Internal Repetition and Intraindividual Variation in the rDNA ITS1 of the Anopheles punctulatus Group (Diptera: Culicidae): Multiple Units and Rates of Turnover

James E. Bower 0 1 2 3 Robert D. Cooper 0 1 2 3 Nigel W. Beebe 0 1 2 3 0 N. W. Beebe (&) School of Integrative Biology, University of Queensland , Room 171B, Goddard Building, St. Lucia, QLD 4072, Australia 1 R. D. Cooper Australian Army Malaria Institute , Gallipoli Barracks, Enoggera, QLD 4052, Australia 2 J. E. Bower Biological Sciences, University of Wollongong , Northfields Avenue, Wollongong, NSW 2522, Australia 3 N. W. Beebe CSIRO, Long Pocket Laboratories , Indooroopilly, QLD 4068, Australia The rapid divergence of repetitive sequences makes them desirable markers for phylogenetic studies of closely related groups, provided that a high level of sequence homogeneity has been maintained within species. Intraspecific polymorphisms are found in an increasing number of studies now, and this highlights the need to determine why these occur. In this study we examined intraindividual variation present in the first ribosomal internal transcribed spacer (ITS1) from a group of cryptic mosquito species. Individuals of the Anopheles punctulatus group contained multiple ITS1 length variants that ranged from 1.2 to 8.0 kb. Nucleotide and copy number variation for several homologous internal repeats is common, yet the intraspecific sequence divergence of cloned PCR isolates is comparable to that of other mosquito species (*0.21.5%). Most of the length variation is comprised of a 50ITS1 repeat that was identified as a duplication of a - conserved ITS2 region. Secondary structure conservation for this repeat is pronounced and several repeat types that are highly homogenized have formed. Significant interspecific divergence indicates a high rate of evolutionary change for this spacer. A maximum likelihood tree constructed here was congruent with previous phylogenetic hypotheses and suggests that concerted evolution is also accompanied by interpopulation divergence. The lack of interindividual differences and the presence of homogenized internal repeats suggest that a high rate of turnover has reduced the overall level of variation. However, the intraindividual variation also appears to be maintained by the absence of a single turnover rate and the complex dynamics of ongoing recombination within the spacer. Concerted evolution DNA turnover Anopheles punctulatus The process of gene duplication is considered a fundamental driving force in evolution because the generation of additional gene copies leads to a relaxation of selective constraint and the possibility for the evolution of new functions (Ohno 1970). The evolution of these repeated sequences forms a range of homologous relationships which requires an awareness of this and their effect on the phylogenetic congruence of data sets (Fitch 2000). For example, gene duplication and speciation events do not always co-occur because paralogous sequences reflect the phylogeny of the gene family and orthologous sequences reflect the duplication of taxa via speciation. Paralogous sequences evolve in the absence of recombinant interactions, via a continual process of birth-by-duplication and death-by-mutation (Nei and Rooney 2005). However, many repeated sequences evolve in a nonindependent fashion, by ongoing intra- and interlocus recombination within a species that homogenizes paralogous sequences into what are called plerologues. This accumulation of species-specific differences is termed concerted evolution (Graur and Li 2000). Here the distinction between orthology and paralogy is blurred by concerted evolution and depending on the level of homogenization, whole families of repeated sequences may appear to evolve like singlecopy genes and so reflect speciation events (Sanderson and Doyle 1992). Homogenization occurs through a variety of non-Mendelian recombination processes that transfer DNA either reciprocally or nonreciprocally within and between chromosomes (Dover 2000). The continual turnover of DNA involved in these exchanges results in the gain or loss of repeated sequences during the lifetime of an individual and includes unequal crossing-over, gene conversion, replication slippage, duplicative transposition, and retroposition (see Graur and Li 2000). The level of homogenization is maintained in a balance between the processes that generate variation and those that eliminate it. This level is expected to decrease with increasing mutation rate, population size, number of repeats, and number of nonhomologous loci, as well as when interchromosomal exchanges are rare (Ohta and Dover 1983). DNA turnover can also generate variation and plays a similar, though not equivalent, role to mutation (Kimura and Ohta 1979). Recombinant exchanges occurring at the length of a repeat or greater may lead to its homogenization, but its rate of turnover in the genome also has to exceed the mutation rate and the turnover of other repeats present within or overlapping it. This is an important consideration because several recombinant processes can operate simultaneously on the same stretch of DNA. More attention is usually given to differences in the rates of turnover that occur within and between chromosomes (i.e., intra- and interlocus recombination) (e.g., Gonzalez and Sylvester 2001), as this forms the core of a debate over the relative importance of population-level versus genomic processes for concerted evolution. Low rates of recombinant exchanges between chromosomes can lead to the formation of independent chromosomal lineages within a population that can be observed by differences between individuals. For example, physical constraints to XY chromosome pairing in Drosophila melanogaster lead to incomplete homogenization of the ITS1, in which a 24-bp deletion is restricted to males (Schlotterer and Tautz 1994). The presence of interindividual differences enables natural selection and random genetic drift to affect the frequency of variant repeats. However, without restrictions to interchromosomal exchange, the coupling of DNA turnover to meiotic recombination (i.e., sex) can reduce interindividual differences and spread variation throughout a population in a genomic-level process called molecular drive (Dover 2000). The ribosomal DNA (rDNA) multigene family, with its contrasting regions of phylogenetically conserved rRNA genes and divergent spacers, has long provided insights into the underlying processes of concerted evolution. Of these, the first internal transcribed spacer (ITS1) diverges rapidly between closely related species, which has enabled it to be utilized for phylogenetic studies at or below the species level (e.g., Fabry et al. 1999; Rodriguez-Perez et al. 2006). Despite some success at investigating population relationships, intraindividual variation is common for this spacer, and unless a high level of sequence homogeneity has been maintained within species, it can obscure the resolution of phylogenetic trees (Miller et al. 1996; Fama` et al. 2000). This variation is often due to internal repetition, in the form of (...truncated)