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Introduction

American trypanosomiasis or Chagas disease is widespread in Latin America from Mexico to Chile and southern Argentina. Although present estimates of 10 to 12 million people infected with the haemoflagellate protozoan species Trypanosoma cruzi represent 6–8 million fewer cases than those reported in the 1980s [1], it remains one of the most serious parasitic diseases of the Americas for its social and economic impact [2]. Although it can also be transmitted by blood transfusion or across the placenta from infected mothers, most human contamination is attributed to insect vectors in poor rural or periurban areas of Central and South America [1].

Chagas disease vectors are haematophagous reduviid (Hemiptera: Heteroptera) insects belonging to the subfamily Triatominae. Species of Triatominae are usually grouped into 17 genera forming five tribes, although other arrangements have been proposed. Of these, Alberproseniini, Bolboderini, Cavernicolini and Rhodniini are considered monophyletic, whereas Triatomini is considered polyphyletic [3]. Among the latter, most of the species (over 70) are included in the genus Triatoma, among which two main clades appear in ribosomal DNA (rDNA) sequence phylogenies, corresponding to species of North and Central America and species of South America separated prior to the closing of the isthmus of Panama about 3 million years ago [4]–[6]. Moreover, Triatoma species are distributed in three main groupings: the Rubrofasciata group (mainly North American and Old World species), the Phyllosoma group (mainly Mesoamerican and Caribbean), and the Infestans group (mainly South American), each including different complexes and subcomplexes in a classification which is progressively updated according to new genetic and morphometric data [7].

A priori, all of the over 130 species currently recognized within Triatominae seem capable of transmitting T. cruzi. Among the species of greatest epidemiological significance as domestic vectors, three belong to the genus Triatoma: T. infestans and T. brasiliensis from South America, and T. dimidiata, distributed in Meso- and Central America from Mexico down to Colombia, Venezuela, Ecuador and northern Peru [3].

Triatoma dimidiata can be found in sylvatic, peridomestic and domestic habitats. Non-domiciliated populations may act as reinfestation sources and become involved in the transmission of the parasite to humans [8],[9]. This species includes morphologically variable populations [10],[11]. A molecular comparison of Triatominae, including many Central American species of the Phyllosoma complex by means of rDNA second internal transcribed spacer (ITS-2) sequences demonstrated an unusual intraspecific sequence variability in a few T. dimidiata populations studied. This study even revealed differences consistent with a specific status for populations from the Yucatan peninsula, Mexico [4]–[6], thus opening a debate. A large number of recent, multidisciplinary studies using RAPD-PCR, genital structures, morphometrics of head characters, and antennal phenotypes have shown that variation within this species seems much greater than previously considered [8], [12]–[16]. Morphometric and cuticular hydrocarbon analyses suggest that a sylvatic population from Lanquin, Guatemala, is undergoing a speciation process [13],[17]. Chromosomal variation and genome size suggest that T. dimidiata may represent a complex of cryptic species (i.e. morphologically indistinguishable, yet reproductively isolated taxa) [18].

The aim of the present work is to analyze the intraspecific variability, haplotype profiling, phylogeography and genetic polymorphism of populations of the species T. dimidiata, to get a new framework able to facilitate the future understanding of the diferring peculiarities of this crucial vector species throughout its broad geographical distribution. This may also help in understanding the related differences in characteristics of Chagas disease transmission and epidemiology, as well as in responses to control initiatives in the countries concerned. After a deep analysis, it was considered that the most convenient approach would be obtained by using an appropriate marker able to furnish significant information about evolutionary trends of variation on which to construct the new baseline. This new baseline should be, whenever possible, of sufficient weight as to allow its conclusions to be reflected at systematic-taxonomic level.

For this purpose, the rDNA was preferred over mitochondrial DNA (mtDNA) because of its mendelian inheritance, evolutionary rates and overall recognized usefulness in systematics in all metazoan organism groups because of including sequences which allow to distinguish between species and between subspecies units. The better fitting of rDNA for molecular systematics has already been emphasized in large reviews on rDNA/mtDNA marker comparisons in insects [19]. Ribosomal DNA includes excellent genetic markers, because (i) the rDNA operon is tandemly repeated and present in sufficiently high quantities among the genome of an individual thus facilitating sequencing procedures; (ii) the different genes and spacers of the rDNA follow a concerted evolution which, with sufficient time, effectively homologizes the many copies of nuclear rDNA within a genome [20]; this gives rise to a uniformity of their sequences within all individuals of a population and becomes extremely useful from an applied point of view, because it is sufficient to obtain the sequence of only one individual to characterize the local population it belongs to, that is, all other individuals of that population will present the same sequence; (iii) the usefulness of rDNA genes and spacers as genetic markers at different evolutionary levels have already been verified on a large number of very different eukaryotic organism groups including insects, and consequently extensive knowledge on the different rDNA fragments is available [21]. rDNA sequence comparisons offer valuable information about the evolutionary events in triatomine lineages and, by deducing the routes of spreading of triatomine populations, they may also shed light on the ability of different species to colonize new areas [5].

Within rDNA, ITS-2 was selected as marker because of its well-known usefulness at species and subspecies levels, including the differentiation of taxa within problematic groups, as is the case of those comprising cryptic or sibling species of other insect groups [22]–[24]. Moreover, the sequences of the ITS-2 have already proved to be a useful tool in the analysis of species, subspecies, hybrids and populations, and for inferring phylogenetic relationships in Triatominae in general [4],[5],[6],[25],[26].

In order to be able to assess the ITS-2 evolutionary processes followed by T. dimidiata populations, the ITS-2 sequences of many members of the Phyllosoma, Rubrofasciata and Infestans groups were obtained and analyzed. For this purpose, a large number of rDNA ITS-2 sequences of Triatoma species from numerous geographic origins in Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil was studied. Thus, the nucleotide divergence limits between taxa within the lineage of the genus Triatoma could be established. The present study on T. dimidiata is the largest interpopulational analysis performed on a triatomine species so far.

Materials and Methods

Triatomine materials

A total of 165 triatomine specimens representing 13 Triatoma species of the Phyllosoma, Rubrofasciata and Infestans groups, among which 137 specimens representing T. dimidiata from 64 different geographic origins, were used for sequencing, genetic variation and phylogenetic analyses (Table 1; Figure 1). The systematic classification recently proposed for the genus Triatoma [7] is used here throughout.

Figure 1. Geographical distribution of the sampling sites furnishing the triatomine materials.

Numbers correspond to sampling sites listed in Table 1. • = Triatoma dimidiata; ▴ = other Triatoma species studied.

doi:10.1371/journal.pntd.0000233.g001

Table 1. Triatoma species and samples studied, including ITS-2 sequence length and AT composition (in percentage).

doi:10.1371/journal.pntd.0000233.t001

Sequencing of rDNA ITS-2

For DNA extraction, one or two legs fixed in ethanol 70% from each specimen were used and processed individually, as previously described [5],[27]. Total DNA was isolated by standard techniques [28] and stored at −20°C until use. The complete ITS-2 fragment was PCR amplified using 4–6 µl of genomic DNA for each 50 µl reaction. Amplifications were generated in a Peltier thermal cycler (MJ Research, Watertown, MA, USA), by 30 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C, preceded by 30 sec at 94°C and followed by 7 min at 72°C. PCR products were purified with Ultra Clean™ PCR Clean-up DNA Purification System (MoBio, Solana Beach, CA, USA) according to the manufacturer's protocol and resuspended in 50 µl of 10 mM TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). Sequencing was performed on both strands by the dideoxy chain-termination method, and with the Taq dye-terminator chemistry kit for ABI 3730 and ABI 3700 capillary system (Perkin Elmer, Foster City, CA, USA), using the same amplification PCR primers [6].

Triatomine haplotype code nomenclature

The haplotype (H) terminology used in the present paper follows the nomenclature for composite haplotyping (CH) recently proposed [25]. Accordingly, ITS-2 haplotypes (H) are noted by numbers (Table 1).

Sequence alignment

Sequences were aligned using CLUSTAL-W version 1.83 [29] and MEGA 3.1 [30], and assembly was made with the Staden Package [31]. The alignment was carried out using the Central, Meso and South American Triatoma species studied together with other species and populations whose sequences are available in GenBank: T. phyllosoma (Accession Number AJ286881), T. pallidipennis (AJ286882), T. longipennis (AJ286883), T. picturata (AJ286884), and T. mazzotti (AJ286885) (Phyllosoma group, Phyllosoma complex); T. barberi (AJ293590) (Rubrofasciata group, Protracta complex) [5],[6]; T. rubrovaria H1 (AJ557258) [32], T. infestans CH1A (AJ576051), and T. sordida (AJ576063) [25]. The ITS-2 sequence of Rhodnius prolixus (Triatominae: Rhodniini) (AJ286882) [6] was used as outgroup.

Data deposition footnote

The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the new ITS-2 rDNA sequences discussed in this paper are: 31 haplotypes of T. dimidiata (AM286693–AM286723), T. bassolssae AM286724, T. bolivari (AM286725), 2 haplotypes of T. hegneri (AM286726, AM286727), T. mexicana (AM286728), 2 haplotypes of T. pallidipennis (AM286729, AM286730), T. ryckmani (AM286731), T. flavida (AM286732), T. gerstaeckeri (AM286734), T. rubida (AM286735), T. nitida (AM286733), T. maculata (AJ582027), and T. arthurneivai (AM286736).

Phylogenetic inference

Phylogenies were inferred by maximum-likelihood (ML) using PAUP*4.0b10 [33] and PHYMLv2.4.4 [34]. Maximum-likelihood parameters and the evolutionary model were determined using the hierarchical Likelihood Ratio Test (hLRTs) and the Akaike Information Criterion (AIC) [35],[36] implemented in Modeltest 3.7 [37] in conjunction with PAUP*4b10. To assess the reliability of the nodes in the ML tree, a bootstrap analysis using 1000 pseudo-replicates was made with PHYML. Since haplotype sequences for T. dimidiata individuals (populations) are quite similar and potentially subject to homoplasy and recombination, alternative procedures to phylogenetic tree reconstruction revealing their relationships were tested. Therefore, a median-joining network analysis [38] was performed using Network version 4.1.1.2 (available from Fluxus Technology Ltd., http://www.fluxus-engineering.com) with the variable positions in the multiple alignment of the different ITS-2 haplotypes from T. dimidiata populations.

Alternative methods of phylogenetic reconstruction allowing an evaluation of the support for each node were also applied. A distance-based phylogeny using the neighbor-joining (NJ) algorithm [39] with the ML pairwise distances was obtained. Statistical support for the nodes was evaluated with 1000 bootstrap replicates, with and without removal of gapped positions. Finally, a Bayesian phylogeny reconstruction procedure was applied to obtain posterior probabilities (BPP) for the nodes in the ML tree. We used the same evolutionary model as above implemented in MrBayes 3.1 [40] with four chains during 1,000,000 generations and trees were sampled every 100 generations. The last 9,000 trees were used to obtain the consensus tree and posterior probabilities.

Genetic variation studies

Genetic variation within and among populations of T. dimidiata was evaluated using DnaSP version 4 [41] and Arlequin 2000 [42]. Summary parameters include those based on the frequency of variants (haplotype number and diversity) as well as some taking genetic differences among variants into account (gene diversity, polymorphic sites). A hierarchical analysis of molecular variance (AMOVA) was performed using Arlequin. This analysis provides estimates of variance components and F-statistics [43] analogs reflecting the correlation of haplotype diversity at different levels of hierarchical subdivision. Unlike other approaches for partitioning genetic variation based on the analysis of variance of gene frequencies, AMOVA takes into account the genetic relatedness between molecular haplotypes. The hierarchical subdivision was made at three levels. At the top level, different groups were defined on the basis of the phylogenetic relationships for the different T. dimidiata haplotypes obtained. The second level corresponded to countries of sampling within each of these groups, and the third level corresponded to the different haplotypes found in each country within group. AMOVA reports components of variance at the three levels under consideration (among groups, among countries within groups, and within countries within groups) as well as F-statistics analogs. Under the present scheme, F_ST is viewed as the correlation of random haplotypes within countries within groups, relative to that of random pairs of haplotypes drawn from the whole species, F_CT as the correlation of random haplotypes within groups, relative to that of random pairs of haplotypes drawn from the whole species, and F_SC as the correlation of the molecular diversity of random haplotypes within countries within groups, relative to that of random pairs of haplotypes drawn from the corresponding group [44]. Although in the program used (only currently available for molecular variance analysis) the choice for establishing an intermediate level is fully arbitrary and has no influence on the final result of the comparison between units at the higher level, these same analyses were repeated by considering each haplotype, which may encompass several individuals, as a separate group for this intermediate level, because it could be argued that geopolitical country borders was not an appropriate choice despite its interest from the point of view of the control of Chagas disease. The statistical significance of fixation indices was tested using a non-parametric permutation approach [44]. Genetic differentiation between pairs of populations was evaluated by means of F-statistics [43]. Exact tests of population differentiation were performed [45]. Slatkin's linearized F_ST's [46],[47] procedure was also followed to obtain estimates of pairwise equilibrium migration rates, both among groups, among countries within groups, and within countries for those cases in which haplotypes from more than one group were present.

Results

Sequence Analyses of Triatoma dimidiata Populations

The 137 ITS-2 sequences revealed the existence of 31 different haplotypes in the T. dimidiata studied (T.dim-H1 to T.dim-H31) (see Tables 1 and 2 for localities and countries). Their length was 489–497 base pairs (bp) (mean, 495.10) with a relative AT-biased nucleotide composition of 75.25–76.85% (75.72%). Sequence similarity analysis of these 31 haplotypes revealed four distinct groupings: grouping 1 (T.dim-H1 to T.dim-H10); grouping 2 (T.dim-H11 to T.dim-H17); grouping 3 (T.dim-H18 to T. dim-H24); and grouping 4 (T. dim-H25 to T. dim-H31) (Figure 2). These four groupings appear linked to concrete wide geographical areas including neighboring countries and regions. The only exception is Providencia Island, which, although part of Colombia, is located 720 km off the northern coast of Colombia but only 240 km off the western coast of Nicaragua. No haplotype presents a very broad geographical distribution.

Figure 2. Interhaplotype sequence differences found in the rDNA ITS-2 of the Triatoma dimidiata populations analyzed.

Numbers (to be read in vertical) refer to positions obtained in the alignments made with CLUSTAL-W 1.8 and MEGA 3.3. . = identical; * = singelton sites (7); • = parsimony informative positions (24); − = insertion/deletion. Rectangled area = microsatellite region. Horizontal lines separate the four major T. dimidiata haplotype groupings according to sequence analyses.

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Table 2. Distribution of Triatoma dimidiata ITS-2 haplotypes (H) per country and locality.

doi:10.1371/journal.pntd.0000233.t002

The alignment of the 31 T. dimidiata haplotype sequences was 501 bp-long, of which 450 characters were constant and 24 were parsimony-informative. The interrupted microsatellite (AT)_4–5 TTT (AT)_5–7 was detected between positions 47 and 73 in all specimens studied. Variability in this microsatellite region and their respective sequence positions are noted in Figure 2.

The 51 nucleotide variable positions detected including gaps represented a 10.18% of polymorphic sites. The seven haplotypes T.dim-H25 to T.dim-H31 are responsible for this high genetic divergence (Figure 2). This genetic divergence decreases considerably when two separate alignments are performed: (i) the first includes T.dim-H1 to T.dim-H24 from all the seven countries shows a divergence of 5.62% in a 498-bp-long alignment, including 28 nucleotide variable positions, of which 6 (1.20%) were transitions (ti), 13 (2.61%) transversions (tv) and 9 (1.81%) insertions/deletions (indels); (ii) the second includes T.dim-H25 to T.dim-H31 from only three countries (Mexico: localities of Yucatan, Chiapas, Cozumel Island and Holbox Island; Guatemala: Peten; Honduras: Yoro Yoro) shows a divergence of 2.42% in a 495-bp-long alignment, with 12 nucleotide variable positions, of which 2 ti (0.40%) and 10 are indels (2.02%).

Sequence Analyses in the Phyllosoma and Rubrofasciata Groups

ITS-2 sequences of T. bassolsae, T. bolivari, T. hegneri, T. mexicana, T. pallidipennis, T. ryckmani, T. flavida, T. nitida, T. gerstaeckeri, and T. rubida, including haplotype length and AT content are listed in Table 1. The comparison analyses which include these ITS-2 sequences and those of the Phyllosoma and Rubrofasciata groups (available in GenBank) provided 48 different haplotypes. Their alignment resulted in a total of 551 characters including gaps, of which 365 sites were constant and 99 parsimony-informative.

All the T. dimidiata haplotypes clearly differed from the Phyllosoma, Flavida, Protacta and Rubrofasciata complex species included in this analysis. Triatoma bassolsae differed in only one deletion in position 489 from T. pallidipennis of Morelos, Mexico (AJ286882). The T. pallidipennis sequence obtained represents a new haplotype (T.pal-H2) differing in only one deletion in position 31 from T. picturata and T. longipennis. The haplotype alignment of T. bassolsae, T. longipennis, T. mazzotti, T. picturata, T. pallidipennis and T. phyllosoma was 490 bp long showing a relatively small genetic diversity of 1.83%, with only 5 mutations (1.02%) and 4 indels (0.81%). The two T. hegneri haplotypes differ between each other in only 1 ti and, when compared with T. dimidiata H18 to H24 from Mexico and Guatemala, nucleotide differences found were only 1 ti and 2 tv.

Sequence Analyses in the Infestans Group

ITS-2 sequences of T. maculata and T arthurneivai, including haplotype length and AT content are listed in Table 1.

The ITS-2 of T. maculata fits very well within sequences of the Infestans complex species studied in the present work, a total of 6–19 (13.7) mutations, namely 6–11 (7.25) ti and 0–10 (6.5) tv, appearing when comparing the five Infestans complex species in question. The material of Triatoma arthurneivai here analyzed is very close to T. rubrovaria H1 (AJ557258), showing only 6 nucleotide differences (1.22%), of which only 1 ti and 5 indels.

Phylogenetic Analyses

Two different phylogenetic approaches were performed with the 31 T. dimidiata haplotypes, both yielding coincident results. A maximum likelihood tree was reconstructed using the best model of evolution as determined by the lowest AIC, which was GTR+I (−Ln = 887.089), being the proportion of invariable sites (I) of 0.166. Three groups appeared with high support values indicating that their differentiation was not due to random sampling of a low variable sequence (tree not shown). The large group 1 encompassed haplotypes from all the countries, whereas groups 2 (Mexico and Guatemala) and 3 (Mexico, Guatemala and Honduras) were more geographically restricted.

Alternatively, a median-joining network was reconstructed with the 31 different T. dimidiata sequences using the variable sites in the multiple alignment (Figure 3). This network showed the same three groups found in the ML tree. Group 1 occupies a central position in the network and is the most widespread and variable group, so that it most likely corresponds to the ancestral or source set. This is further reinforced by the direct relationship between this group and the two others, more geographically restricted and encompassing fewer variants, group 2 including samples from Mexico and Guatemala, and group 3 including samples from these two countries and Honduras. The group 1 source set would in turn be derived from group 3, which might be interpreted as a geographically restricted relict according to the phylogeographic results. Moreover, sequence variants in group 1 are clustered in two different subgroups, with genetic and geographical borders: subgroup 1A includes sequences from Colombian Providencia island, Ecuador, Guatemala, Honduras, Mexico (only South of Chiapas) and Nicaragua; subgroup 1B encompasses sequences from continental Colombia and Panama. The two closest sequences of each subgroup differ in two sites, which might correspond to haplotypes not found in this sampling.

Figure 3. Median network for Triatoma dimidiata haplotypes based on rDNA ITS-2 sequences.

The area of each haplotype is proportional to the total sample. Small black-filled circles represent haplotypes not present in the sample. Mutational steps between haplotypes are represented by a line. More than one mutational step is represented by numbers. H = haplotype. Blue: Colombia; orange: Panama; yellow: Mexico; red: Honduras; lilac: Ecuador; ocher: Nicaragua; green: Guatemala.

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The relevance of the ITS-2 differences among these T. dimidiata groups and subgroups was assessed by comparison with other Triatoma species. Therefore, a multiple, 562-nucleotide-long alignment was obtained by incorporating 22 additional ITS-2 sequences. This set includes 53 ITS-2 sequences of Triatoma species and, using R. prolixus as outgroup, a ML tree was obtained (−Ln = 2648.5129) using the HKY+G model, according to the AIC results with a gamma distribution shape parameter = 0.58. This tree (Figure 4) shows that:

• the 31 T. dimidiata haplotypes appear within a highly supported clade (95/97/100 in ML/NJ/BPP), distributed as follows: a first large subclade, also very well supported (99/97/100), comprising subgroup 1A, subgroup 1B, group 2, and group 3 of the network analysis; subgroup 1A (sequence grouping 1 = T.dim-H1 to T.dim-H10) includes populations from Central America (Honduras, Nicaragua, Guatemala and scattered haplotypes from Mexico, Ecuador and Providence Island); interestingly, the haplotype T.dim-H10 corresponding to phenetically peculiar specimens found in cave-dwellings of Lanquin, Guatemala, appears independent although related to the rest with very high supports; subgroup 1B (sequence grouping 2 = T.dim-H11 to T.dim-H17) comprises populations from continental Colombia and Panama and appears as a monophyletic haplotype cluster; group 2 (sequence grouping 3 = T.dim-H18 to T.dim-H24) shows a well supported branch (91/92/100) and comprises populations from Mexico (Gulf coast, high plains, and Cozumel island) and Guatemala, including the two T. hegneri haplotypes; the second large clade is also highly supported (97/96/100), corresponding to group 3 (sequence grouping 4 = T.dim-H25 to T.dim-H31) and includes populations from the Yucatan peninsula, Holbox and Cozumel islands and northern Chiapas (Mexico), northern Honduras and northern Guatemala;

• T. bassolsae clusters together with T. phyllosoma, T. mazzotti, T. longipennis, T. picturata and T. pallidipennis with very high support (99/91/100 in ML/NJ/BPP) in a sister clade of T. dimidiata; the separated location of the two T. pallidipennis haplotypes indicates the marked similarity of all these taxa;

• T. mexicana and T. gerstaeckeri cluster together in a group basal to both T. dimidiata and T. phyllosoma clades; the extremely high values (100/99/100) supporting the monophyletic clade including T. mexicana, T. gerstaeckeri, T. phyllosoma and close species, and T. dimidiata, are worth emphasizing;

• T. barberi, T. nitida, T. rubida, T. ryckmani and T. bolivari cluster in an unresolved branch, within which only T. ryckmani and T. bolivari appear related with a high support; the insular species T. flavida from Cuba appears as a basal lineage although with insufficient support values;

• finally, the South American species T. rubrovaria, T. arthurneivai, T. sordida, T. maculata and T. infestans cluster together with the highest support.

Figure 4. Phylogenetic ML tree of Triatoma species and haplotypes within the Phyllosoma, Rubrofasciata and Infestans groups.

The scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with ML reconstruction using PhyML with 1000 replicates; values larger than 70%; b: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; values larger than 70%; c: Bayesian posterior probability with ML model using MrBayes; values larger than 90%.

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Triatoma dimidiata groupings appeared well supported, with very high bootstrap proportions (BP>90%) using ML and neighbor-joining reconstruction and the highest Bayesian posterior probabilities (BPP = 100%). Similar levels were found for other well established Triatoma species, many of which showed substantially lower support values in the three statistical measurements employed. However, other species presented no ITS-2 nucleotide differences (T. picturata and T. longipennis; T. mazzotti and T. phyllosoma).

Genetic Variation Analyses

The phylogenetic analyses showed that samples from the same country may belong to different clusters. This result, on its own, is not enough to demonstrate the biological distinctiveness of the corresponding populations. Sampled individuals may represent a minor fraction of the total genetic variability in a highly heterogeneous population and the sampling procedure might have resulted, by pure chance, in the observed clustering of some variants. Given that each of these clusters holds some genetic variability of its own, the first task was to evaluate whether the observed groupings were significantly different from each other, in terms of genetic variation, by partitioning the observed genetic variability at three different levels: among groups, among populations (countries) within groups, and within populations. A hierarchical analysis of molecular variance was used to test the null hypothesis of no genetic differentiation among groups considering variation at lower levels. This procedure was first applied to T. dimidiata sequences using three levels as defined above (Table 3a). Most of the genetic variation found was allocated to the among groups level (80.24% of the total variation), with much lower portions of variation assigned to differences among populations within groups level (11.71%) and within populations level (8.05%), although both were still statistically significant after 1000 pseudo-random samples generated for testing. This indicates that, despite genetic variation within and among populations at these three levels, there is a substantial amount of genetic differentiation among them that justifies their consideration as separate groupings for further analysis. The same results were obtained, notwithstanding small numerical differences due to the different numbers of groups, when haplotypes instead of countries were considered at the intermediate level (Table S1). The geographical fitting represents in fact no surprise at all, taking into account that the distribution of T. dimidiata covers different countries which are more or less aligned following a north-south axis because of the relatively slenderness of the Central American bridge. Hence, as any of the two versions of the analyses conveys the same information and leads to the same conclusions, and which one should be reported is simply a matter of opinion, the first considering countries becomes practically more useful because Chagas disease control measures are organized at national level.

Table 3. Summary of analysis of molecular variance for Triatoma dimidiata populations.

doi:10.1371/journal.pntd.0000233.t003

The median-joining network reconstructed with the 31 different T. dimidiata ITS-2 sequences revealed the existence of three distinct groups (groups 1, 2 and 3), the first of which further subdivided into two subgroups 1A and 1B. The same AMOVA procedure was applied to ascertain whether these two subgroups could be considered as distinct populations or not. The results (Table 3b) indicate that a significant fraction (60.15%) of the total genetic variation corresponds to differences between these two subgroups which, correspondingly, could be considered as separate populations for the ensuing analyses.

Based on the four groups/subgroups previously described in the median-joining network, a summary of relevant population genetic parameters for T. dimidiata is presented in Table 4. Genetic variation in T. dimidiata populations was quite evenly distributed, with similar levels of nucleotide and haplotype diversities in the four groups/subgroups considered. Nevertheless, for all the parameters studied, subgroup 1A presented higher values than the rest, although significance of the differences was only obtained for haplotype diversity. A similar summary is shown for each country sample within groups in Table S2.

Table 4. Summary of population genetic variation parameters from ITS-2 haplotypes in the Triatoma dimidiata populations.

doi:10.1371/journal.pntd.0000233.t004

Different estimates of θ were obtained based on the expected heterozygosity, the expected number of alleles, the number of polymorphic sites and the nucleotide diversity. The four estimates were quite consistent for the four groups/subgroups and they agreed in assigning a larger value to subgroup 1A.

Differences in the genetic composition of the four groups/subgroups 1A, 1B, 2 and 3 have previously been shown to be statistically significant according to analyses of molecular variance. A further evaluation of this distinctiveness was made (Table 3c), in which the four groups/subgroups were considered for the AMOVA, in correspondence with the previous results. In this case, the amount of among-group variation rose to 86.84% of the total variation, whereas among population within groups and within population levels they were substantially lower, 3.21% and 9.95% respectively.

Genetic differences within and among the ITS-2 locus for T. dimidiata samples were further explored through pairwise comparisons, and estimates of average pairwise differences within and among the four groups/subgroups considered were obtained (Table 5). Subgroup 1A presented the largest value for within-group pairwise differences. The within-population values were much lower than among-populations comparisons. Among the latter, the smallest number of differences was found between subgroup 1A and 1B, in correspondence with their close phylogenetic relationship. Subgroup 1B was the one with the lowest overall number of pairwise differences, slightly below 1A. On the contrary, the highest value of pairwise differentiation corresponds to group 3, with almost 20 differences (corrected estimate) when compared with any other group.

Table 5. Population average pairwise differences in Triatoma dimidiata populations.

doi:10.1371/journal.pntd.0000233.t005

Within groups genetic differentiation was evaluated by computation of pairwise F_ST values for populations defined by country of origin (Table S3). Since all groups/subgroups, with the only exception of subgroup 1A, are characterized by one large (n>10) and several small (n<10) populations, significance values for test of genetic differentiation have to be interpreted cautiously. Hence, there is no apparent differentiation between two populations in subgroup 1B (Colombia2, n = 30, and Panama, n = 4) and similarly in group 2 (Mexico2, n = 23, and Guatemala2, n = 4). The only significant value found in group 3 corresponds to Honduras3 (n = 2) and Guatemala3 (n = 7), for which F_ST = 0.529, P<0.05. None of these two populations presented significant differentiation with respect to the largest population in this group, Mexico3 (n = 15). Subgroup 1A includes two large populations, Honduras1 (n = 18) and Guatemala1 (n = 26), which presented a highly significant F_ST = 0.193, P<0.001. Although this value, under the assumption of migration-drift equilibrium, corresponds to an estimate of 2.1 migrants per generation between both populations, which would be enough to prevent their complete differentiation, such estimations shall be verified by using larger samples and markers better suited for population genetics analyses. Comparisons between each of these two populations and the smaller ones in subgroup 1A revealed that Honduras1 differed from Mexico1, Guatemala1 was different from Ecuador and Nicaragua, and none of them differed from the only two individuals from Providencia island. Similar comparisons for all pairs of populations assigned to different groups/subgroups resulted in highly significant F_ST values (Table S4).

Discussion

Triatoma dimidiata, T. sp. aff. dimidiata and T. hegneri

The highest intraspecific ITS-2 variability (absolute nucleotide differences including indels) known in Triatomini members is 2.70% (13/482) in T. infestans specimens collected throughout the very wide geographical distribution of this species [25]. Hence, the result of 10.18% ( = 51/501) detected in T. dimidiata (Figure 2) appears to be pronouncedly outside the limits of the intraspecific variability range known for Triatoma species. Group 3 is the main responsible for such differences (Table 5) and shows a high 2.42% divergence within itself, suggesting an old origin in the light of the relatively reduced geographical area of distribution of these haplotypes in Mexico (Yucatan, Chiapas, Cozumel Island and Holbox Island), Guatemala (Peten) and Honduras (Yoro) only. The time of divergence between group 3 and other T. dimidiata populations was estimated to be of 5.9–10.5 million years ago (Mya) according to a molecular clock analysis based on rDNA evolutionary rates [4].

The divergence of 5.62% shown by the other 24 ITS-2 haplotypes (Figure 2) also appears to be too large, in spite of the wide geographical area they occupy from Mexico down to Ecuador, suggesting a speciation process. However, population average pairwise differences between subgroup 1A, subgroup 1B and group 2 are markedly lower than between these three and group 3 (Table 5), and intragroup differences do fall within the above-mentioned Triatomini range: 2.61% within subgroup 1A, 2.41% within subgroup 1B, and 2.01% within group 2.

Results indicate that several T. dimidiata populations are following different evolutionary divergences in which geographical isolation appears to have had an important influence (Figure 5). A phenotypic consequence of that process had been observed by other specialists before, who wrote about an assemblage of morphologically variable populations [10]. More recently, significant head shape differences between populations showed a separation between northern, intermediate and southern collections of T. dimidiata and also support an evolutionary divergence of populations within this species [13].