Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta)

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Abstract

The organization, structure, and nucleotide variability of the ribosomal repeat unit was compared among families, genera, and species of cockroaches (Insecta: Blattodea). Sequence comparisons and molecular phylogenetic analyses were used to describe rDNA repeat unit variation at differing taxonomic levels. A ∽1200 bp fragment of the 28S rDNA sequence was assessed for its potential utility in reconstructing higher-level phylogenetic relationships in cockroaches. Parsimony and maximum likelihood analyses of these data strongly support the expected pattern of relationships among cockroach groups. The examined 5′ end of the 28S rDNA is shown to be an informative marker for larger studies of cockroach phylogeny. Comparative analysis of the nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among closely related species of Blattella and Periplaneta reveals that ITS sequences can vary widely in primary sequence, length, and folding pattern. Secondary structure estimates for the ITS region of Blattella species indicate that variation in this spacer region can also influence the folding pattern of the 5.8S subunit. These results support the idea that ITS sequences play an important role in the stability and function of the rRNA cluster.

Introduction

Ribosomal DNA (rDNA) has long been considered a useful marker for comparative evolutionary and phylogenetic studies (Hillis and Davis, 1986, Mindell and Honeycutt, 1990, Wesson, McLain, Oliver, Piesman and Collins, 1993, Wesson, McLain, Oliver, Piesman and Collins, 1993, Vogler and DeSalle, 1994, Tang, Toe, Back and Unnasch, 1996). This utility results largely from the differential conservation and variability of different genes and regions that make up the rDNA repeat unit (Hillis and Dixon, 1991, Hamby and Zimmer, 1992). The basic organization of rDNA is conserved throughout eukaryotes. Eukaryotic ribosomal RNA genes are arranged in tandemly repeated clusters, with each cluster containing the genes for 18S-, 5.8S-, and 28S-like ribosomal RNAs. The genes are separated by several spacers, namely, the NTS (nontranscribed spacer), and ITS1 and ITS2 (internal transcribed spacers). The NTS separates neighboring repeat units; ITS1 is located between the 18S- and 5.8S-like coding regions; ITS2 lies between the 5.8S- and 28S-like genes (Fig. 1A) (Gerbi, 1985). Ribosomal repeats are usually localized to one or a few chromosomes and form part of the nucleolar organizers (NO). Because the coding regions and spacers differ widely in their rate of evolution, they can reveal phylogenetic relationships ranging from the level of major phyla of living organisms to the population level (Hillis and Dixon, 1991, Wesson, Porter and Collins, 1992, Kuperus and Chapco, 1994, Honda, Nakashima, Yanase, Kawarabata and Hirose, 1998, Muccio, Marinucci, Frusteri, Maroli, Pesson and Gramiccia, 2000, Wiegmann, Mitter, Regier, Friedlander, Wagner and Nielsen, 2000).

Additional information about relationships and rDNA evolution can be obtained by examining the secondary structure of the ribosomal RNA. In the rDNA coding regions (18S-, 5.8S-, and 28S-like), sequence conservation is reflected in the high similarity of secondary structures in a variety of distantly related organisms (Gerbi, Gourse and Clark, 1982, Raué, Klootwijk and Musters, 1988, Wesson, Porter and Collins, 1992). Sequence tracts that are highly variable between species may still retain certain, apparently functionally important, components of secondary structure. For example, estimated secondary structures of the ITS1 sequences are nearly identical in human, chimpanzee, and gorilla (Gonzales et al., 1990), and those from mouse and rat also show high similarity (Michot et al., 1983). Integrity of the rDNA secondary structure is maintained through compensatory nucleotide changes to preserve base pairing in stems, and through indels which change stem length but do not alter the overall folding pattern. Structural analysis of the ITS in yeast has shown that the spacer regions also play a role in the maturation of precursor rRNA molecules (Musters, Boon, Van der Sande, Van Heerikhuizen and Planta, 1990, Van der Sande, Kwa, Van Nues, Van Heerikhuizen, Raue and Planta, 1992, Schulenburg, Englisch and Wagele, 1999).

Despite the importance of rDNAs in phylogenetic and molecular evolutionary studies of insects, very little is known about the rDNA of cockroaches. Because of their pest status and the ease with which they are cultured in the laboratory, cockroaches have served as model organisms for studies of insect physiology, molecular genetics, and chemical ecology, but relatively few of these studies have been explicitly comparative or evolutionary in their focus. Our studies of the nuclear ribosomal genes in cockroaches are aimed at understanding the dynamics of sequence and structural variation in these genes and assessing the utility of that variation for comparative evolutionary studies. In this paper, we examine the organization, structure, and nucleotide variability of the ribosomal repeat unit of cockroaches (Insecta: Blattodea). Our comparison focuses on the ITS1 and ITS2 spacer regions, as well as on portions of the rDNA subunit coding regions that immediately flank them. These regions are compared across a broad range of taxonomic divergences within cockroaches.

Divergences between extant genera of cockroaches may be as old as 75–100 my before present (Labandeira, 1994, Nalepa and Bandi, 1999), but some genus and species-level divergences could be much more recent (Nalepa and Bandi, 1999). Phylogenetic relationships among cockroach groups are the subject of current debate in the insect systematics literature (McKittrick, 1964, Grandcolas , 1994, Grandcolas, 1996, Grandcolas, 1999, Kambhampati, 1995, Kambhampati, 1996, Klass, 1997, Klass, 1998, Nalepa and Bandi, 1999). Despite renewed interest in cockroach phylogenetics and a wealth of new data, major differences remain among proposed phylogenetic arrangements for cockroach families (Fig. 2). For divergences as old as those hypothesized for cockroach families, it is likely that the more slowly evolving regions of the nuclear ribosomal DNA (18S rDNA, Lo et al., 2000; 28S rDNA, this study) and conserved nuclear protein encoding genes could be important sources of new evidence on cockroach relationships.

Our sequence comparisons and molecular phylogenetic analyses are used to describe rDNA repeat unit variation at differing taxonomic levels. First, we evaluate the phylogenetic utility of a ∽1200 bp fragment of the 28S rDNA for reconstructing higher-level cockroach relationships. Our findings show that the 28S rDNA is highly informative for higher-level cockroach phylogeny. Second, comparative analysis of the nucleotide sequences and the secondary structures of the ITS1 and ITS2 among closely related Blattella and Periplaneta species reveals major structural and sequence-level constraints.

Section snippets

Cockroach taxa sampled

rDNA sequences were obtained from 11 cockroach species from three families: Blattellidae — Blattella germanica, B. vaga, B. lituricollis, B. asahinai, Parcoblatta latta; Blattidae — Periplaneta americana, P. fuliginosa, P. brunea; Blaberidae — Diploptera punctata, Blaberus atropus, B. giganteus. Specimens were obtained from laboratory cultures (CS lab, NCSU), or from colleagues, and frozen at −80 °C to preserve nucleic acids.

Laboratory methods

Total genomic nucleic acids were extracted using a standard DNA

Phylogenetic utility of the 28S rDNA in Blattodea

Alignment of the 28S rDNA sequence data for the 11 cockroach species used in this study resulted in 883 sites included in the phylogenetic data set. Of these, 267 were variable and 156 were parsimony informative (see http://www2.ncsu.edu/unity/users/b/bwiegman/public_html/align.html). Analysis of the entire alignment generated in Clustal-W 1.7 with all positions included gave identical phylogenetic results to those reported below.

The length of the fragment was nearly identical for all of the

Acknowledgements

We thank the North Carolina State University DNA Sequencing Facility for help with automated DNA sequencing. This study was supported in part by the Blanton J. Whitmire Endowment and the W. M. Keck Center for Behavioral Biology at NCSU, and by the Russian State Program “Frontiers in Genetics”. We thank two anonymous reviewers for valuable suggestions.

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