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

CSIRO, Long Pocket Laboratories Indooroopilly QLD 4068 Australia
Journal of Molecular Evolution (Impact Factor: 1.68). 12/2008; 68(1):66-79. DOI: 10.1007/s00239-008-9188-z


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 8.0kb. 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.

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Available from: Nigel W Beebe, Mar 11, 2015
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    • "In the genus Anopheles the ITS2 is characterized by a very low level of polymorphism between species, even between cryptic species [24]. Despite reports of intra-specific and intra-individual variation [21], ITS2 is still widely used to infer phylogenetic relations among species [30,31]. In the Barbirostris Group, the presence of repeats did not affect tree topology, and this was consistent with results from COI analysis (10). "
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    ABSTRACT: The Anopheles barbirostris group is widely distributed in Southeast Asia. Although seven species have been formally described, a molecular analysis of the rDNA ITS2 and the mitochondrial cytochrome oxidase I gene suggests that the group includes species that are morphologically very similar or identical.We have previously shown that species in the Anopheles barbirostris Subgroup have an exceptionally large ITS2 (>1.5 kb), greater than in any other Anopheline group. However, the molecular processes responsible for generating such a large ITS2 have not previously been explored. To determine the processes by which this large ITS2 is generated, we examined the sequence and secondary structure of the ITS2 of 51 specimens from five species of the Anopheles barbirostris Subgroup. These include the anthropophilic species An. campestris and three morphospecies of the Barbirostris Complex: An. vanderwulpi, An. barbirostris I and III, together with a previously undescribed member of this group (Clade IV).Results and conclusions: All the specimens were found to have an ITS2 greater than 1.5 kb in length. The possibility that the spacer sequences amplified were pseudogenes was examined and discarded. The large size of ITS2 in the species studied is due to the presence of internal repeats of approximately 110 bp in length, confined to the central region of the spacer. Repeats varied markedly between the species examined, with respect to their organization, number and sequence similarity. The nucleotide diversity increased in direct relation to size variation and the presence of non-repeated elements.A secondary structure analysis showed that the repeats form hairpin structures with a wide range of free energy values. These hairpin structures are known to facilitate the subsequent processing of mature rRNA. An analysis of the repeats from the different species suggests they originate from a common ancestor, with the repeats appearing before speciation of the Barbirostris Group.
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    • "The division of populations into arrangements (1), (2) and (3) is marked by slashes *P<0.01, significant percentage values a High values but nonsignificant possibly due to low df species, such as fagaceans (Mayol and Rosselló 2001), pinaceans (Wei et al. 2003; Wei and Wang 2004), boxes (Roselló et al. 2007), mosquitoes (Fairley et al. 2005; Bower et al. 2009), lice (Leo and Barker 2002), etc. In cestodes, intragenomic ITS sequence variation has been detected in the cyclophyllideans Echinococcus spp. "
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    ABSTRACT: Sequence structure of complete internal transcribed spacer 1 and 2 (ITS1 and ITS2) of the ribosomal DNA region and partial mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences were studied in the monozoic tapeworm Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965) (Cestoda: Caryophyllidea), a parasite of common carp (Cyprinus carpio carpio L.). Intraindividual sequence diversity was observed in both ribosomal spacers. In ITS1, a total number of 19 recombinant clones yielded eight different sequence types (pairwise sequence identity, 99.7-100%) which, however, did not resemble the structure typical for divergent intragenomic ITS copies (paralogues). Polymorphism was displayed by several single nucleotide mutations present exclusively in single clones, but variation in the number of short repetitive motifs was not observed. In ITS2, a total of 21 recombinant clones yielded ten different sequence types (pairwise sequence identity, 97.5-100%). They were mostly characterized by a varying number of (TCGT)(n) repeats resulting in assortment of ITS2 sequences into two sequence variants, which reflected the structure specific for ITS paralogues. The third DNA region analysed, mitochondrial cox1 gene (669 bp) was detected to be 100% identical in all studied A. sagittatus individuals. Comparison of molecular data on A. sagittatus with those on Atractolytocestus huronensis Anthony, 1958, an invasive parasite of common carp, has shown that interspecific differences significantly exceeded intraspecific variation in both ribosomal spacers (81.4-82.5% in ITS1, 74.4-75.2% in ITS2) as well as in mitochondrial cox1, which confirms validity of both congeneric tapeworms parasitic in the same fish host.
    Full-text · Article · Oct 2011 · Parasitology Research
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    • "The ITS2 PCR-RFLP patterns were better defined and more clearly distinguishable among species than those for the ITS1. Also, various studies have found greater levels of sequence and length variation in the ITS1 compared with the ITS2 (Fairley et al., 2005; Bower et al., 2008; 2009). The Zapata et al. (2007) assay was subsequently optimized to include other species of the Oswaldoi Group reported in the country (Cienfuegos et al., 2008). "
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    ABSTRACT: Anopheles mosquitoes are routinely identified using morphological characters of the female that often lead to misidentification due to interspecies similarity and intraspecies variability. The aim of this work was to evaluate the applicability of a previously developed PCR-RFLP-ITS2 assay for accurate discrimination of anophelines in twelve localities spanning three Colombian malaria epidemiological regions: Atlantic Coast, Pacific Coast, and Uraba-Bajo Cauca-Alto Sinu region. The evaluation of the stability of the PCR-RFLP patterns is required since variability of the ITS2 has been documented and may produce discrepancies in the patterns previously reported. The assay was used to evaluate species assignation of 939 mosquitoes identified by morphology. Strong agreement between the morphological and molecular identification was found for species Anopheles albimanus, Anopheles aquasalis, Anopheles darlingi and Anopheles triannulatus s.l. (p≥0.05, kappa=1). However, disagreement was found for species Anopheles nuneztovari s.l., Anopheles neomaculipalpus, Anopheles apicimacula and Anopheles punctimacula (p≤0.05; kappa ranging from 0.33 to 0.80). The ITS2-PCR-RFLP assay proved valuable for discriminating anopheline species of northern and western Colombia, especially those with overlapping morphology in the Oswaldoi Group.
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