Incomplete concerted evolution and reproductive isolation at the rDNA locus uncovers nine cryptic species within Anopheles longirostris from Papua New Guinea

Abstract

Background: 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. 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.

Results: Genetic 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.

Conclusion: The 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.

Figure 1

Map of Papua New Guinea showing collections sites of A. longirostris.

Figure 2

ITS2 genotypes of A. longirostris. Panel A: ITS2 PCR products run through a 1.0% agarose gel indicate some small size variation. Panel B: The same ITS2 PCR products cut with Msp I and run through a 3.0% agarose gel reveals eight distinct RFLP genotype profiles. Panel C: Most ITS2-RFLP genotypes revealed the same heteroduplex profile when PCR products were run through a 7.0% acrylamide gel suggesting copy variants are fixed within individuals and within interbreeding populations. However, genotype C contained two distinct heteroduplex profiles (designated C1 and C2) within its RFLP profile revealing the presence of two independently evolving ITS2 genotypes.

Figure 3

Phylogenetic assessment of A. longirostris based on DNA sequence from the ITS2 (Panel A) and mtDNA COI (Panel B) reveal nine lineages. Both Bayesian and Maximum Likelihood procedures produced trees of similar topology with branch support values above 70% shown - Bayesian posterior probability above the branch (converted to percentage) and Maximum Likelihood bootstrapping (percentage) below the branch. The ITS2 showed a well resolved tree with all clades well separated into genotypes. All ITS2 genotypes were evident as mtDNA COI divergent lineages with cloned individuals co- assessed for the COI.