18 S rRNAVARIABILITY MAP FOR GASTROPODA

One of the most extensively used single-locus molecular markers for phylogenetic studies in general, but also for Gastropoda in particular (Winnepenninckx, Backeljau & De Wachter, 1996; Wollscheid-Lengeling et al., 2001; Vonnemann et al., 2005; Klussmann-Kolb et al., 2008; Dayrat et al., 2011), is the gene for the 18S ribosomal RNA (rRNA) molecule. From an historical point of view and due to its molecular properties (e.g. nuclear-encoded, not protein-coding) this marker is traditionally used in molecular phylogenetics, especially in studies of deep phylogeny (Aguinaldo et al., 1997; Field et al., 1988; Halanych, 2004). The primary sequence of rRNA folds into a specific secondary structure, which is crucial for conservation of the three-dimensional structure and molecular function within the ribosome (Noller et al., 1990; Moore & Steitz, 2002). The secondary structure is maintained by hydrogen bonds between RNA nucleotides, which form stems (paired regions) or loops and bulges (unpaired regions). Due to different functional aspects, we can assume different selective constraints in paired and unpaired regions, leading to different degrees of variability (Caetano-Anollés, 2002). It is this particular feature that makes rDNA markers popular in phylogenetics, because different questions with different time scales of diversification can be investigated (Higgs, 2000). Here we present a core structure (Fig. 1) and a coloured variability map (Fig. 2) of the 18S rRNA for the Gastropoda. The latter gives more detailed and quantitative information of positional variability than the simple distinction between variable and conserved areas that is often made by visual inspection of sequence alignments alone and which is based too much on intuition (Van de Peer, Chapelle & De Wachter, 1996a; Ben Ali et al., 1999; Van de Peer et al., 2000). As an easy-to-handle tool a variability map can be used in many ways, e.g. for primer design, the process of alignment optimization and for the study of molecular evolution of the phylogenetic marker. For construction of the variability map, we included 45 complete 18S rDNA gastropod sequences comprising representatives of all major gastropod clades: Patellogastropoda, Cocculiniformia, Neritopsina, Vetigastropoda, Caenogastropoda and Heterobranchia (Table 1). The 18S rDNA sequences were prealigned with the software MAFFT v. 6 (Katoh & Toh, 2008) using the L-INS-I option (MAFFT alignment). Individual secondary structures of the 18S rRNA were calculated in RNAsalsa (Stocsits et al., 2009) and used to create the final alignment (RNA alignment). As a constraint sequence (a sequence with an already estimated secondary structure) we used the 18S rRNA primary and corresponding secondary structure of the outgroup taxon Acanthopleura japonica (Polyplacophora), which was downloaded from the European ribosomal RNA database (Wuyts, Perriere, & Van de Peer, 2004). The MAFFT alignment and constraint sequence served as input for the RNAsalsa analysis, which was conducted using the default parameters. The RNA alignment yielded a length of 2,217 bp. The secondary structure model of the 18S rRNA of Bursa rana (Fig. 1) is a representative for most of the secondary structures (gastropod core structure) and on that account was used as the backbone on which we plotted the variability map for gastropods (Fig. 2). Furthermore, we estimated relative substitution rates which serve as a basis for the construction of an evolutionary-rate spectrum of the gastropod 18S rRNA marker (Fig. 3), as well as for the gastropod variability map by applying the substitution rate calibration (SRC) method (Van de Peer et al., 1996a, 2000; Ben Ali et al., 1999), using the software package TREECON for Windows (Van de Peer & De Wachter, 1997) and our RNA alignment. With the SRC method, substitution rates of individual sites (vi) can be measured relative to the average substitution rate of the 18S rRNA molecule (Van de Peer, Van der Auwera & De Wachter, 1996b). Rates were estimated for each alignment position containing a nucleotide (instead of a gap) in at least 25% of the sequences in the RNA alignment. The spectrum was obtained by 10 iterations. The variability of each site in the map is indicated by means of a coloured dot. A coloured variability map was constructed by dividing nucleotides into five variability subsets following Ben Ali et al. (1999): 0 , vi , 10 (blue), 10 , vi , 10 –0.425 (green), 10, vi , 10 þ0. 075 (yellow), 10þ0. 075 , vi , 10 þ0. 575 (orange) and vi 10þ0. 575 (red). Positions that were conserved (vi 1⁄4 0) are indicated in purple, while variable positions with more than 75% absence data are depicted in grey. The secondary structure diagrams reconstructed with RNAsalsa as well as the gastropod variability map were edited with the software XRNA v. 1.2. 0 beta (http://rna.ucsc.edu/rnacenter/xrna/xrna. html) and CorelDRAW v. 11. The evolutionary-rate spectrum of the gastropod 18S rRNA (Fig. 3) shows a nonrectangular rate distribution, i.e. the nucleotide-specific values are not uniformly distributed across the rate spectrum. The most frequent category of the evolutionary-rate spectrum comprised positions with relative substitution rates between 10þ0. 4 and 10þ0. 45. The most variable nucleotide sites have a substitution rate c. 400 times