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

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.

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

“…ITS intragenomic variation has also been reported in other species of grasshoppers, such as C. parallelus (Parkin and Butlin, 2004) and Podisma pedestris (Keller et al, 2006), and in other groups of organisms, such as nematodes (Hugall et al, 1999), crabs (Harris and Crandall, 2000), crustaceans (Gandolfi et al, 2001), sponges (Wörheide et al, 2004), mosquitoes (Alquezar et al, 2010), tapeworms (Orosová et al, 2010) and plants (Nieto-Felliner, Roselló, 2007;Song et al, 2012). Such a high intragenomic variation between paralogous copies of rDNA units could lead to highly divergent phylogenetic results, depending on the specific copy or copies sampled.…”

Molecular differentiation of Central European blowfly species (Diptera, Calliphoridae) using mitochondrial and nuclear genetic markers

“…This is a highlighted issue considering that many phylogenetic analyses become counterproductive with sequence lengths of about 300 bp. It is worth mentioning that this nuclear locus, as mitochondrial markers, exists in high copy number [44]. This feature added to the small size of the target sequence may be a great advantage, especially in degraded samples where occurs a significant reduction of the fragment size.…”

Population structure, mitochondrial polyphyly and the repeated loss of human biting ability in anopheline mosquitoes from the southwest Pacific

“…ITS2 data were only used for phylogenetic analysis because it is part of the multicopy rDNA gene family that evolves through a poorly described non‐Mendelian process (Beebe et al. 1999; Alquezar et al. 2010).…”