Graphical representation of the distributions of the <i>Pvama1</i> and <i>Pfama1</i> allele frequencies from the Venezuelan Amazon.

<p>A. Distribution of 18 <i>Pvama1</i> haplotypes across surveys in 1996 and 1997. Sequences from the additional village sampled in 1997 (Koshiro) are presented separately from those of the villages sampled in both surveys. B. Haplotype frequency distribution for <i>Pfama1</i> and <i>Pvama1</i> from the 1996 survey only.</p

Differential staining of gametocytes during gametocytogenesis.

<p>Pfg377 and α-tubulin II double-stained IFAT slides prepared on: A) day 2 (stage II); B) day 5 (stage III); C) day 7 (stage IV); D) day 11 (stage V); E) after gametocyte activation (AG). Parasites were magnified ×1000. Early stage parasites were more likely to be damaged during the purification process, and appear as stained with DAPI alone (panels A, B).</p

‘Apparent’ sex ratio during gametocytogenesis.

<p>IFAT counts were performed on gametocyte preparations of stage II, stage III, stage IV, stage V, and 30 minutes after induction of activation (AG). Standard error is estimated for the ratio based on error calculated for the mean estimates of the proportion of males across the three independent cultures analysed.</p><p>% Alpha-tub. II+, % Pfg377+: indicate the percentage of gametocytes reacting with the given α-tubulin II and Pfg377 antibodies respectively.</p

Sex ratios established using light microscopy.

<p>Purified stage V gametocytes stained with Giemsa. A.) 3D7 magnet-purified gametocytes magnified ×1000. B.) (Detail) The cytoplasm of male gametocytes (MG) can be seen to stain pink and that of female gametocytes (FG) to stain purple; male gametocytes are smaller than females, the nucleus is bigger in males than in females, the granules of the malaria pigment are centrally located in female gametocytes and more widely scattered in males gametocytes The other 4 discriminatory characters can also be discerned.</p

Stage III and later gametocytes visualized with anti-Pfg377 and anti-α-tubulin II antibodies.

<p>Fluorescent staining of α-tubuIin (red) generated a characteristic striated pattern, particularly in earlier gametocytes. Expression of Pfg377 (green) was not seen prior to stage III. A) stage III; B) late stage III; C) stage IV; D) late stage IV; E) stage V; F) activated female gametocyte. Nuclear material was stained with DAPI, appearing blue in colour. Parasites were magnified ×1000.</p

Linkage disequilibrium indices of <i>Pvama</i>1 and <i>Pfama</i>1.

<p>Linkage disequilibrium plots of D' and R² for <i>Pvama1</i> (Graphs A and B) and <i>Pfama1</i> (Graphs C and D). Sites with significant linkage (P<0.05) are shown as solid circles; non-significant sites are shown as for <i>Pvama1</i> only. No non-significant sites for <i>Pfama1</i> were found.</p

Within-population analyses of <i>Pvama</i>1 and <i>Pfama</i>1 from the Venezuelan Amazon.

<p>^* = 0.10>P>0.05; ^** = P>0.10; ^*** = P<0.02.</p>†<p> = Effective population size, <i>Ne</i>, = C/4<i>r</i>, where <i>r</i> is the recombination rate and <i>r</i> of <i>P. falciparum</i> is used for all, approximately 6×10⁻⁷, as the recombination rate for <i>P. vivax</i> has not yet been determined (Joy <i>et al.</i>, 2006).</p

Geographical differences in <i>Pvama1</i> and <i>Pfama1</i> sequences across all domains.

<p>Pairwise F_ST values between Venezuelan sequences and other sites are highlighted. Sample sizes are in parenthesis. ¹ = all Venezuela <i>Pvama1</i>data from both 1996 and 1997; ² = all Venezuela <i>Pfama1</i> data from 1996 only; ² = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Gunasekera1" target="_blank">[15]</a>; ³ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Rajesh1" target="_blank">[35]</a> one sequence was ignored in the whole gene and DI analysis as it was not full length; ⁴ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Polley2" target="_blank">[19]</a>; ⁵ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Polley1" target="_blank">[18]</a>. Significant difference (P<0.05) indicated by bold text. Slightly negative numbers, indicated by italics, are biologically meaningless and are equivalent to F_ST = 0.000.</p

McDonald-Kreitman analyses of <i>Pvama</i>1 and <i>Pfama</i>1.

1<p> = Neutrality index indicates the extent to which the levels of amino acid polymorphism depart from the expected in the neutral model.</p>2<p> = Fisher's exact test.</p><p>*0.001</p

Sliding window plots of Tajima's D and Fu and Li's F* for <i>Pvama</i>1 and <i>Pfama</i>1.

<p>A and B – <i>Pvama1</i>; C and D – <i>Pfama1</i>. Midpoints where the sequence significantly departs from zero (P<0.05) are shown as solid points. Domains, DI, DII, DIII, are marked.</p