heraty 2003; Heraty et al. 2004), and at the generic level or below (Campbell, Steffen-Campbell, and Werren 1993; Babcock and Heraty, 2000; De Barro et al. 2000; Babcock et al. 2001; Manzari et al. 2002; Pedata and Polaszek, 2003). All these studies have sequenced either or both the D2 and D3 expansion segments as well as related core elements in this region of the 28S rRNA.

    The 28S rRNA molecule of insects is composed of a series of highly conserved core elements and 13 expansion segments (Hancock and Dover 1988; Hancock, Tautz, and Dover 1988; Tautz et al. 1988). While the core regions of the LSU rRNA are highly conserved structurally across all domains of life (Clark et al. 1984; Hadjiolov et al. 1984; Hogan, Gutell, and Noller 1984; Michot, Hassouna, and Bachellerie 1984), the expansion segments can vary greatly, even across recently diverged lineages (e.g., Michot, Qu, and Bachellerie 1990; Hillis and Dixon 1991; Schnare et al. 1996; Gillespie, Yoder, and Wharton 2005). Thus, severe deviations from a common structure in eukaryotic expansion segments are expected (Schnare et al. 1996), especially among taxa that have diverged over a large evolutionary timescale. The 13 expansion segments (D1–D7a, D7b–D12) of the 28S rRNA vary greatly among insect orders (Hwang et al. 1998; Gillespie, unpublished data), as well as within Diptera (Tautz et al. 1988; Kjer, Baldridge, and Fallon 1994; Schnare et al. 1996) and Hymenoptera (Belshaw and Quicke 2002; cf., Gillespie, Yoder, and Wharton 2005). As in other eukaryotes, the expansion segments of the insect 28S rRNA are more variable than the core elements but are constrained structurally, with deleterious mutations often accommodated by compensatory base changes that maintain helical formation (Hancock, Tautz, and Dover 1988; Tautz et al. 1988; Rousset, Pelandakis, and Solignac 1991; Kjer, Baldridge, and Fallon 1994). This variation in expansion segments is typically associated with a wide range of phylogenetically informative characters among higher taxonomic levels (De Rijk et al. 1995; Schnare et al. 1996).

    The 28S rRNA expansion segments consist of one or a series of putative helical and nonpairing regions that are useful for assessing different levels of taxonomic divergence. The 28S ribosomal rDNA (rDNA) (28S rRNA transcript) is considered useful for positing divergences of between 60 and 200 MYA (Larsen 1991). The D2 expansion segment (300–700 bp) is more commonly used in analyses, with the more conserved D3 expansion segment (350 bp) used in fewer studies and typically providing supporting evidence for relationships inferred by the D2 region (Nunn et al. 1996; Rasplus et al. 1998; De Barro et al. 2000; Lopez-Vaamonde et al. 2001; Manzari et al. 2002; Heraty 2003; Kim 2003; Pedata and Polaszek 2003; Schulmeister 2003; Heraty et al. 2004). Divergence rates for the D2 and D3 expansion segments are proportionally the same within groups, with the D3 region being more conserved than the D2 region (Heraty 2003). However, there can be a great deal of divergence of rates for similar regions across different taxonomic groups. For example, the divergence within Eulophidae for D2 (17.4%) is comparable to the divergence in Eucharitidae for D3, and the divergence rates between species of Encarsia (29.0%) and Aphytis (23.1%) are much higher than found within most families of Chalcidoidea (Heraty 2003). Various primers have been developed to amplify different portions of the D2 and D3 regions of the 28S rRNA for use in Hymenoptera (table 1).

    Table 1 Primer Sequences Commonly Used to Amplify Portions of the D2 and D3 Expansion Segments in Hymenoptera

    FIG. 1.— Secondary structure of the expansion segments D2 and D3 of the LSU 28S rRNA from the chalcidoid Encarsia hispida (Aphelinidae). The boxed regions show the expansion segments D2 and D3 (regions 545 and 650, respectively, of Schnare et al. [1996]) that were analyzed in this study. A third variable region identified in this study, H604, is also boxed. Base pairing is indicated as follows: standard canonical pairs 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); and other noncanonical pairs by filled circles (e.g., C ? A). Tertiary interactions within the LSU are shown as described in Cannone et al. (2002). Missing nucleotides are described in the text and are shown as Ns. Structures consisting solely of Ns were modeled after the predicted structure for the beetle Tenebrio (Coleoptera: Tenebrionidae) (Gillespie et al. 2004b). Core helices, for which at least one strand was sequenced, are numbered following Cannone et al. (2002). The diagram was generated using the program XRNA (B. Weiser and H. Noller, University of California at Santa Cruz).

    Because the expansion segments of the 28S rRNA are less constrained structurally than core regions of the LSU, they are ideal as phylogenetic markers; however, their use in phylogeny reconstruction of Insecta is often problematic due to the difficulty of alignment of multiple sequences from divergent taxa (e.g., De Rijk et al. 1995). It has been known for at least a decade that reference to secondary structure can improve the assignment of positional homology in length heterogeneous data sets (e.g., Gutell et al. 1985, 1992; Gutell, Larsen, and Woese 1994; Kjer 1995; Hickson et al. 1996), and structure-based alignments have also been shown to increase phylogenetic accuracy over automated approaches (Dixon and Hillis 1993; Kjer 1995; Titus and Frost 1996; Morrison and Ellis 1997; Uchida et al. 1998; Mugridge et al. 1999; Cunningham, Aliesky, and Collins 2000; Gonzalez and Labarere 2000; Hwang and Kim 2000; Lydeard et al. 2000; Morin 2000; Xia 2000; Xia, Xie, and Kjer 2003). Despite this, structural models for the 28S rRNA expansion segments generated from many sequences within insect taxa are rare (reviewed in Gillespie et al. 2004b), and few studies within Hymenoptera have attempted to elucidate global structures that guide the assignment of nucleotide homology in local regions of these molecules. Using the program foldRNA in the Wisconsin Computer Package (GCG 1994), Belshaw and Quicke (1997) generated secondary structure models of the D2 for two braconid wasps and also provided alignments from conserved regions of the molecule. Belshaw and Quicke (2002) also provided a structure-based alignment of the D2 and D3 generated from the alignment editor SSE (Page 2000), which performs the search for potential helices in rRNA based on maximum weighted matching, with the most optimal folding structure used as a guide, as based on a folding algorithm (Matthews et al. 1999; Zuker, Mathews, and Turner 1999). While both these studies provide structural models for the expansion segments D2 and D3, no subsequent studies have used these structural alignments as templates for guiding the comparison of newly generated sequences. Possible reasons for this, including issues of repeatability and data exclusion, are discussed by Gillespie, Yoder, and Wharton (2005).

    Aside from these few studies using structure as a guide to assigning nucleotide positional homology, alignments for 28S rRNA in Hymenoptera are usually either optimized with a "by-eye" approach or subjected to automated alignment programs, such as ClustalW (Thompson, Higgins, and Gibson 1994). For Chalcidoidea, an explicit structural model for any of the rRNAs has not been proposed. If divergence rates are low, then a structural-based approach is a feasible strategy, but often there are numerous insertions in some groups (i.e., Trichogrammatidae, Encyrtidae) that make these alignments very difficult and time consuming. These divergent sequences, with extreme length heterogeneity observed across even the closest of taxa, also make it difficult to use automated alignment programs, especially because the assignment of gap cost and extension penalties are entirely subjective and no single set of costs are likely to be universally optimal for any data set (Kjer 2004; Petersen et al. 2004). Methods for optimizing the alignment of length-variable regions based on other data sources (i.e., alignable regions from the same gene, characters from other genes, morphology) are considered to be biased by us because the evolutionary properties of these partitions may not be similar to those of hypervariable regions of rRNA molecules (see Simmons 2004).

    We agree with Belshaw and Quicke (2002) that subjective alignments are not to be preferred over objective methods, if they are indeed available. In this study, we present a structural model for the expansion segments D2 and D3 of the 28S rRNA from 527 chalcidoid wasps (Hymenoptera: Chalcidoidea), representing 187 genera in 18 of the 19 extant families (Rotoitidae not included). This model is a refined annotation from previous studies that incorporated secondary structure to improve homology assignment in the D2 and D3 regions of chrysomelid beetles (Gillespie et al. 2004b), Amphiesmenoptera (Kjer, Blahnik, and Holzenthal 2001), and ichneumonoid Hymenoptera (Gillespie, Yoder, and Wharton 2005). We use compensatory base change evidence to objectively define conserved regions of the molecule, resulting in a custom chalcidoid model for this region of the 28S rRNA. Our characterization of regions of alignment ambiguity (RAA), slipped-strand compensation (RSC), and expansion and contraction (REC) from structural homology is discussed within a phylogenetic context. This model will be useful for future studies on related apocritan groups that utilize the D2 and D3 expansion segments for phylogeny reconstruction and for studies that address rRNA structural evolution across higher level insect taxa (e.g., Page, Crulckshank, and Johnson 2002; Misof and Fleck 2003).

    Materials and Methods

    Taxon Sampling

    All species analyzed in this study are summarized in Supplementary Material 1, with taxa organized according to current chalcid taxonomy (Viggiani 1971, 1984; Noyes 1978, 1990; Gibson, Heraty, and Woolley 1999). A total of 527 taxa representing 194 genera in 18 of the 19 extant families of the Chalcidoidea were compiled. The out-group is represented by the one extant genus of Mymarommatoidea (Mymarommatidae) (Supplementary Material 1).

    Sequence Generation

    For new sequences generated in this study, all species are represented, as indicated, by a primary (remains of actual specimen sequenced) or secondary (compared specimen from same collection series) molecular voucher in the University of California, Riverside (UCR) Entomology Research Museum. Sequences used from previous studies are indicated as either having come from GenBank or J.-W. Kim (KD numbers) in the UCR CODE column (Supplementary Material 1). All adults were killed in 75%–95% ethanol and stored in 95% ethanol at –80°C. Adults were either removed from 95% to 100% ethanol and dried in open Eppendorf tubes at 32°C for 30 min prior to a phenol-chloroform extraction (Campbell, Steffen-Campbell, and Werren 1993) or Chelex? extraction (modified from Walsh, Metzger, and Higuchi 1991) or dried prior to sequencing using hexamethyldisilazane (Heraty and Hawks 1998). Two rDNA regions were independently sequenced (28S-D2 and 28S-D3) corresponding to the variable regions 545 and 650 of the Escherichia coli 23S LSU rRNA model (Cannone et al. 2002). The following forward (F) and reverse (R) primer combinations were used: 28S-D2, D2-3551F, and D2-4057R (table 1); 28S-D3, D3-4046F, and D3-4413R (table 1). Polymerase chain reaction (PCR) protocols followed Babcock et al. (2001). PCR products were purified using GeneClean? PCR Purification Kits (Q-BIOgene). Both sense and antisense strands were sequenced at either the San Diego Microchemical Core Facility or the UCR Genomics facility. Sequences were deposited in GenBank, and accession numbers for all sequences used in this study are provided in Supplementary Material 1.

    Multiple Sequence Alignment Utilizing Secondary Structure

    The 28S-D2, D3 sequences were aligned manually according to secondary structure, with the notation following Kjer, Baldridge, and Fallon (1994) and Kjer (1995), with slight modifications (see Supplementary Material 2). Alignment initially followed the secondary structural models of Gutell, Larsen, and Woese (1994), which were obtained from the comparative RNA Web site http://www.rna.icmb.utexas.edu (Cannone et al. 2002), and was further modified according to chrysomelid (Gillespie et al. 2003, 2004a, 2004b) and ichneumonoid (Gillespie, Yoder, and Wharton 2005) D2 models and a trichopteran D3 model (Kjer, Blahnik, and Holzenthal 2001). Individual sequences, especially hairpin-stem loops, were evaluated in the program mfold (version 3.1; http://www.bioinfo.rpi.edu/applications/mfold), which folds rRNA based on free energy minimizations (Matthews et al. 1999; Zuker, Mathews, and Turner 1999). These free energy–based predictions were used to facilitate the search for potential base-pairing stems, which were confirmed only by the presence of compensatory base changes across a majority of taxa.

    Regions in which positional homology assessments were ambiguous across all taxa were defined according to structural criteria as in Kjer (1997) and described as RAAs or RSCs (Levinson and Gutman 1987; for reviews regarding rRNA sequence alignment see Schultes, Hraber, and LaBean 1999; Hancock and Vogler 2000). Briefly, ambiguously aligned regions in which base pairing was not identifiable were characterized as RAAs. For ambiguously aligned regions wherein base pairing was observed (RSCs), compensatory base change evidence was used to confirm structures that were not consistent across the alignment due to the high occurrence of adjacent insertion and deletion events (indels). For eight ambiguously aligned regions in the alignment caused by the expanding and contracting of hairpin-stem loops, RSCs were further characterized as RECs based on structural evidence used to identify separate nonpairing ambiguously aligned regions of the alignment (terminal bulges). Gillespie (2004) addresses the characterization of RAAs, RSCs, and RECs with a discussion on phylogenetic methods accommodating these regions.

    Structure-Based Summary Statistics

    Our structural alignment was modified as a PAUP* v.4.0b10 (Swofford 1999) executable Nexus file, and an associated index file identifying all elements (subblocks) separated by white spaces in the alignment was created. A pairing mask similar to that utilized in the program PHASE (Jow, Hudelot, and Higgs 2002; Hudelot et al. 2003) was assigned to all characters in the alignment, distinguishing between pairing and nonpairing regions of the sequences. Several modifications to the Nexus format were made to allow for integration of the index file, the pairing mask statement, and the implied delimitation of pairing, nonpairing, and ambiguously aligned regions taken from the notation used to construct the alignment (see above). Using the freely available cross-platform scripting language Perl (scripts titled "jRNA," source available at http://hymenoptera.tamu.edu/rna), the data were parsed into several input files, allowing for pairing and nonpairing regions to be analyzed separately, as well as combined. These jRNA scripts were further used to export the combined index and Nexus input files to several Web-viewable formats that include color-highlighted alignments, summary statistics on base pair composition and covariation, and column and region base composition (http://hymenoptera.tamu.edu/rna). The covariation tables (Supplementary Material 3) were obtained using our jRNA scripts, with subsequent calculations of base pair frequencies scored as percentages within each position of the alignment. The jRNA scripts were also implemented to produce Nexus files for the calculation of nucleotide frequencies and transition-transversion ratios (table 2) using PAUP*.

    Table 2 Some Summary Statistics of Pairing Versus Nonpairing Regions of the D2 and D3 Expansion Segments and Related Core Elements of the 28S rRNA Gene from 527 Sampled Chalcidoid Taxaa

    Secondary structure diagrams were generated with the computer program XRNA (developed by B. Weiser and H. Noller, University of Santa Cruz). Individual secondary structure diagrams are available at http://hymenoptera.tamu.edu/rna. The reader is encouraged to check for continuing updates to the alignment and availability of secondary structure diagrams.

    Results and Discussion

    Predicted Secondary Structure

    A predicted secondary structural model of part of the 5' half of the LSU rRNA from a chalcidoid is shown here (fig. 1) in concordance with the conserved 23S- and 23S-like structures of the LSU rRNA from the literature (Wool 1986; Gutell and Fox 1988; Gutell, Schnare, and Gray 1990, 1992; Gutell, Gray, and Schnare 1993; Schnare et al. 1996). For purposes of showing the interactions between helices H15 and H35 with the 5.8S as well as the 5' strand of helix H265, we depict the entire 5.8S and positions 1–374 of the conserved E. coli model (Cannone et al. 2002) with Ns. Additionally, the 3' strands of helices H579 and H812 plus related core elements are also shown as Ns to portray a complete structure on this region of the 28S rRNA. The expansion segments D2 and D3 are boxed, as well as a third region, H604, identified as highly variable in this and another recent study (Gillespie, Yoder, and Wharton 2005). A multiple sequence alignment spanning the expansion segments D2 and D3 of the 28S rRNA and related core elements was generated from 527 chalcidoid taxa; however, seven sampled taxa are listed for brevity (Supplementary Material 2). The entire alignment in a variety of formats is posted at the jRNA Web page, http://hymenoptera.tamu.edu/rna.

    Expansion Segment D2

    The 28S-D2, corresponding to the 545 variable region of the 23S-like LSU (Schnare et al. 1996), is composed of four main compound helices that are flanked by highly conserved elements of the 28S rRNA core structure. These motifs are labeled "helix 1," "helix 2," "helix 3–1," and "helix 3–2," while the subcomponents of the compound helices are named a, b, c, etc. (fig. 2A). A total of 30 conserved helical elements comprise the D2 in chalcidoids (but see below regarding helix 3q in many taxa). The innermost helix of the D2, named here as helices 1a and 1b (helix A in Schnare et al. 1996), is highly conserved in nucleotide composition; however, higher level comparisons in insects reveal substantial evidence for covariation within this helix (Gillespie, unpublished data).

    FIG. 2.— Secondary structure diagram of the D2 expansion segment (synonymous with the 545 region of Schnare et al. [1996]) shown for Encarsia hispida. Notation for the four compound helices follows the convention of Gillespie et al. (2004b) and Gillespie, Yoder, and Wharton (2005). Helices are boxed in (A), and ambiguously aligned regions are boxed in (B). The notation for RAAs, RSCs, and RECs is explained in Supplementary Materials 2 and 4. The explanations of base pair symbols and reference for software used to construct structure diagrams are given in figure 1.

    Helix 2 in the D2 region is at the base of the second compound helix and is composed of six base pairs across nearly all holometabolous insects (most Diptera have nine; Gillespie, unpublished data). The chalcidoids contain seven helices that are apical to helix 2 (2a–2g). Many of the base pairs within these helices are supported with positional covariation. The terminal helix in this motif, helix 2g, has the potential to form additional base pairings other than the five boxed positions; however, homology assignment is not clear beyond the fifth base pair in 2g due to the lack of conservation in base composition and the high occurrence of adjacent indels in this region (see REC 1 below). One REC, one RSC, and six RAAs occur in helix 2 (fig. 2B).

    Helix 3 (H2 of Michot and Bachellerie 1987; E of Schnare et al. 1996) is highly conserved in the higher eukaryotes and is the most basal helix to several compound helices (Schnare et al. 1996; Gillespie et al. 2004b). Helix 3 is 6-bp long in the chalcidoids and most holometabolous insect lineages (Gillespie, unpublished data). The chalcidoids have two compound helices distal to helix 3, helix 3–1 (helices 3a–3f–3) and helix 3–2 (helices 3g–3p) (fig. 2A). The terminal helix in helix 3–1, 3f–3, has the potential to form additional base pairings beyond the five boxed positions; however, homology assignment is ambiguous distal to the 3f–3 boxed base pairs in figure 2A due to the lack of sequence conservation and the variation in sequence lengths. Helix 3–1 has one REC, two RSCs, and four RAAs (fig. 2B).

    Unlike compound helices 2 and 3–1 in the D2 expansion segment, which contain some length variation, the terminal helices of helix 3–2, 3o, and 3p are very conserved in length and base sequence. This is consistent with recent studies on chrysomelid beetles (Gillespie et al. 2003, 2004a, 2004b) and ichneumonoid Hymenoptera (Gillespie, Yoder, and Wharton 2005) and may reflect a highly conserved structure across Holometabola. In contrast, helix 3i is variable in length and sequence across all taxa (fig. 2B). Length variation is also located in the unpaired nucleotides between strands 3h' and 3g', ranging from 0 to 17 bp (= helix 3q, fig. 2A), with additional length heterogeneity associated with flanking, unpaired nucleotides (fig. 2B). Gillespie et al. (2004b) observed this putative structure in the chrysomelid Agelastica coerulea and labeled the helix 3q. Despite being characterized as an REC, we find strong compensatory evidence within helix 3q in many of the sampled chalcidoids, suggesting that this region of the D2 is indeed a likely position to accumulate insertions and support a coevolving helix (fig. 3). Helix 3–2 has 14 RAAs, 1 RSC, and 2 RECs (fig. 2B), with an additional ambiguously aligned region (RAA (11)) linking compound helices 3–1 and 3–2.

    FIG. 3.— Portion of our multiple sequence alignment of the D2 expansion segment illustrating the expansion and contraction of a putative helix (3q) inserted between the 3' strands of helices 3h and 3g. See Supplementary Material 2 legend for alignment notation and other descriptions.

    Expansion Segment D3

    The 28S-D3, corresponding to the 650 region of the nuclear LSU (Schnare et al. 1996), contains three compound helices in chalcidoids, labeled "D3–1," "D3–2," and "D3–3" (fig. 4A), following the notation of Kjer, Blahnik, and Holzenthal (2001). Compound helix D3–1 is subdivided into three helices (D3–1a, D3–1b, and D3–1c) that are separated by two lateral bulges (fig. 4A). Base pairing apical to the third position of helix D3–1c is possible in many taxa; however, alignment beyond the three boxed positions in figure 4A is ambiguous. Compound helix D3–2 is subdivided into two helices (D3–2a and D3–2b) by insertions in several taxa, with a 3-bp extension of helix D3–2a present in the trichogrammatid Megaphragma sp. 1 (Supplementary Material 2). Helix D3–2b has the potential to form additional base pairings beyond the three boxed positions (fig. 4B); however, homology assignment is ambiguous distal to this helix due to the lack of sequence conservation and the variation in sequence lengths. The final compound helix in expansion segment D3, D3–3, is also subdivided into two helices (D3–3a and D3–3b) that are separated by a region of slippage. Three taxa (Gonatocerus triguttatus [Mymaridae], Trechnites sp. [Encyrtidae], and Centrodora sp. 2 [Aphelinidae]) form an extension of the conserved helix D3–3b that is difficult to homologize (REC (8) in fig. 4B). Interestingly, one taxon with this expansion of helix D3–3b, Trechnites sp., has the entire D3–1 compound helix deleted (fig. 4C). In Diptera (Kjer, Baldridge, and Fallon, 1994; Schnare et al. 1996; Hwang et al. 1998), the machilid Petrobius sp. (Archaeognatha) (Hwang et al. 1998) and other apterygote insects (Gillespie, unpublished data), the compound helix D3–1 is shortened or completely deleted, resulting in only two helices (D3–2 and D3–3) in the D3 expansion segment. This deletion in a hymenopteran, however, is the first report of a deleted D3–1 compound helix in a nondipteran holometabolous insect. Five RAAs, two RSCs, and three RECs occur in the D3 expansion segment in chalcidoids (fig. 4B).

    FIG. 4.— Secondary structure diagram of the D3 expansion segment (synonymous with the 650 region of Schnare et al. [1996]) shown for Encarsia hispida (A, B) and Trechnites sp. (C). Notation for the three compound helices follows the convention of Kjer, Blahnik, and Holzenthal (2001), Gillespie et al. (2004b), and Gillespie, Yoder, and Wharton (2005), with the exception of the subdivision of helix D3–3 into three subhelices. Helices are boxed in (A), and ambiguously aligned regions are boxed in (B). The notation for RAAs, RSCs, and RECs is described in Supplementary Materials 2 and 4. The explanations of base pair symbols and reference for software used to construct structure diagrams are given in figure 1.

    Helix H604

    The hairpin-stem loop forming between positions 604 and 624 of the E. coli model (Gutell, Gray, and Schnare 1993; Cannone et al. 2002) is highly variable across the alignment. The variability described in this area of our multiple sequence alignment has also been documented in a recent study on Ichneumonoidea LSU rRNA structure (Gillespie, Yoder, and Wharton 2005). Our structural alignment identified nine homologous base pairings in helix 604, with a conserved A ? A bulge occurring between base pairs 3 and 4. Additional pairing occurs distal to base pair 9, with expansion and contraction of this helix ranging from 1 to 14 nucleotides (fig. 5A–F). The observed length heterogeneity in helix H604 is found throughout the entire Chalcidoidea.

    FIG. 5.— A gallery of diverse secondary structure diagrams from variable helix H604 is shown for the following chalcidoid taxa: (A) Polstonia pelagocorypha; (B) Cheiloneurus fulvescens; (C) Plagiomerus ?diaspidis; (D) Acerophagus sp.; (E) Tanaostigmodes albiclavus (b); (F) Neolosbanus palgravei (a). Helices are boxed in A, and ambiguously aligned regions are boxed in D. The notation for RAAs and RECs is described in Supplementary Materials 2 and 4. The explanations of base pair symbols and reference for software used to construct structure diagrams are in figure 1.

    Core Elements

    The D2 and D3 expansion regions are flanked by segments of the highly conserved core rRNA structure. In contrast with the D2 and D3 regions, the core region usually has less insertions and deletions and more sequence conservation. The sequence between D2 and D3, including the 5' and 3' halves of helices H563, H604, and H628, and the 5' halves of helices H579 and H589 were determined with the D2 and D3 sequences. Additionally, sequences flanking the D2 on the 5' side, including the 5' and 3' halves of helices H375, H406, H461, and H484, the 5' half of helix H413, and the 3' half of helices H265, H35, H31, and H15, were used to delimit this second expansion segment. Finally, sequences flanking the D3 on the 3' side, including the 5' and 3' halves of helices H671, H687, H700, H736, and H777, the 5' half of helix H812, and the 3' half of helix H589, were used to determine the position of this expansion segment within the core RNA. The positions of these conserved core elements are illustrated on the structural diagram in figure 1.

    Helical Conservation

    Characteristic patterns of nucleotide substitutions and positional covariation in the expansion segments D2 and D3 reveal 37 conserved helices in the secondary structure model in the chalcidoids (Supplementary Material 3). A total of 34.4% of the base pairs within the helical regions of the D2 and D3 chalcidoid expansion segments (not including the core elements sequenced) exhibit some degree of covariation (37.9% in D2, 17.6% in D3; calculated manually from Supplementary Material 3). Within the chalcidoid data set, the more variable positions within helices usually have more positional covariation at a larger percentage of the proposed base pairs, while the positions that are more conserved have a minimal amount of covariation among the two positions that are base paired. While many of the base pairs in the helices in the D2 and D3 secondary structure model have extensive amounts of positional covariation, some of the sequences underlying the helices, including 1a, 2, 2d, 3, 3a, 3b, 3g, 3h, 3i, 3l, 3o, 3p, D3–1b, D3–3a, and D3–3b, are conserved within the chalcidoids and thus have minimal or no comparative support. However, sequence variation between the chalcidoids and other insect taxa D2 and D3 sequences contains positional covariations that substantiate the proposed base pairs in the structure model (http://www.rna.icmb.utexas.edu/).

    The frequency of the four nucleotides in the paired and unpaired regions of the chalcidoid hymenopteran D2 and D3 sequences reveals an elevated G content in pairing regions and an elevated A content in nonpairing regions (table 2). The G bias in helices can be attributed to the ability of guanine to base pair with both cytosine and uracil in RNA molecules (reviewed in Gutell, Larsen, and Woese 1994). The elevated frequency of adenine in unpaired regions, as found in other studies on rRNA (Vawter and Brown 1993; Springer, Hollar, and Burk 1995; Ortí et al. 1996; Springer and Douzery 1996; Bakke and Johansen 2002), supports the evidence that unpaired adenines have a wide range of structural motifs with significant function (Gutell et al. 1985; Gutell et al. 2000; Doherty et al. 2001; Elgavish et al. 2001; Nissen et al. 2001). Given that a good portion of unpaired positions are within ambiguously aligned regions, and hence not included in these calculations, the percentage of unpaired adenines is likely even higher.

    An analysis of the ratio of transitions to transversions in paired and unpaired regions reveals a bias for more transitions in paired regions (table 2). This is consistent with a mutational mechanism under selection for compensatory base changes repairing deleterious substitutions (Wheeler and Honeycutt 1988; Rousset, Pelandakis, and Solignac 1991; Kraus et al. 1992; Vawter and Brown 1993; Gatesy et al. 1994; Nedbal, Allard, and Honeycutt 1994; Douzery and Catzeflis 1995; Springer, Hollar, and Burk 1995; Springer and Douzery 1996; Gillespie et al. 2004b). Specifically, U C transitions are elevated in pairing regions (Marshall 1992) and reflect the transition pathways, A ? C to A–U and G ? U to G–C, that restore noncanonical pairings to Watson-Crick base pairs after slightly deleterious mutations. In summary, our covariation analyses within these putative helices strongly support our predicted model (fig. 1) for the expansion segments D2 and D3 from these sampled chalcidoid taxa.

    Regions of Ambiguous Alignment

    Positional nucleotide homology could not be confidently assigned to 45 regions of our multiple sequence alignment (Supplementary Material 4). Thirty-one of these unalignable regions are defined as RAA, wherein single insertion and deletion events cannot be assessed as homologous characters across all the sequences in the alignment, and consistent positional covariation (base pairing) is not found. Without secondary structure base pairing to guide the establishment of columnar homology in regions with many insertions and deletions (Kjer, 1995, 1997; Hickson et al. 1996), we did not establish homology statements within RAAs. These nucleotides in the alignment were contained within brackets and were justified to the left (5' strand) or right (3' strand). Within the RAA regions, gaps do not represent insertion and deletion events as they do in the unambiguously aligned data. Instead they reveal size variation within each RAA.

    Regions of Slipped-Strand Compensation

    The sequence alignment in six regions in the D2 and D3 expansion segments cannot be aligned with high confidence due to the inconsistent base pairing in their helices (Supplementary Material 4). These helices are flanked on both sides by conserved base pairs wherein positional homology assessment is unambiguous. Patterns of covariation were used to confirm inconsistent base pairing across the alignment within these six RSCs, as suggested by Gillespie (2004). As with RAAs, nucleotides in RSCs were bracketed and aligned to approximate homologous base pairs (when base pairs are proposed) or left or right justified, with gaps inserted to adjust for length heterogeneity as in the RAA regions (see above).

    Regions of Expansion and Contraction

    The sequence alignment in eight additional helical regions in the D2 and D3 expansion segments cannot be aligned with confidence due to the inconsistent base pairing within their helices (Supplementary Material 4). These regions have variation in the length of hairpin helices 2g, 3f–3, 3i, 3q, H604, D3–1c, D3–2b, and D3–3b, making the precise placement of nucleotides and indels in the alignment uncertain. While consistent homology statements could not be made in these ambiguously aligned regions across all sequences in the alignment, secondary structure base pairing was used to differentiate between the helical component and the terminal bulge (see Gillespie, 2004). After bracketing, nucleotides in RECs were treated the same as RSCs (see above).

    Utility for Phylogeny Reconstruction

    The assignment of positional homology in length heterogeneous data sets based on biological criteria has been shown to improve phylogeny estimation (Dixon and Hillis 1993; Kjer 1995; Titus and Frost 1996; Morrison and Ellis 1997; Uchida et al. 1998; Mugridge et al. 1999; Cunningham, Aliesky, and Collins 2000; Gonzalez and Labarere 2000; Hwang and Kim 2000; Lydeard et al. 2000; Morin 2000; Xia 2000; Xia, Xie, and Kjer 2003). While the alignment of rDNA sequences becomes progressively more difficult as the sequence and length variation increases, conserved secondary structures are useful for guiding the assignment of positional homology (e.g., Kjer 1995). The expansion segments of the eukaryotic LSU rRNA are unique because they accumulate an extreme amount of nucleotide insertions (Veldman et al. 1981; Michot, Hassouna, and Bachellerie 1984; Michot, Qu, and Bachellerie 1990) and yet presumably have little impact on the function of the ribosome in translation (Musters et al. 1989, Sweeney and Yao 1989; Musters et al. 1991), with the exception of expansion segment D8, which is thought to interact with small nucleolar RNA E2 (Rimoldi et al. 1993, Sweeney, Chen, and Yao 1994). Extraordinary differences in sequence length (Gutell 1992; De Rijk et al. 1994) and secondary structure in expansion segments, even in recently diverged organisms, are not uncommon (e.g., Hillis and Dixon 1991; Schnare et al. 1996); yet, the majority of the expansion segments contain conserved features that are comparable across high levels of divergence (Gillespie, Yoder, and Wharton 2005; Gillespie, unpublished data). These conserved regions involved in hydrogen bonding can be used to delimit regions wherein primary assignment of homology is uncertain and indefensible (Kjer 1997; Lutzoni, Wagner, and Reeb 2000; Kjer, Blahnik, and Holzenthal 2001). In previous studies on Hymenoptera, these hypervariable regions typically were either subjectively aligned by eye or excluded from subsequent phylogenetic analysis (e.g., Cameron et al. 1992; Unruh and Woolley 1999). Our study identified 45 alignment-ambiguous regions in the 28S D2, D3 expansion segments using a global chalcidoid structural model that is supported by compensatory base changes in many of the proposed base pairs (Supplementary Material 3). The potential retention of phylogenetic information from these regions is discussed below.

    Unlike the alignment of highly conserved core regions of rRNA molecules, the expansion segments require inspection for compensatory base changes that facilitate the alignment of highly divergent sequences. Coevolving helices and highly conserved single-stranded regions provide empirical homology assignments that delimit unalignable regions (Kjer 1995, 1997). After initial exclusion, these subsequent ambiguously aligned regions can be incorporated into phylogeny reconstruction in a variety of ways. They can be recoded as multistate characters based on nucleotide identity (Lutzoni, Wagner, and Reeb 2000; Kjer, Blahnik, and Holzenthal 2001; Gillespie et al. 2003, 2004a) and further subjected to a step matrix that implements unequivocal weighting to character transformations (FSO, Wheeler 1999; INAASE, Lutzoni, Wagner, and Reeb 2000). These weighted, multistate characters can be analyzed in combination with the unambiguously aligned data (INAASE) or combined with the dynamic optimization alignment process in POY (Gladstein and Wheeler 1997). However, we caution use of this approach, given that alternative data sources (unambiguously aligned regions of the same rDNA partition, different gene regions, morphology, and so on) bias the tree building in FSO and that ts/tv ratios and gapcosts are not likely to be similar across all regions of rDNA genes. Unalignable regions can also be recoded as morphological characters based on the differences these regions impose on the secondary structure of the molecule (Billboud et al. 2000; Collins, Moulton, and Penny 2000; Lydeard et al. 2000; Ouvrard et al. 2000). Across taxa, transformations from one structure to another can be calculated as a measure of structural variability (Fontana et al. 1993; Notredame, O'Brien, and Higgins 1997; Moulton et al. 2000; Misof and Fleck 2003). Homologous, yet unalignable structures can even be characterized as phylogenetic trees, with differences in tree topology representing transformations across variable structures (Shapiro and Zhang 1990; Hofacker et al. 1994).

    Model Applicability

    The structural model presented here for the D2 and D3 expansion segments of the 28S rRNA gene from chalcidoids is consistent with several insect groups, including ichneumonoid, proctotrupoid, and cynipoid Hymenoptera, scarabaeoid, curculionoid, and chrysomeloid Coleoptera, and lower level studies on adephagous and other polyphagous beetles, including cassidine Chrysomelidae. All of these insect lineages contain the seven compound helices described in our model, with the majority of the length and structure variation occurring in the most distal regions of these compound helices (Gillespie, unpublished data). Our model is highly consistent with the predicted structures of other eukaryotic groups (reviewed in Gillespie et al. 2004b) and adds to the evidence that at least some regions of the D2 and D3 expansion segments will be comparable across higher insect taxa given that covariation analysis is performed to isolate homologous regions. However, with increased sequence divergence, it is likely that many regions of the D2 and D3 expansion segments will prove unalignable and noncomparable at the nucleotide level, with the birth and death of hypervariable helices confounding objective homology assignment (see Page, Crulckshank, and Johnson 2002; Misof and Fleck 2003).

    With its objective assignment of nucleotide homology based only on structural evidence, our multiple sequence alignment should be considered highly conservative at best. Our structure of the D2 and D3 expansion segments predicts the motifs that are homologous across these sampled chalcidoid taxa; hence, it is a global model. Lower level alignments within Chalcidoidea, particularly at the family and subfamily levels, will likely provide alignments with less ambiguously aligned regions in which further homology assignments can be made with confidence. Additionally, methods for retaining information from ambiguously aligned regions (see above) will certainly lend more characters to local chalcidoid phylogeny reconstructions given that the degree of sequence length and heterogeneity should be lower than in the present study.

    Conclusion

    Our purpose with this investigation was to provide a predictive global secondary structural model that will be useful for homology assignment in future phylogenetic studies of chalcidoid and related hymenopteran taxa that utilize the D2 and D3 expansion segments of the 28S LSU rRNA gene region. Given that we sampled adequately from all but one of the extant families of Chalcidoidea (Rotoitidae), we believe our predicted structural model reflects the historical divergence across these taxa by characterizing the variability associated with this region of the 28S rRNA. Additionally, by including the closely related family Mymarommatidae in our analysis, the likely sister group of the chalcidoids (Gibson 1986), our model may potentially provide a template for aligning sequences from taxa above the superfamily level within the Aculeata. Indeed, many features of our model are comparable to the recently predicted structures for the D2 and D3 expansion segments from Ichneumonoidea (Gillespie, Yoder, and Wharton 2005). Only with the inclusion of more sequences from in-depth structural analyses of other aculeatan superfamilies will the utility of our model for higher level phylogenetic analyses within Aculeata be revealed.

    Supplementary Material

    Supplementary Materials 1–4 are available at the laboratory Web site (http://hymenoptera.tamu.edu/rna) and at Molecular Biology and Evolution online (www.mbe.oupjournals.org).

    Acknowledgements

    We are grateful for the suggestions that greatly improved this manuscript from Spencer Muse and two anonymous reviewers. This work was supported in part by National Science Foundation Research Grants BSR-9978150, DEB 0108245 and DEB-0341149 to J.M.H. J.J.G. is especially grateful to Anthony Cognato for start-up funds provided by the TAMU Entomology Department and grant DEB-0328920. MJY was supported by NSF DEB 0328922 to Robert Wharton. Roger Burks (UCR) and Jung-Wook Kim (TAMU) provided some of the sequence data. John Noyes (NHM, UK) identified the Encyrtidae. UCR would also like to thank the many people who provided us with specimens (too numerous to list herein).

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