Internal repetition and intraindividual variation in the rDNA ITS1 of the anopheles punctulatus group (Diptera: Culicidae): multiple units and rates of turnover

Abstract

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 (~0.2-1.5%). Most of the length variation is comprised of a 5'-ITS1 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.

Fig. 1

PCR products of the ITS1 from primers F-ITS/18S and R-5.8S resolved on 1.2% agarose gel. Samples are shown in the following order: An. farauti (lanes 1–3; Rabaul, Northern Territory and Central Province), An. irenicus (lane 4), An. farauti 6 (lane 5), An. hinesorum (lanes 6–7; Central Province and QLD), An. torresiensis (lane 8), An. koliensis (lanes 9–10; Central and Madang Provinces), An. punctulatus (lane 11), An. sp. nr punctulatus (lane 12), An. annulipes s.l. (lane 13), and An. clowi (lane 14). Fragment sizes of the flanking DNA ladders are in kilobases

Fig. 2

Southern blot of genomic DNA digested with EcoR47III and PstI, probed with a ³²P-labeled 18S rDNA fragment. Samples are shown from An. farauti (lanes 1–3; Rabaul, Northern Territory, and Central Province), An. irenicus (lane 4), An. farauti 6 (lanes 5–7), An. hinesorum (lanes 8 and 9, QLD; and lane 10, Central Province), An. torresiensis (lanes 11–13), An. koliensis (lanes 14 and 15, Central and Madang Provinces), plus EcoR47III and BlnI digested DNA from An. sp. nr punctulatus (lanes 16–18). Fragment sizes of the flanking DNA ladders are in kilobases

Fig. 3

Secondary structure prediction of a 5′-subrepeat from An. irenicus showing the dual A- and B-type repeats and their ITS2 paralogue (inset) (GenBank accession no. EF042725). Identical nucleotides between the ITS structures are shown in boldface for both ITS1 repeats. Black arrows show the sequence match boundaries to the ITS2 structure. Gray arrows denote the beginning and end of the dual repeat. The downstream ITS1 sequence, or ‘core’ region, is drawn in black as a generalized Y-shaped structure with two long central stems

Fig. 4

Scaled representations of the ITS1 from members of the An. punctulatus group (using the longest clone) alongside the most parsimonious tree of the 18S rDNA (taken from Beebe and Cooper 2002). The 5′-subrepeat and core region are shown for each species (except the two basal taxa). Different shading patterns illustrate the internal repeats and a conserved core region. *Additional part of the translocated ITS2 sequence

Fig. 5

Phylogram showing phylogenetic relationships of the ITS1 (50% majority-rule consensus tree from 500 bootstrap replicates using ML analysis) from a sample of the An. farauti complex. Bootstrap proportions are shown above each node of the phylogram and branch lengths are proportional to the number of changes (see scale bar). Individuals are denoted ‘A’ to ‘F’ after species name, and clones are numbered 1 to 5. The An. farauti populations are denoted R = Rabaul (wherein m = male and f = female), NT = Northern Territory, and CP = Central Province