Abstract
BackgroundNuclear ribosomal DNA (rDNA) genes and transcribed spacers are highly utilized as taxonomic markers in metazoans despite the lack of a cohesive understanding of their evolution. Here we follow the evolution of the rDNA second internal transcribed spacer (ITS2) and the mitochondrial DNA cytochrome oxidase I subunit in the malaria mosquito Anopheles longirostris from Papua New Guinea (PNG). This morphospecies inhabits a variety of ecological environments indicating that it may comprise a complex of morphologically indistinguishable species. Using collections from over 70 sites in PNG, the mtDNA was assessed via direct DNA sequencing while the ITS2 was assessed at three levels - crude sequence variation through restriction digest, intragenomic copy variant organisation (homogenisation) through heteroduplex analysis and DNA sequencing via cloning.ResultsGenetic evaluation of over 300 individuals revealed that A. longirostris comprises eight ITS2 PCR-RFLP genotypes and nine ITS2 heteroduplex genotypes showing distinct copy variant organization profiles after PCR amplification. Seven of these nine genotypes were found to be sympatric with other genotypes. Phylogenetic analysis of cloned ITS2 PCR products and mtDNA COI confirmed all nine clades with evidence of reproductive isolation at the rDNA locus. Compensatory base changes in the ITS2 secondary structure or in pseudoknots were absent when closely related species were assessed. Individuals from each ITS2 genotype showed the same copy variant heteroduplex profile suggesting that the rDNA array is fixed within each genotype.ConclusionThe centromere-proximal position of the rDNA array in Anopheles mosquitoes has probably reduced interchromosomal recombination leaving intrachromosomal events responsible for the observed pattern of concerted evolution we see in these mosquitoes. The stability of these intragenomic ITS2 copy variants within individuals and interbreeding populations suggests that rDNA is moving as a single evolutionary unit through natural populations to fixation and has provided a complementary diagnostic tool to the restriction digest for studying genetic discontinuities and species boundaries. In this, the utility of the ITS2 as a universal taxonomic marker is probably contingent on several factors pertaining to spacer dimensions and the genomic location of the rDNA array with respect to recombination and proximity to regions potentially under selection.
Highlights
Nuclear ribosomal DNA genes and transcribed spacers are highly utilized as taxonomic markers in metazoans despite the lack of a cohesive understanding of their evolution
The aim of this study is to determine if A. longirostris constitutes a cryptic species group by showing reproductive isolation at the ribosomal DNA (rDNA) ITS2 locus, while co-investigation of the cytochrome oxidase subunit I (COI) locus is used to determine if the identified cryptic species exist as divergent mitochondrial DNA (mtDNA) lineages [5]
We examine the evolution of the ITS2 at three levels: 1) PCR-restriction fragment length polymorphism (RFLP) to view crude sequence variation; 2) PCR-copy variant analysis to view the homogenization of potential ITS2 copy variants within the rDNA array; and 3) DNA sequence analysis via cloning and sequencing
Summary
Nuclear ribosomal DNA (rDNA) genes and transcribed spacers are highly utilized as taxonomic markers in metazoans despite the lack of a cohesive understanding of their evolution. The ITS2 is part of the rDNA gene family tandemly organized head to tail, often hundreds of times in the nucleolar organizer regions (NORs) This family of structural RNA genes and spacers are observed to evolve through a pattern of concerted evolution using DNA turnover machinery that operates to spread or remove sequence variant copies within individuals (homogenization), and operates to spread or remove variants within an interbreeding population (fixation). In metazoans we observe that each gene sequence in the array is generally the same, producing sequence similarity within a species and sequence diversity between species [14,15] The practicality of this rDNA turnover machinery for resolving closely related species has been that the less functionally restricted regions, including the ITS2, can accumulate mutations within reproductively isolated populations relatively quickly and can be the first indicators of genetic discontinuity between populations [3,7,16]
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