Molluscan Studies

Journal of The Malacological Society of London Molluscan Studies

Journal of Molluscan Studies (2012) 78: 151–156. doi:10.1093/mollus/eyr047 Advance Access publication date: 2 December 2011

RESEARCH NOTE 18S rRNA VARIABILITY MAP FOR GASTROPODA

Alexander M. Weigand, Angela Dinapoli and Annette Klussmann-Kolb Downloaded from Institute for Ecology, Evolution and Diversity, Goethe University, Max-von-Laue-Str.13, 60438 Frankfurt am Main, Germany

Correspondence: A.M. Weigand; e-mail: [email protected]

One of the most extensively used single-locus molecular structure of the outgroup taxon Acanthopleura japonica http://mollus.oxfordjournals.org/ markers for phylogenetic studies in general, but also for (Polyplacophora), which was downloaded from the European Gastropoda in particular (Winnepenninckx, Backeljau & De ribosomal RNA database (Wuyts, Perriere, & Van de Peer, Wachter, 1996; Wollscheid-Lengeling et al., 2001; Vonnemann 2004). The MAFFT alignment and constraint sequence served et al., 2005; Klussmann-Kolb et al., 2008; Dayrat et al., 2011), is as input for the RNAsalsa analysis, which was conducted using the gene for the 18S ribosomal RNA (rRNA) molecule. From the default parameters. The RNA alignment yielded a length of an historical point of view and due to its molecular properties 2,217 bp. The secondary structure model of the 18S rRNA of (e.g. nuclear-encoded, not protein-coding) this marker is trad- Bursa rana (Fig. 1) is a representative for most of the secondary itionally used in molecular phylogenetics, especially in studies structures (gastropod core structure) and on that account was of deep phylogeny (Aguinaldo et al., 1997; Field et al., 1988; used as the backbone on which we plotted the variability map

Halanych, 2004). The primary sequence of rRNA folds into a for gastropods (Fig. 2). at Stanford University Libraries on October 10, 2012 specific secondary structure, which is crucial for conservation Furthermore, we estimated relative substitution rates of the three-dimensional structure and molecular function which serve as a basis for the construction of an within the ribosome (Noller et al., 1990; Moore & Steitz, evolutionary-rate spectrum of the gastropod 18S rRNA 2002). The secondary structure is maintained by hydrogen marker (Fig. 3), as well as for the gastropod variability bonds between RNA nucleotides, which form stems (paired map by applying the substitution rate calibration (SRC) regions) or loops and bulges (unpaired regions). Due to differ- method (Van de Peer et al., 1996a, 2000; Ben Ali et al., ent functional aspects, we can assume different selective con- 1999), using the software package TREECON for Windows straints in paired and unpaired regions, leading to different (Van de Peer & De Wachter, 1997) and our RNA align- degrees of variability (Caetano-Anolle´s, 2002). It is this par- ment. With the SRC method, substitution rates of individ- ticular feature that makes rDNA markers popular in phyloge- ual sites (vi) can be measured relative to the average netics, because different questions with different time scales of substitution rate of the 18S rRNA molecule (Van de Peer, diversification can be investigated (Higgs, 2000). Here we Van der Auwera & De Wachter, 1996b). Rates were esti- present a core structure (Fig. 1) and a coloured variability mated for each alignment position containing a nucleotide map (Fig. 2) of the 18S rRNA for the Gastropoda. The latter (instead of a gap) in at least 25% of the sequences in the gives more detailed and quantitative information of positional RNA alignment. The spectrum was obtained by 10 itera- variability than the simple distinction between variable and tions. The variability of each site in the map is indicated conserved areas that is often made by visual inspection of by means of a coloured dot. A coloured variability map sequence alignments alone and which is based too much on was constructed by dividing nucleotides into intuition (Van de Peer, Chapelle & De Wachter, 1996a; Ben five variability subsets following Ben Ali et al. (1999):0, vi – 0.925 – 0.925 – 0.425 –0.425 Ali et al., 1999; Van de Peer et al., 2000). As an easy-to-handle , 10 (blue), 10 , vi , 10 (green), 10 , þ0.075 þ0.075 þ0.575 tool a variability map can be used in many ways, e.g. for vi , 10 (yellow), 10 , vi , 10 (orange) and þ0.575 primer design, the process of alignment optimization and for vi 10 (red). Positions that were conserved (vi ¼ 0) are the study of molecular evolution of the phylogenetic marker. indicated in purple, while variable positions with more than For construction of the variability map, we included 45 75% absence data are depicted in grey. The secondary complete 18S rDNA gastropod sequences comprising structure diagrams reconstructed with RNAsalsa as well as representatives of all major gastropod clades: Patellogastropoda, the gastropod variability map were edited with the software Cocculiniformia, Neritopsina, Vetigastropoda, Caenogastropoda XRNA v. 1.2.0 beta (http://rna.ucsc.edu/rnacenter/xrna/xrna. and Heterobranchia (Table 1). The 18S rDNA sequences were html) and CorelDRAW v. 11. prealigned with the software MAFFT v. 6 (Katoh & Toh, The evolutionary-rate spectrum of the gastropod 18S rRNA 2008) using the L-INS-I option (MAFFT alignment). (Fig. 3) shows a nonrectangular rate distribution, i.e. the Individual secondary structures of the 18S rRNA were calcu- nucleotide-specific values are not uniformly distributed across lated in RNAsalsa (Stocsits et al., 2009) and used to create the the rate spectrum. The most frequent category of the final alignment (RNA alignment). As a constraint sequence (a evolutionary-rate spectrum comprised positions with relative sequence with an already estimated secondary structure) we substitution rates between 10þ0.4 and 10þ0.45. The most vari- used the 18S rRNA primary and corresponding secondary able nucleotide sites have a substitution rate c. 400 times

# The Author 2011. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved RESEARCH NOTE Downloaded from http://mollus.oxfordjournals.org/ at Stanford University Libraries on October 10, 2012

Figure 1. Core structure of gastropod 18S rRNA, based on secondary-structure model for the nuclear 18S rRNA of Bursa rana as a representative for most of the gastropod sequences. Helix numbering is according to Wuyts et al. (2001). higher than the least variable ones. With few exceptions, the sequences. Nevertheless, some general conclusions can be two corresponding nucleotides of a stem-forming base pair gen- drawn. erally have the same or a neighbouring colour. This implies The gastropod variability map (Fig. 2) can be utilized in that they are more or less equally variable and evolve at various ways. For instance, the calculation of site variability approximately the same rate. It has to be noted that all values can be used in phylogenetic inference within the Gastropoda. have to be considered as speciﬁc for our dataset and vary as a Nine highly variable areas (orange, red and grey) can be dis- function of the number and evolutionary relatedness of the tinguished within the following domains: 6, 10, E10, 17, E23,

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Figure 2. 18S rRNA variability map for gastropods. The colour code is plotted on the 18S rRNA secondary structure model of the gastropod core structure. Nucleotides are subdivided into ﬁve groups of increasing variability; the most conserved positions are in blue, the most variable ones in red; hypervariable positions are indicated in grey (present in ,25% of the data); 100% conserved positions are indicated in purple. Helix numbering is according to Wuyts et al. (2001). For enlargement of this ﬁgure, see Supplementary material.

29, 43, 46 and 49. All other areas show a more (e.g. domains conserved regions (Fig. 2). The phylogenetic signal based on 19–21, 35 and 50) or less conserved structure (e.g. domains sequential variations within these regions is enhanced com- 16, 37 and 48). Using conserved regions (e.g. domains 19, 35 pared to a signal within variable areas, because of the strong and 50) deep (old) evolutionary relationships can be investi- functional constraints at these sites. These sequence positions gated, while the more variable areas (e.g. domains 6, 10 and should be investigated in more detail and are potential autapo- 46) are helpful for the study of relationships between closely morphies for certain clades. Hence, the 18S rRNA gene can related organisms (Van de Peer et al., 1996a). Various nucleo- serve as a ‘molecular chronometer’ to investigate phylogenetic tide substitutions can be observed within the otherwise problems at different depths of the evolutionary tree (Woese,

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Table 1. Classiﬁcation of gastropods in dataset (after Bouchet & Table 1. Continued Rocroi, 2005) and their GenBank accession numbers. Taxon Family 18S rDNA Taxon Family 18S rDNA Cima sp. Cimidae FJ917206 GASTROPODA Orbitestella vera Orbitestellidae FJ917207 PATELLOGASTROPODA UMBRACULOIDEA PATELLOIDEA Umbraculum umbraculum Tylodinidae AY427499 Patella rustica Patellidae DQ013352 AKEROIDEA NACELLOIDEA Akera bullata Akeridae AY427502 Cellana nigrolineata Nacellidae DQ013353 CAVOLINIOIDEA Nacella magellanica Nacellidae DQ013349 Cavolinia uncinnata Cavoliniidae DQ237964 ACMAEOIDEA CLIONOIDEA Niveotectura pallida Lottiidae AF308645 Spongiobranchaea australis Pneumodermatidae DQ237969 Lottia digitalis Lottiidae DQ248942 POLYBRANCHIOIDEA COCCULINIFORMIA Cyerce nigricans Polybranchiidae AY427500

COCCULINOIDEA HAMINOEOIDEA Downloaded from Cocculina messingi Cocculinidae AY090796 Haminoea hydatis Haminoeidae AY427504 NERITOPSINA PLEUROBRANCHOIDEA NERITOIDEA Tomthompsonia antarctica Pleurobranchidae AY427492 Nerita albicilla Neritidae X91971 ANADORIDOIDEA Theodoxus ﬂuviatilis Neritidae AF120515 Goniodoris nodosa Goniodorididae AJ224783 http://mollus.oxfordjournals.org/ VETIGASTROPODA SIPHONARIOIDEA FISSURELLOIDEA Siphonaria alternata Siphonariidae AY427523 Diodora graeca Fissurellidae AF120513 CHILINOIDEA TROCHOIDEA Latia neritoides Latiidae EF489339 Bathymargarites symplector Trochidae DQ093433 ELLOBIOIDEA Turbo cornutus Turbinidae AF165311 Ophicardelus ornatus Ellobiidae DQ093442 LEPETODRILOIDEA OTINOIDEA Lepetodrilus elevatus Lepetodrilidae AY145381 Otina ovata Otinidae EF489344 PLEUROTOMARIOIDEA Smeagol philippensis Smeagolidae FJ917210

Sinezona confuse Scissurellidae AF120512 ONCHIDIOIDEA at Stanford University Libraries on October 10, 2012 HALIOTOIDEA Onchidella floridana Onchididae AY427522 Nordotis discus Haliotidae AF082177 Onchidium verruculatum Onchididae AY427521 ‘Hot-vent Taxa’ PUNCTOIDEA Cyathermia naticoides Neomphalidae DQ093430 Discus rotundatus Endodontidae FJ917212 Depressigyra globulus Peltospiridae DQ093431 CAENOGASTROPODA CYCLOPHOROIDEA Aperostoma palmeri Cyclophoridae DQ093435 1987). But this molecular property also contains a risk—the VIVIPAROIDEA valuable phylogenetic information content of conserved Viviparus georgianus Viviparidae AF120516 domains can be masked by random similarity and noise of the LITTORINOIDEA highly variable domains. Littorina littorea Littorinidae X91970 The gastropod core structure (Fig. 1) and variability map TONNOIDEA (Fig. 2) are comparable to the eukaryotic core structure and Bursa rana Bursidae X94269 map (Van de Peer et al., 1997, 2000; Wuyts, Van de Peer & De Wachter, 2001). The fact that the two nucleotides of a stem- BUCCINOIDEA forming base pair have the same or a neighbouring colour is Neptunea eulimata Buccinidae EU236266 expected, since the substitution of a paired nucleotide generally MURICOIDEA needs a compensating substitution in the opposite strand (Ben Bolinus brandaris Muricidae DQ279944 Ali et al., 1999). Eukaryotic 18S rRNA shows the largest vari- CONOIDEA ation in size due to specific insertion events, which can also be Conus orbignyi Conidae EU015496 observed for gastropods. There are several insertion events that Terebra textilis Terebridae EU015525 result in either new branches in the structure of the molecule HETEROBRANCHIA and/or in a lengthening of existing hairpins (e.g. within PYRAMIDELLOIDEA Patellogastropoda: domains E23, 45 and 46; and Turbonilla sp. Pyramidellidae FJ917216 Vetigastropoda: by domain 6; data not shown). However, due to a high variability in the respective motifs no phylogenetic GLACIDORBOIDEA signal could be detected and convergent evolution is likely. If Glacidorbis rusticus Glacidorbidae FJ917211 the insertion events are of independent origin, then these ACTEONOIDEA sequences of the molecule should not be considered in the final Pupa solidula Acteonidae AY427516 analyses. As a result of the various insertion events, the 18S UNASSIGNED TO SUPERFAMILY rRNA of the Patellogastropoda is extended (by about 200–250 nucleotides) in comparison to the 18S rRNA gastropod core Continued molecule. This elongation of the 18S rRNA molecule has been

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noted that a simplistic binary division into paired and unpaired nucleotides for use in evolutionary models is not correct, because these regions show a wide range of nucleotide substitution rates (Fig. 3). The alternation of variable and conserved domains and the contrasting degree of variability among stem and loop nucleotides are clearly visible in Figure 2. Therefore, entire domains or individual nucleotide positions should be considered independently. In contrast, two base-pairing nucleotides generally exhibit a more or less equal variability and thus evolve interdependently at approximately the same rate (Fig. 2). Thus it is desirable to apply specific evolutionary models for RNA, with trade-off weighting schemes for stem and loop nucleotides. In practice, RNA mod- Figure 3. Evolutionary-rate spectrum of gastropod 18S rRNA. Rates elling is highly complex, because it simultaneously has to deal were estimated for all positions that contain a nucleotide in more than with two potential pitfalls. On one hand, the phylogenetic 25% of alignments. Totally conserved nucleotide positions are not information of homoplastic loop regions has to be down- – 0.925 – 0.925 shown (vi ¼ 0). Colour code: 0 , vi , 10 (blue), 10 , vi , weighted. On the other, the weighting scheme should also be – 0.425 – 0.425 þ0.075 þ0.075 Downloaded from 10 (green), 10 , vi , 10 (yellow), 10 , vi , appropriate to overcome the problem of interdependence of þ0.575 þ0.575 10 (orange) and vi 10 (red). paired sites, which violates the basic assumption of independent evolution of sites; if this is not considered the phylogenetic signal of paired positions will be overestimated. This is normal- ly achieved by applying DNA models to loops and RNA models to stem regions (Dixon & Hillis, 1993; Tillier & Collins, 1998; Keller et al., 2010; Letsch & Kjer, 2011). http://mollus.oxfordjournals.org/ However, more elaborate partitioning strategies have recently been proposed, to account for the varying substitution rates between stem and loop regions and particularly among nucleotides of the same class (Letsch & Kjer, 2011). The present study strongly supports this initiative (Figs 2, 3). A variability map can be also interpreted in terms of function and evolution of the molecule (Van de Peer et al., 2000) and applied to a range of approaches. For instance, highly variable sequence regions can be identified and used for the Figure 4. Consensus profile of the Aliscore check for randomly similar development of taxon-specific PCR primers or hybridization at Stanford University Libraries on October 10, 2012 characters of 18S rDNA. Alignment positions are shown on x-axis, scores on y-axis. Positions with negative scores indicate random probes (Ben Ali et al., 1999), whereas conserved regions can be similarity. utilized for the design of universal primers. Invariant positions are helpful in finding homologous regions during the process of sequence alignment. In general, highly variable domains are spatially related to the periphery of the 18S rRNA tertiary recognized earlier in several phylogenetic studies dealing with structure, whereas conserved domains are predominantly this taxon (Colgan et al., 2003; Grande, Templado & Zardoya, located in the core of the molecule (Wuyts et al., 2001). 2008; Meyer et al., 2010). It is now common practice to mask In summary the gastropod variability map will serve as an and exclude positions of random similarity (Talavera & easy and fast a priori tool to explore the 18S rDNA marker in Castresana, 2007; Criscuolo & Gribaldo, 2010). Our test for the light of molecular evolution, leading to the improvement of random similarity, using Aliscore (Misof & Misof, 2009) and gastropod phylogenetic studies. the RNA alignment, detected 401 nucleotide positions as puta- tively randomly similar (18.09%), as depicted in the Aliscore histogram (Fig. 4, values 0 . x . –1). The remaining 1,816 SUPPLEMENTARY MATERIAL nucleotide positions show a nonrandom similarity and there- Supplementary material is available at Journal of Molluscan fore a less noisy phylogenetic signal. The identification of ran- Studies online. domly similar sites can be used to improve the process of phylogenetic tree reconstruction. After the alignment is opti- mized, randomly similar sequence positions can be excluded ACKNOWLEDGEMENTS from the dataset. Such ambiguously aligned sequence sections We are very grateful to three reviewers for highly constructive can interfere negatively with the estimation of substitution input and especially to Harald Letsch and Roman Stocsits model parameters (Talavera & Castresana, 2007; Misof & who helped us with the software RNAsalsa. We thank Patrick Misof, 2009; Criscuolo & Gribaldo, 2010). The excluded Ku¨ck for his support with the software Aliscore. sequence blocks (Fig. 4) correspond to highly variable domains in the variability map (Fig. 2), e.g. the insertion hot- spots of the taxa Patellogastropoda and Vetigastropoda. REFERENCES Knowledge about differences in nucleotide substitution rates can be important for developing improved phylogenetic AGUINALDO, A.M.A., TURBEVILLE, J.M., LINEFORD, L.S., RIVERA, M.C., GAREY, J.R., RAFF, R.A. & LAKE, J.A. 1997. tree-reconstruction methods (von Reumont et al., 2009; Keller Evidence for a clade of nematodes, arthropods and other moulting et al., 2010). 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