Short Communication
Molecular phylogenetics in 2D: ITS2 rRNA evolution and sequence-structure barcode from Veneridae to Bivalvia

https://doi.org/10.1016/j.ympev.2012.07.017Get rights and content

Abstract

In this study, we analyzed the nuclear ITS2 rRNA primary sequence and secondary structure in Veneridae and comparatively with 20 Bivalvia taxa to test the phylogenetic resolution of this marker and its suitability for molecular diagnosis at different taxonomic levels.

Maximum likelihood and Bayesian trees based on primary sequences were congruent with (profile-) 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/nuclear) gene fragments 2–5 times longer. Structural elements of the ITS2 folding, such as specific mismatch pairing and compensatory base changes, provided further support for the monophyly of some groups and for their phylogenetic relationships. Veneridae ITS2 folding is structured in six domains (DI–VI) and shows five striking sequence-structure features. Two of them, the Basal and Apical STEMs, are common to Bivalvia, while the presence of both the Branched STEM and the Y/R stretches occurs in five superfamilies of the two Heterodonta orders Myoida and Veneroida, thus questioning their reciprocal 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.

Highlights

► We analyzed ITS2 rRNA sequence and secondary structure in Veneridae and Bivalvia. ► Phylogenetic trees based on ITS2 sequences and on sequence-structure are congruent. ► ITS2 sequence-structure approach is suitable for phylogeny and taxonomy of venerids. ► We discuss important sequence-structure features of the ITS2 folding unique to venerids and Bivalvia.

Introduction

Molecular phylogenetics and molecular taxonomy have traditionally focused on analyzing DNA (or RNA) sequences for phylogenetic and taxonomic assessments. However, in the last few years the exploitation of secondary structure information of ribosomal RNA (rRNA) molecules such as the nuclear ribosomal internal transcribed spacer 2 (ITS2) has revealed a promising approach not only in phylogenetic reconstruction (Coleman, 2003, Schultz et al., 2006, Schultz and Wolf, 2009, Salvi et al., 2010) but also in species diagnosis (Coleman, 2003, Coleman, 2009, Müller et al., 2007).

The ITS2 is generally organized in four main helix domains (DI–IV) of secondary structure and the combined information of both folding and primary sequence has been exploited for phylogenetic inference, taxonomic classification, and species delimitation, providing an increased phylogenetic resolution and reliability than approaches based on primary sequence data alone (Coleman, 2003, Schultz et al., 2006, Bologna et al., 2008, Song et al., 2008, Schultz and Wolf, 2009, Keller et al., 2010, Salvi et al., 2010). Compensatory base changes (CBCs) – mutations in both nucleotides of a paired position in a double-stranded structure of the transcribed RNA – in conserved regions of the eukaryote ITS2 sequence-structure have been shown to correlate with interbreeding incompatibility between species. Several studies on animal, plants, and fungi demonstrated that the presence of at least one CBC in ITS2 secondary structures is a good classifier (reliability higher than 90%) of two organisms belonging to distinct species (Coleman, 2000, Coleman, 2003, Coleman, 2009, Müller et al., 2007). Moreover, some conserved features of the ITS2 secondary structure such as stem-loops domains have shown to be diagnostic of higher taxonomic grouping such as tribes, subfamilies, families, and orders (Oliverio et al., 2002, Bologna et al., 2008, Keller et al., 2008, Salvi et al., 2010). Thus, the information from the ITS2 folding and sequence-structure variation is a useful tool in molecular taxonomy for species and higher-taxa diagnosis.

In this study we analyzed the primary sequence and the secondary structure information from the nuclear ITS2 rRNA in the phylogenetic context of the family Veneridae. This family is a large group of bivalves with approximately 800 species, with a largely unresolved phylogenetic history and unstable taxonomy despite recent molecular and morphological studies (Canapa et al., 1996, Canapa et al., 2003, Kappner and Bieler, 2006, Mikkelsen et al., 2006, Chen et al., 2011). Indeed, while on the one hand the use of morphological traits is problematic since most of them are either uniform or homoplastic (Mikkelsen et al., 2006), on the other hand the use of molecular markers can be problematic due to (i) double uniparental inheritance of mitochondrial genomes in some venerids (Mikkelsen et al., 2006); (ii) inherent difficulties in amplifying and sequencing “the barcoding gene” COI in venerids (Kappner and Bieler, 2006), and (iii) the very low resolution of nuclear markers such as the H3 gene and the ribosomal 18S/28S rRNAs (Chen et al., 2011). To overcome these problems, the use of an extremely variable and easy to amplify nuclear marker such as the ITS2 is highly advisable, but despite a promising test on four species (Cheng et al., 2006), ITS2 has never been used in phylogenetic and taxonomic studies of Veneridae.

The main aim of this study was to integrate the information of individual RNA secondary structures with primary sequences in venerids phylogeny and taxonomy, by employing a combined model of sequence-structure evolution as well as identifying conserved elements of the ITS2 folding. By including several representatives from Bivalvia subclasses we further test the phylogenetic resolution of the ITS2 marker and its suitability as a barcode tool for molecular taxonomy of venerid species to higher taxonomic levels among bivalves.

Section snippets

Sampling, DNA extraction and sequencing

ITS2 sequences were obtained from live specimens (60%), frozen specimens from trade (4%), or retrieved from Genbank (36%). A total of 45 specimens from 24 venerid species were employed in the molecular analyses. Twenty additional Bivalvia ITS2 sequences from the subclasses Heterodonta, Paleoheterodonta, and Pteriomorphia were also analyzed (see Supplementary Table S1 for taxonomic references, locality data, and Genbank accession numbers). Taxonomic nomenclature in the text, tables and figures

ITS2 sequence variation

The venerid ITS2 sequences ranged in length from 241 (Saxidomus gigantea) to 417 (Meretrix meretrix) base pairs (bp). For the eight species for which multiple specimens were available no differences in ITS2 sequences length were observed except in Ruditapes philippinarum. In this species, specimens from four different localities showed ITS2 sequences length from 310 to 379 bp with an average length of 363 bp. Intra-individual variation of the ITS2 sequences was observed only in two species,

Discussion

The suitability of the ITS2 as a barcode marker for venerids is highly supported by the present study as assessed against several criteria: (1) the presence of a barcode gap between inter- and intra-specific variation; (2) the clustering of conspecific sequences in distinct groups with high bootstrap supports; and (3) the presence of sequence-structure diagnostic character differences between congeneric species. Based on the ITS2 sequence-structure analyses, it was possible to identify all the

Acknowledgments

We thank the Associate Editor Suzanne Williams, Mattias Wolf and Marco Oliverio for useful suggestions and people from “Gruppo Malacologico Mediterraneo” (Rome, Italy) for their help during the sampling. PM wishes to thank the University of Roma Tre for financial support. DS is supported by the FCT post-doctoral grant SFRH/BPD/66592/2009 and by the SYNTHESYS Project http://www.synthesys.info/ which is financed by European Community Research Infrastructure Action under the FP7 Capacities

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