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Molecular Phylogenetics and Evolution 65 (2012) 792–798
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Molecular Phylogenetics and Evolution
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Short Communication Molecular phylogenetics in 2D: ITS2 rRNA evolution and sequence-structure barcode from Veneridae to Bivalvia ⇑ Daniele Salvi a, , Paolo Mariottini b a CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal b Dipartimento di Biologia, Università ‘‘Roma Tre’’, Viale G. Marconi 446, 00146 Rome, Italy article info abstract
Article history: In this study, we analyzed the nuclear ITS2 rRNA primary sequence and secondary structure in Veneridae Received 7 November 2011 and comparatively with 20 Bivalvia taxa to test the phylogenetic resolution of this marker and its suit- Revised 11 July 2012 ability for molecular diagnosis at different taxonomic levels. Accepted 19 July 2012 Maximum likelihood and Bayesian trees based on primary sequences were congruent with (profile-) Available online 27 July 2012 neighbor-joining trees based on a combined model of sequence-structure evolution. ITS2 showed higher resolution below the subfamily level, providing a phylogenetic signal comparable to (mitochondrial/ Keywords: nuclear) gene fragments 2–5 times longer. Structural elements of the ITS2 folding, such as specific mis- ITS2 rRNA match pairing and compensatory base changes, provided further support for the monophyly of some Secondary structure Phylogeny groups and for their phylogenetic relationships. Veneridae ITS2 folding is structured in six domains Molecular taxonomy (DI–VI) and shows five striking sequence-structure features. Two of them, the Basal and Apical STEMs, Veneridae are common to Bivalvia, while the presence of both the Branched STEM and the Y/R stretches occurs Bivalvia in five superfamilies of the two Heterodonta orders Myoida and Veneroida, thus questioning their reci- procal monophyly. Our results validated the ITS2 as a suitable marker for venerids phylogenetics and taxonomy, and underlined the significance of including secondary structure information for both applications at several systematic levels within bivalves. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction and Wolf, 2009; Keller et al., 2010; Salvi et al., 2010). Compensa- tory base changes (CBCs) – mutations in both nucleotides of a Molecular phylogenetics and molecular taxonomy have tradi- paired position in a double-stranded structure of the transcribed tionally focused on analyzing DNA (or RNA) sequences for phyloge- RNA – in conserved regions of the eukaryote ITS2 sequence-struc- netic and taxonomic assessments. However, in the last few years ture have been shown to correlate with interbreeding incompati- the exploitation of secondary structure information of ribosomal bility between species. Several studies on animal, plants, and RNA (rRNA) molecules such as the nuclear ribosomal internal tran- fungi demonstrated that the presence of at least one CBC in ITS2 scribed spacer 2 (ITS2) has revealed a promising approach not only secondary structures is a good classifier (reliability higher than in phylogenetic reconstruction (Coleman, 2003; Schultz et al., 90%) of two organisms belonging to distinct species (Coleman, 2006; Schultz and Wolf, 2009; Salvi et al., 2010) but also in species 2000, 2003, 2009; Müller et al., 2007). Moreover, some conserved diagnosis (Coleman, 2003, 2009; Müller et al., 2007). features of the ITS2 secondary structure such as stem-loops do- The ITS2 is generally organized in four main helix domains (DI– mains have shown to be diagnostic of higher taxonomic grouping IV) of secondary structure and the combined information of both such as tribes, subfamilies, families, and orders (Oliverio et al., folding and primary sequence has been exploited for phylogenetic 2002; Bologna et al., 2008; Keller et al., 2008; Salvi et al., 2010). inference, taxonomic classification, and species delimitation, pro- Thus, the information from the ITS2 folding and sequence-struc- viding an increased phylogenetic resolution and reliability than ap- ture variation is a useful tool in molecular taxonomy for species proaches based on primary sequence data alone (Coleman, 2003; and higher-taxa diagnosis. Schultz et al., 2006; Bologna et al., 2008; Song et al., 2008; Schultz In this study we analyzed the primary sequence and the second- ary structure information from the nuclear ITS2 rRNA in the phylo- genetic context of the family Veneridae. This family is a large group ⇑ Corresponding author. Address: CIBIO, Centro de Investigação em Biodiversid- of bivalves with approximately 800 species, with a largely unre- ade e Recursos Genéticos, Campus Agrário de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal. Fax: +351 252661780. solved phylogenetic history and unstable taxonomy despite recent E-mail address: [email protected] (D. Salvi). molecular and morphological studies (Canapa et al., 1996, 2003;
1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.07.017 Author's personal copy
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Kappner and Bieler, 2006; Mikkelsen et al., 2006; Chen et al., 2011). tween intraspecific and interspecific congeneric distance by Indeed, while on the one hand the use of morphological traits is constructing a histogram based on Kimura 2-parameter distances problematic since most of them are either uniform or homoplastic using the program TaxonDna 1.7.8 (Meier et al., 2006). (Mikkelsen et al., 2006), on the other hand the use of molecular markers can be problematic due to (i) double uniparental inheri- 2.3. Phylogenetic analysis based on primary and secondary sequence tance of mitochondrial genomes in some venerids (Mikkelsen information et al., 2006); (ii) inherent difficulties in amplifying and sequencing ‘‘the barcoding gene’’ COI in venerids (Kappner and Bieler, 2006), Flanking sequence from 5.8S and 28S were trimmed from align- and (iii) the very low resolution of nuclear markers such as the ments and the complete ITS2 sequences were used in phylogenetic H3 gene and the ribosomal 18S/28S rRNAs (Chen et al., 2011). To analyses. According to traditional classification and previous phy- overcome these problems, the use of an extremely variable and logenies, Arctica islandica (Veneroida, Arcticidea, Arcticidae) was easy to amplify nuclear marker such as the ITS2 is highly advisable, used as the outgroup taxon (see Mikkelsen et al., 2006). In order but despite a promising test on four species (Cheng et al., 2006), to test for the effect of the outgroup choice on the Veneridae topol- ITS2 has never been used in phylogenetic and taxonomic studies ogy, additional phylogenetic analyses were performed using Cor- of Veneridae. bicula japonica (Veneroida, Corbiculidea, Corbiculidae) or the The main aim of this study was to integrate the information of divergent taxon Potamocorbula amurensis (Myoida, Myoidea, Corb- individual RNA secondary structures with primary sequences in ulidae) as alternative outgroups. venerids phylogeny and taxonomy, by employing a combined Phylogenetic analyses of ITS2 sequences were performed based model of sequence-structure evolution as well as identifying con- on primary sequences alone as well as combined analyses includ- served elements of the ITS2 folding. By including several represen- ing both primary sequences and secondary structure information. tatives from Bivalvia subclasses we further test the phylogenetic Phylogenetic analyses based on primary sequence were carried resolution of the ITS2 marker and its suitability as a barcode tool out using Maximum likelihood (ML) and Bayesian (BA) approaches for molecular taxonomy of venerid species to higher taxonomic using the GTR + G model selected by JModeltest (Posada, 2008) levels among bivalves. according to the AIC criterion. The best ML tree was calculated using heuristic searches (TBR branch swapping, 100 replicates 2. Materials and methods and random stepwise addition) using Paup 4.0b (Swofford, 2002) and Treefinder (Jobb, 2008). Nodal support was calculated using 2.1. Sampling, DNA extraction and sequencing 1000 bootstrap replicates (BP) in Treefinder. Bayesian analyses (BA) was conducted in MrBayes 3.1 (Huelsenbeck and Ronquist, ITS2 sequences were obtained from live specimens (60%), frozen 2001). Two independent Markov chain Monte Carlo (MCMC) simu- 7 specimens from trade (4%), or retrieved from Genbank (36%). A to- lations were run in parallel for 10 generations, sampling every tal of 45 specimens from 24 venerid species were employed in the 1000 generations. MCMC chains convergence was assessed using molecular analyses. Twenty additional Bivalvia ITS2 sequences the program AWTY (Wilgenbusch et al., 2004) (burn-in = 2500) from the subclasses Heterodonta, Paleoheterodonta, and Pterio- and the trees sampled after burn-in were used to assess posterior morphia were also analyzed (see Supplementary Table S1 for tax- probabilities for nodal support (BPP). Phylogenetic analysis based onomic references, locality data, and Genbank accession numbers). on primary sequence and secondary structure was performed Taxonomic nomenclature in the text, tables and figures follows employing a combined model of sequence-structure evolution Mikkelsen et al. (2006). Total DNA purification and PCR amplifica- with the software ProfDistS (Wolf et al., 2008). ProfDistS employs tion were performed according to the standard procedures previ- a tree construction algorithms suited for RNA sequence-structure ously described by Oliverio and Mariottini (2001). Purification alignment including the 4 nucleotides (A, G, C, and U) in three and sequencing of Plasmid DNA from positive clones and PCR prod- structural states (single strand, stem left, stem right) coded as in ucts were carried out by an external service (Genechron Centre of Vienna format [‘‘.’’, ‘‘(‘‘,’’)’’, respectively]. The substitution model Sequencing, ENEA, La Casaccia, Rome). of sequence and structure evolution employed combines a general time evolution model (GTR) on the sequence level with a substitu- tion model on morphological features of the structure (Wolf et al., 2.2. Secondary structure modeling, sequence alignment, and barcode 2008). We produced ITS2 sequence-structure alignments by trans- gap analysis lating the secondary structure information of individual sequences (previously obtained, see above) in a Vienna format. Phylogenetic Preliminary ITS2 sequences alignments were performed by trees were calculated with neighbor-joining (NJ) and profile neigh- Clustal X 2.0 (Larkin et al., 2007). Afterwards, the alignment was bor-joining (PNJ) approaches (strNJ and strPNJ, respectively), progressively optimized according to secondary structure employing the automatic profile definition procedure imple- homology. mented in ProfDistS. The robustness of the phylogenies produced The best-supported folding models were obtained combining was tested by 1000 bootstrap replicates. thermodynamic and comparative approaches. On a thermody- namic basis, folding was predicted for each sequence using the software package RNA Structure version 3.71 (available at the 3. Results Turner Lab Homepage, http://rna.chem.rochester.edu; Mathews et al., 1999). The lowest structure energy alone does not guarantee 3.1. ITS2 sequence variation the best-model prediction (Wolf et al., 2005), thus the folding was optimized by contrasting several candidate low free energy folding The venerid ITS2 sequences ranged in length from 241 (Saxido- models with ITS2 folding models from molluscan taxa (Oliverio mus gigantea) to 417 (Meretrix meretrix) base pairs (bp). For the et al., 2002; Salvi et al., 2010). The maximized structural homolo- eight species for which multiple specimens were available no dif- gies were retained and checked for the presence of compensatory ferences in ITS2 sequences length were observed except in Rudi- base changes (CBCs). tapes philippinarum. In this species, specimens from four different Conserved and variable sites were counted using MEGA 5 (Tam- localities showed ITS2 sequences length from 310 to 379 bp with ura et al., 2011). We assessed the presence of a barcode gap be- an average length of 363 bp. Intra-individual variation of the ITS2 Author's personal copy
794 D. Salvi, P. Mariottini / Molecular Phylogenetics and Evolution 65 (2012) 792–798 sequences was observed only in two species, Ruditapes decussatus Ruditapes, and, together with the absence of DV, in the genus Sax- and Tapes aureus, with one or two variable sites discriminating idomus. Outside Veneridae, either the ‘‘six domains’’ folding or the the two alleles. Multiple sequence alignment resulted in a total ‘‘four domains’’ structure standard to most eukaryotes were ob- of 625 nucleotide positions including indels, among which 125 served in the ITS2 secondary structure of eight Veneroida super- positions were constant, 419 variable, and 324 parsimony families analyzed, while in the superfamily Tellinoidea both the informative. two folding architectures are present in different families (data The presence of a barcode gap was evident in the comparison of not shown). interspecific and intraspecific pairwise distances between ITS2 se- In venerids, the variable regions of the ITS2 RNA are located in quences (Supplementary Fig. S2). Intraspecific genetic distances the DI and DIV–VI stems that are poorly conserved in their primary (K2P) ranged between 0–0.025 (Mean = 0.006; SD = 0.006). Inter- sequences, but still highly conserved in their folding architecture. specific genetic distances ranged between 0.035 and 0.767 The basal portion of DII – here defined as Basal STEM – is highly (Mean = 0.514; SD = 0.185) using all the congeneric species com- conserved in all venerids and most Bivalvia and consists of two 4 parisons, and 0.035–0.222 (Mean = 0.138, SD = 0.069) excluding to 6 G/C rich base-pairing stems separated by a segment including comparisons within the genera Ruditapes and Venerupis as these a small asymmetric bulge, a 50-CG/CG-30 base-pairing, and the mis- genera were not monophyletic in our study (see discussion). matching of two pyrimidines (Fig. 1 and Supplementary Fig. S3). The apical portion of DIII – previously defined as Apical STEM (Salvi et al., 2010) – is confirmed to be present in all the Bivalvia species 3.2. ITS2 secondary structure of Veneridae, Heterodonta and other examined and in venerids is characterized by the fourth adenine Bivalvia invariantly exposed, a protruding dinucleotide UU in the opposite base-paired strand, and an apical bulge containing the tetranucle- The typical venerid ITS2 RNA folding along with conserved sec- otide 50-GAAC-30 (see Fig. 1 and Table 1 for details). Finally, the two ondary structure elements across Veneroida and Myoida super- conserved pyrimidine and purine sequences (potentially capable to families and Bivalvia subclasses are represented in Fig. 1. The base pairing) located 50 upstream of the DIII and 30 downstream of common derived Veneridae ITS2 rRNA structure is generally orga- the DIV – here defined Y and R stretch motifs respectively – and the nized in six stems, defined as DI-VI (see Salvi et al., 2010 for sec- extra stem branching out from the very conserved median bulge of ondary structure nomenclature). The six domains are always DIII – here defined as Branched STEM – are structural elements of identifiable in terms of sequence and position except in three the ITS2 folding remarkably present in all venerids as well as in cases: the loss of the DIV was observed in the genera Protothaca,
Fig. 1. ITS2 secondary structure model for Veneridae showing the typical six domains folding (exemplified in the type-species Venus verrucosa, Vve1). Conserved sequence- structure elements across Veneroida, Myoida, and Bivalvia subclasses are boxed and named as in the text. Author's personal copy
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Table 1 Comparison of Apical STEM sequences among Veneridae (nomenclature according to Mikkelsen et al., 2006). See Table 1 for details on sample data and accessions numbers. Superscript letters are nucleotides invariantly exposed; in bold Veneridae Apical STEM consensus sequence under 90% minimum identity criterion (see text for details).
Species Apical STEM sequence Chamelea gallina (Chioninae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUUU-30 Mercenaria mercenaria (Chioninae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Protothaca staminea (Chioninae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Timoclea ovata (Chioninae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Cyclina sinensis (Cyclininae) 50-GGGACAGGGCUCGA UGGGCC UUCUGUCU-30 Dosinia exoleta (Dosiniinae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Gouldia minima (Gouldiinae) 50-GGGACUGGGCUCGAACGGGCC UUCAGUUC-30 Meretrix meretrix (Meretricinae) 50-GGGACAGGGCUCG CGAGCCCAG UGUCC-30 Callista chione (Pitarinae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Saxidomus gigantea (Pitarinae) 50-UGGACAGGGCUCGAACGGGCCA CUGUCU-30 Paphia undulata (Tapetinae) 50-GGGACAGGGCUCGAACGGGCC UUAUGUCU-30 Ruditapes decussatus (Tapetinae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Ruditapes philippinarum (Tapetinae) 50-GAGACAGGGCUCGAACGGGCCCGC ACGUCC-30 Ruditapes variegata (Tapetinae) 50-GGGACAGGGCUCGAACGGGCUCGUUCUGUCC-30 Tapes aureus (Tapetinae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Venerupis lucens (Tapetinae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUCU-30 Venus verrucosa (Venerinae) 50-GGGACAGGGCUCGAACGGGCC UUCUGUUU-30 Veneridae Apical STEM Consensus90% 50-GGGACAGGGCUCGAACGGGCC UUCUGUYY-30 50-______STEM______bulge______STEM___-30
representatives of other superfamilies of Veneroida and Myoida quence-structure synapomorphies with T. aureus (Supplementary (Fig. 1). Fig. S4) and species assigned to the genus Ruditapes show divergent ITS2 secondary structures with six domains in R. decussatus and 3.3. ITS2 sequence/structure phylogenetic information from Veneridae five domains in R. variegata and R. phylippinarum (Fig. 2). to Bivalvia Multiple species of the subfamilies Chioninae and Tapetinae were analyzed, which provided the opportunity to test whether Phylogenetic analyses based on primary sequence alone (ML the combined sequence-structure alignment is also informative and BA) and combined with secondary structure (strNJ and strPNJ) at the subfamily ranking. In all the phylogenetic analyses, the spe- yielded analogous topologies with no supported branch in conflict cies assigned to the subfamily Chioninae were placed in different except for the sister relationship between Ruditapes variegata and clades and showed divergent ITS2 secondary structures (with five R. decussatus (supported in ML and BA analyses, not shown, but domains in the genus Protothaca and six domains in the other spe- not in the sequence-structure strNJ analyses, Fig. 2). Analyses car- cies), indicating that this taxon is polyphyletic. On the other hand, ried out using additional outgroups from Veneroida and Myoida the monophyly of the subfamily Tapetinae (not including Paphia recovered the same overall topology relative to the analyses using undulata) was supported by the sequence-structure phylogenetic the primary outgroup taxa A. islandica (Veneroida), but with lower analyses and by the mismatch in the DII and two CBCs in the DI, bootstrap support (data not shown). The profile neighbor-joining that define even more tightly the relationships between some of procedures (strPNJ) produced virtually the same results both in them (Fig. 2 and Supplementary Fig. S4). terms of topologies and node support relative to the initial strNJ. The resolution of the ITS2 marker in reconstructing the phylo- Therefore, only results obtained by strNJ method are shown. genetic relationships between the nine venerid subfamilies in- The results from ITS2 sequence/structure analyses based on (i) cluded in our analyses was very low, both as concerns the primary sequence alone, (ii) combined primary sequence-second- sequence-structure phylogenetic analyses and the inspection of ary structure and (iii) secondary structure alone were highly con- the secondary structure models (Fig. 2). Indeed, phylogenetic anal- gruent at any taxonomic level. yses returned a basal polytomy, and failed to disentangle the rela- At the species level, all the eight species for which multiple tionships between venerid subfamilies, while secondary structures specimens were available constituted monophyletic supported were not informative since at deeper nodes most of the taxa clades (BP > 82; BPP P 99). Moreover, in the three species for showed the plesiomorphic ‘‘six domains’’ folding. which specimens from different geographic localities were ana- Above the family Veneridae, while the phylogenetic resolution lyzed divergent intra-specific phylogroups were recovered of the ITS2 sequence-structure alignments was lower, several con- (Fig. 2), suggesting a resolution power of ITS2 sequence data also served features of ITS2 secondary structure can be identified as at the intra-specific level. The ITS2 secondary structure from con- diagnostic characters of several groups from Veneroida, Myoida, specific individuals of any species was strictly conserved and strik- and Bivalvia (see Section 3.2). ing sequence-structure autoapomorphies were observed in four species (Supplementary Fig. S4). 4. Discussion At the generic level, in ITS2 sequence/structure analyses Dosin- ia, Meretrix, and Protothaca formed monophyletic and well sup- The suitability of the ITS2 as a barcode marker for venerids is ported clades (BP > 84; BPP P 99) with a conserved ITS2 highly supported by the present study as assessed against several secondary structure (showing six domains in Dosinia and Meretrix, criteria: (1) the presence of a barcode gap between inter- and in- and five domains in Protothaca, see Fig. 2). On the other hand, the tra-specific variation; (2) the clustering of conspecific sequences genus Venerupis was paraphyletic relative to Tapes and the genus in distinct groups with high bootstrap supports; and (3) the pres- Ruditapes was paraphyletic with respect to Tapes and Venerupis ence of sequence-structure diagnostic character differences be- (Fig. 2). The paraphyly of the genera Venerupis and Ruditapes was tween congeneric species. Based on the ITS2 sequence-structure confirmed by ITS2 structure data: V. lucens shares two striking se- analyses, it was possible to identify all the venerid species studied Author's personal copy
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Fig. 2. ITS2 sequence-structure neighbor-joining tree (strNJ) based on evolutionary distances simultaneously calculated on sequences and secondary structures of 24 Veneridae species and rooted with A. islandica (Veneroida, Arcticidea, Arcticidae). Statistical support values from phylogenetic analyses based on primary sequence and secondary structure (strNJ) and primary sequences alone (Maximum likelihood/Bayesian inference) are reported above and below the nodes respectively. Number and uppercase letter after species’ name refer to different individuals; minor case letter after species’ name indicate different alleles from heterozygote individuals. Taxonomic nomenclature follows Mikkelsen et al. (2006). Author's personal copy
D. Salvi, P. Mariottini / Molecular Phylogenetics and Evolution 65 (2012) 792–798 797 as reciprocally monophyletic units, which in some cases were fur- nomic groups such as Veneridae and Pectinidae (this study; Salvi ther supported by striking apomorphies (CBCs and/or a mismatch et al., 2010) could provide a molecular (sequence-structure) bar- pairing) shared by the ITS2 sequence-structure of conspecific indi- code tool for their diagnosis. viduals. Moreover, ITS2 seems to be a promising marker also for Interestingly, when comparing the phylogenetic reconstruction phylogeographic studies as intraspecific monophyletic phylo- of Veneridae attained in this study with those recovered in previ- groups, clustering individuals from distinct localities, were identi- ous molecular studies, we found that the ITS2 provides the same fied in most of the species for which individuals from multiple phylogenetic resolution as much longer mitochondrial and nuclear localities were analyzed. fragments including the 16S rRNA (Canapa et al., 1996, 2003) and In Venus clams both the ITS2 primary sequences and secondary the COI (Mikkelsen et al., 2006; Kappner and Bieler, 2006; Chen structure have proven valuable for reconstructing the phylogenetic et al., 2011) mitochondrial fragments, as well as the combined relationships providing the highest phylogenetic resolution and 16S + COI + 28S + H3 (Mikkelsen et al., 2006) and 16S + COI + H3 robustness at the genus level. The genera Dosinia, Meretrix, and (Kappner and Bieler, 2006; Chen et al., 2011) mitochondrial-nucle- Protothaca formed monophyletic units in the ITS2 (sequence and ar datasets. Taking into account that the use of mitochondrial sequence-structure) phylogenetic reconstructions, in accordance markers can be problematic due to double uniparental inheritance with previous molecular studies, although the taxon composition of mitochondrial genomes in some venerids (Mikkelsen et al., of the genus Dosinia needs to be re-assessed (Kappner and Bieler, 2006), and given the very low resolution of nuclear markers such 2006; Mikkelsen et al., 2006; Chen et al., 2011). The ITS2 secondary as the H3 gene and the ribosomal 18S/28S rRNAs (Chen et al., structures of Protothaca species showed a ‘‘five-domains’’ folding 2011), the ITS2 gene emerges as a candidate optimal marker for architecture, unique within Veneridae, which emerges as a synapo- accurate and wide-range taxonomic and phylogenetic studies in morphy of this genus. On the other hand, the monophyly of Ven- venerids. erupis and Ruditapes is not supported either by ITS2 data or by previous molecular studies based on mitochondrial and nuclear Acknowledgments markers (Canapa et al., 2003; Kappner and Bieler, 2006; Mikkelsen et al., 2006; Chen et al., 2011). Indeed, the closer phylogenetic rela- We thank the Associate Editor Suzanne Williams, Mattias Wolf tionship of V. lucens to T. aureus rather than to V. senegalensis is fur- and Marco Oliverio for useful suggestions and people from ‘‘Gru- ther supported by a double CBC in the DI shared by the first two ppo Malacologico Mediterraneo’’ (Rome, Italy) for their help during species. Likewise, the paraphyly of the genus Ruditapes was sup- the sampling. PM wishes to thank the University of Roma Tre for ported by phylogenetic and structural ITS2 data, suggesting that financial support. DS is supported by the FCT post-doctoral grant these three genera need revision as proposed by previous studies SFRH/BPD/66592/2009 and by the SYNTHESYS Project http:// (references above). The monophyly of the subfamilies Venerinae, www.synthesys.info/ which is financed by European Community and Tapetinae and the inclusion of Chamalea gallina in the subfam- Research Infrastructure Action under the FP7 Capacities Pro- ily Venerinae (rather than in the Chioninae) were strongly sup- gramme at the Museo Museo Nacional de Ciencias Naturales of ported in agreement with previous molecular phylogenies Madrid (CSIC). (Canapa et al., 2003; Kappner and Bieler, 2006; Mikkelsen et al., 2006; Chen et al., 2011). Contrary to previous studies (Coleman, 2003; Song et al., 2008; Appendix A. Supplementary material Salvi et al., 2010), in venerids the inclusion of the ITS2 secondary structure information did not extend at higher taxonomic levels Supplementary data associated with this article can be found, in the phylogenetic applicability of this marker attained by compari- the online version, at http://dx.doi.org/10.1016/j.ympev.2012.07. sons of primary sequences. 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