Multilocus Analysis of Divergence and Introgression in Sympatric and Allopatric Sibling Species of the <i>Lutzomyia longipalpis</i> Complex in Brazil

<div><p>Background</p><p><i>Lutzomyia longipalpis</i>, the main vector of visceral leishmaniasis in Latin America, is a complex of sibling species. In Brazil, a number of very closely related sibling species have been revealed by the analyses of copulation songs, sex pheromones and molecular markers. However, the level of divergence and gene flow between the sibling species remains unclear. Brazilian populations of this vector can be divided in two main groups: one producing Burst-type songs and the Cembrene-1 pheromone and a second more diverse group producing various Pulse song subtypes and different pheromones.</p><p>Methodology/Principal Findings</p><p>We analyzed 21 nuclear loci in two pairs of Brazilian populations: two sympatric populations from the Sobral locality (1S and 2S) in northeastern Brazil and two allopatric populations from the Lapinha and Pancas localities in southeastern Brazil. Pancas and Sobral 2S are populations of the Burst/Cembrene-1 species while Lapinha and Sobral 1S are two putative incipient species producing the same pheromone and similar Pulse song subtypes. The multilocus analysis strongly suggests the occurrence of gene flow during the divergence between the sibling species, with different levels of introgression between loci. Moreover, this differential introgression is asymmetrical, with estimated gene flow being higher in the direction of the Burst/Cembrene-1 species.</p><p>Conclusions/Significance</p><p>The results indicate that introgressive hybridization has been a crucial phenomenon in shaping the genome of the <i>L. longipalpis</i> complex. This has possible epidemiological implications and is particularly interesting considering the potential for increased introgression caused by man-made environmental changes and the current trend of leishmaniasis urbanization in Brazil.</p></div

Haplotype networks from sympatric and allopatric comparisons of selected loci with extreme <i>F</i>_ST values.

<p>(A) The network of the loci <i>ζcop</i> and <i>rpL17A</i> from the sympatric species of the <i>Lutzomyia longipalpis</i> complex shows mixed a haplotype distribution, unlike the two well-separated cluster for the allopatric species. This is in agreement with the low degree of divergence between sympatric and high divergence between allopatric species. (B) The loci <i>per</i>, <i>sec22</i>, <i>up</i> and <i>para</i> presented sympatric and allopatric networks with haplotypes separated by species group, which also corroborates the high values of pairwise <i>F</i>_ST (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002495#pntd-0002495-t002" target="_blank">Table 2</a>). Each <i>L. longipalpis</i> population is represented by one color: Sobral 1S (orange), Sobral 2S (green), Lapinha (red) and Pancas (light green). Colored circles represent unique haplotypes, and their sizes are proportional to their frequencies. Black circles are hypothetical haplotypes. Curved lines represent alternative branching between haplotypes.</p

Tajima's neutrality test of 21 loci for the four siblings of <i>L. longipalpis</i> from Brazil.

<p>S1S, Sobral 1S; S2S, Sobral 2S; Lap, Lapinha; Pan, Pancas.</p

Differentiation between sympatric and allopatric siblings of <i>L. longipalpis</i> complex from Brazil.

<p><i>F</i>_ST, pairwise fixation index. Significance evaluated with 1000 permutations;</p>***<p>, significant at P<0.001;</p>**<p>, significant at P<0.01;</p>*<p>, significant at P<0.05;</p>ns<p>, non-significant P>0.05.</p><p><i>Ss</i>, shared sites; <i>Sf</i>, fixed sites.</p

Distribution of marginal posterior probabilities of the migration rates at each locus.

<p>Probability densities (P) of migration estimates for 21 nuclear loci for two comparisons, sympatric - Sobral 1S (orange) <i>vs</i>. Sobral 2S (green) - and allopatric - Lapinha (red) <i>vs</i>. Pancas (light green).</p

Estimates of parameters from IM analysis of sympatric and allopatric comparisons.

<p><i>θ</i>, the estimated population mutation rates; <i>t</i>, the estimated number of mutations since the time of splitting; <i>m</i>, the estimated migration rates; N, the estimated effective population sizes; t, the estimated time in years at which the ancestral population split into the sampled populations; 2Nm, the estimated effective number of migrant gene copies per generation in each direction. The 90% lowest and highest posterior density intervals for each parameter are shown in brackets. _S1S, Sobral 1S; _S2S, Sobral 2S; _Pan, Pancas; _Lap, Lapinha; <i>_A</i>, ancestral species.</p

Geographic locations of the Brazilian populations of <i>Lutzomyia longipalpis</i> species complex studied.

<p>The circles show the approximate location of the two sympatric sibling species from Sobral (Ceará State) and the two allopatric sibling species from Pancas (Espírito Santo State) and Lapinha (Minas Gerais State). Divided circle represents the two sympatric species Sobral 2S (green) and Sobral 1S (orange), with Burst and Pulse (subtype 3) types of copulation songs, respectively. Circles in light green and red correspond to the allopatric species Pancas and Lapinha that produce Burst and Pulse (subtype 2) types of copulation songs, respectively.</p

Distributions of marginal posterior probabilities of model parameters of sympatric and allopatric species.

<p>Probability densities (P) are shown in curves, which present single narrow peaks for six parameters of the isolation with migration model: (A) theta (<i>Θ</i> = 4Nμ), (B) divergence time between species (<i>t</i> = tμ) and (C) migration rate (<i>m</i> = m/μ).</p

Neighbor joining tree of <i>Lutzomyia longipalpis</i> populations based on the pairwise <i>F</i>_ST of 21 loci.

<p>The bootstrap value is based on 100 replicates in which the 21 loci were bootstrapped.</p

Correlation between species' <i>F</i>_ST value ranks and the normalized <i>F</i>_ST differences ranks.

<p>(A) Correlation between the ranks of the normalized differences in <i>F</i>_ST values [(<i>F</i>_ST Allopatric−<i>F</i>_ST sympatric)/<i>F</i>_ST allopatric] and the ranks of <i>F</i>_ST values for the sympatric species pair at 21 markers. High significant negative correlation was observed, and the trend line for all data calculated by ordinary least squares regression is shown by a solid line. (B) Correlation between the ranks of the normalized differences in <i>F</i>_ST and the ranks of <i>F</i>_ST values for the allopatric species pair at 21 markers.</p