Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia

Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a possibility with profound implications for the evolution of virulence, host range and drug resistance in these species

Sliding window analysis of nucleotide diversity in the IGS and flanking 16S and 5.8S rDNA regions of <i>N. bombycis</i> (Part A), <i>N. granulosis</i> (Part B) and <i>V. cheracis</i> (Part C).

<p>A sliding window of 50 base pairs is used, with an increment of 25 base pairs. Nucleotide diversity is calculated as the average heterozygosity per site (<i>π</i>) and the average number of nucleotide differences per site (<i>θ_W</i>). Error bars show the standard error for each window. Regions in which sequences could not be aligned due to multiple insertions and deletions are indicated by missing data (breaks in the line).</p

Recombination events detected in alignments of cloned sequences using RDP4.

<p>Only recombination detection methods providing statistically significant support for a given recombination event are listed. P-values are for Bonferroni-corrected multiple comparisons.</p

Sliding window analysis of nucleotide diversity in the ITS and flanking 18S and 16S rDNA regions of <i>N. bombycis</i> (Part A), <i>N. granulosis</i> (Part B) and <i>V. cheracis</i> (Part C).

<p>A sliding window of 50 base pairs is used, with an increment of 25 base pairs. Nucleotide diversity is calculated as the average heterozygosity per site (<i>π</i>) and the average number of nucleotide differences per site (<i>θ_W</i>). Error bars show the standard error for each window.</p

Pairwise comparisons of <i>RPB1</i> sequences between <i>N. bombycis</i> isolates, showing frequency of shared polymorphisms, unique polymorphisms and fixed differences.

<p>Pairwise comparisons of <i>RPB1</i> sequences between <i>N. bombycis</i> isolates, showing frequency of shared polymorphisms, unique polymorphisms and fixed differences.</p

Assignment of <i>Nosema</i> species to life cycle groups based on transmission and sexuality.

<p>Assignment of <i>Nosema</i> species to life cycle groups based on transmission and sexuality.</p

Genetic diversity in cloned sequences from <i>Nosema</i> samples.

<p>Nucleotide diversity is calculated as the average heterozygosity per site (<i>π</i>) and the average number of nucleotide differences per site (<i>θ_W</i>).</p

Haplotype network of cloned <i>EF-1α</i> sequences from <i>N. bombycis</i> isolates.

<p>Branch lengths are proportional to the number of mutations separating haplotypes. Areas of circles are proportional to the number of clones containing each haplotype. Isolates are labelled as follows: E = England, U = USA, C = China.</p

Pairwise comparisons of <i>RPB1</i> sequences between <i>N. granulosis</i> isolates, showing frequency of shared polymorphisms, unique polymorphisms and fixed differences.

<p>Pairwise comparisons of <i>RPB1</i> sequences between <i>N. granulosis</i> isolates, showing frequency of shared polymorphisms, unique polymorphisms and fixed differences.</p

Population structure of ribosomal IGS sequences from <i>N. bombycis</i>, <i>N.granulosis</i> and <i>V. cheracis</i> isolates.

<p>Comparisons were made using Wright’s index of fixation (<i>F_ST</i>) and net nucleotide substitutions per isolate (<i>D_a</i>). Levels of significance: *0.05, **0.01, ***0.001. Isolates are labelled as follows: A = <i>V. cheracis</i> (Australia), E = <i>N. bombycis</i> (England), U = <i>N. bombycis</i> (USA), C = <i>N. bombycis</i> (China), W = <i>N. granulosis</i> (Wales), Sc = <i>N. granulosis</i> (Scotland), Sw = <i>N. granulosis</i> (Sweden).</p