Supplementary Data 2. Determination of ITS2 secondary structure for Ophion

There are two main analytical methods used to obtain the secondary structure of ITS2 (Schultz and Wolf 2009). The first is to fold sequences using homology modeling based on available structures in a public ITS2 database (Schultz et al. 2006; Selig et al. 2008; Koetschan et al. 2010). However template structures are only identified if the sequences are highly similar, with at least 75% of the structural elements being transferrable from the template to the sequence (Schultz et al. 2006). If no sufficiently similar sequences are available, then the sequence can be folded using structure prediction algorithms (Schultz and Wolf 2009). The ITS2 database did not contain any sequences that were sufficiently similar to use as templates; therefore we used two different RNA folding programs to obtain a template secondary structure.

Comparative methods that simultaneously fold multiple homologous RNA sequences have been found to improve accuracy over single sequence folding algorithms (Gardner and Giegerich 2004). We therefore initially conducted a multiple sequence folding analysis using the program MXScarna (Tabei et al. 2008). We simultaneously folded and aligned six Ophion sequences along with ITS2 sequences from six other species of Ichneumonidae in 4 subfamilies, obtained from GenBank (Table S2). The aligned structure was examined for structural motifs and compared to the conserved structure of ITS2 described by Coleman (2007). We also folded the same six Ophion sequences individually using the RNAfold webserver (Hofacker 2003), with the minimum free energy method and default settings. The structure that was obtained from RNAfold was consistent with the combined structure from MXScarna; we therefore chose to use RNAfold to obtain the template structure for all sequences, as the output from this program facilitated the use of downstream programs.

To obtain the final sequence-structure dataset, we first folded an arbitrarily chosen single sequence in RNAfold. We then imported this structure into the ITS2 database, and used it as a template for folding the remaining sequences. If any sequences had less than 90% similarity to the existing template, one of the anomalous sequences was directly folded in RNAfold, and then added as an additional template to the database. Using this iterative process, the final dataset consisted of eight sequences that were folded directly and 386 sequences that were folded using homology. Twenty-three base pairs of the flanking 28S and 5.8S genes were retained, as this has been shown to improve the accuracy of the secondary structure folding algorithms (Morgan and Blair 1998). For the majority of sequences, the complete 28S flanking region was not successfully sequenced. The missing bases were therefore manually added, as this region of 5.8S was invariant in all Ichneumonidae sequences available on GenBank. The 28S and 5.8S flanking regions were not included in the phylogenetic analyses.

Table S2. Ichneumonidae species included in multiple folding of ITS2 in MXScarna.

Subfamily / Species (species-group) / GenBank / Length
(base pairs)
Campopleginae / Diadegma semiclausum Hellén / AJ885183 / 624
Campopleginae / Meloboris sp. / AJ888025 / 599
Diplazontinae / Sussaba aciculata (Ruthe) / JN626397 / 709
Diplazontine / Tymmophorus erythrozonus (Förster) / JN626423 / 805
Mesochorinae / Mesochorus sp. / AY588968 / 718
Pimplinae / Scambus calobatus (Gravenhorst) / JN243123 / 667
Ophioninae / Ophion ocellaris Ulbricht / KF616299 / 821
Ophioninae / Ophion flavidus Brullé / KF616301 / 865
Ophioninae / Ophion sp. 1 / KF615947 / 867
Ophioninae / Ophion sp. 2 / KF615980 / 977
Ophioninae / Ophion sp. 3 / KF615977 / 1013
Ophioninae / Ophion sp. 4 / KF615984 / 864

References

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Hofacker, I.L., 2003. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431.

Koetschan, C., Förster, F., Keller, A., Schleicher, T., Ruderisch, B., Schwarz, R., Müller, T., Wolf, M., Schultz, J., 2010. The ITS2 Database III: sequences and structures for phylogeny. Nucleic Acids Res. 38, D275–D279.

Morgan, J.A.T., Blair, D., 1998. Trematode and Monogenean rRNA ITS2 secondary structures support a four-domain model. J. Molec. Evol. 47, 406-419.

Schultz, J., Müller, T., Achtziger, M., Seibel, P.N., Dandekar, T., and Wolf, M. 2006. The internal transcribed spacer 2 database: a web server for (not only) low level phylogenetic analyses. Nucleic Acids Res. 34, W704–W707.

Schultz, J., Wolf, M., 2009. ITS2 Sequence-structure analysis in phylogenetics: a how-to manual for molecular systematics. Molec. Phylogenet. Evol. 52, 520 – 523.

Selig, C., Wolf, M., Müller, T., Dandekar, T., Schultz, J., 2008. The ITS2 Database II: homology modelling RNA structure for molecular systematics. Nucleic Acids Res. 36, D377–D380.

Tabei, Y., Kiryu, H., Kin, T., Asai, K., 2008. A fast structural multiple alignment method for long RNA sequences. BMC Bioinformatics 9, 33.