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The nucleotide sequences of the cytochrome B gene and the antennal phenotypes were analyzed for the following triatomine species: Triatoma longipennis, Triatoma pallidipennis, and Triatoma picturata, which belong to the Phyllosoma complex. These species inhabit sympatric areas from Talpa de Allende, Autlan de Navarro, and Teocuitatlan de Corona in Jalisco, Mexico. Molecular marker analysis showed that the sympatric individuals are the natural crossbred descendents of different individuals living in close proximity in these natural areas that resulted in mixed populations. The antennal phenotype results are coincident with these genetic findings, which point to the high similitude between all Phyllosoma complex populations analyzed. These data support the hypothesis that these species are morphotypes with chromatic and genetic varieties, which preserves the possibility of natural breeding with fertile descent. In conclusion, our results strongly support the hypothesis that T. pallidipennis, T. longipennis, and T. picturata are subspecies of the Phyllosoma complex.
The Triatominae (Insect: Hemiptera: Reduviidae) are the vectors of the protozoan parasite Trypanosoma cruzi that is responsible for Chagas disease or American trypanosomiasis, which is a disease that affects between 10 and 18 million people on the American Continent.1,2 Approximately 137 triatomine species have been reported worldwide. More than 33 species have been identified in Mexico, and many of them are endemic.3–5 Among the endemic species present in Mexico are those that belong to the Phyllosoma complex: Triatoma pallidipennis, Triatoma longipennis, Triatoma bassolsae, Triatoma phyllosoma, Triatoma mazzottii, Triatoma mexicana, and Triatoma picturata.6 This complex is principally distributed in the center and southern part of the country. Because of their parasitic infection and colonization indexes, these vectors are of epidemiologic importance. Variable percentages of T. cruzi infection have been reported, ranging from 7.4% to 52% in T. picturata; 14% to 88% in T. pallidipennis; and 18% to 55% in T. longipennis.7–12
Originally, the members of this complex had been considered subspecies of the T. phyllosoma species.13 Hybrid forms have been observed in laboratory crossbreeding experiments between some of the species that integrate this complex.14,15 Nevertheless, Lent and Wygodzinsky,16 using qualitative morphologic characters, classified them as a defined species apparently because the typical specimen of every species could be recognized.
The proposal of the subspecies classification of the members of the Phyllosoma complex has again been considered because after their molecular analysis a high phylogenetic similarity was shown to exist between the species that integrate the complex. For example, genetic proximity and little variation between the species T. longipennis and T. picturata or T. pallidipennis and T. bassolsae were found using Cyt B and ITS-2 markers (Kimura distances between 0.002 and 0.017 in the case of ITS-2 and 0.034 and 0.172 in the case of the Cyt B).17 Furthermore, the observations that the Phyllosoma complex species are included in compact clades suggest the subspecies status with the existence of morphologic varieties within one species.6,18,19
Species recognition traditionally follows the morphologic qualitative characteristics. Nevertheless, the phenotypic plasticity of the triatomines and the similarity of the nymphal state limit the appropriate recognition of the species and their interbreeding descendants.20 More than 25 years ago, Lent and Wygodzinsky16 pointed out the need to develop intensive collection activity over the entire Phyllosoma complex distribution area with rigorously planned and executed rearing and crossbreeding experiments to provide the definitive answer regarding the taxonomic rank of the group components. Zárate and Zárate3 agreed and pointed out the need to develop an exhaustive collection of the complex in the wild to establish continuity between currently disjointed populations and to detect those areas with overlapping distributions.
Even though several researchers have sampled overlapping areas, little recent information about individuals with mixed characteristics is available.8,10,21,22 Apart from Mazzotti and others,14 there have been only three observations of the presence of individuals who present morphologic characteristics of two species; these were located in sympatric areas for some species of the Phyllosoma complex.15,23,24 In the later work, Martínez-Ibarra and others conducted laboratory hybridization studies between T. longipennis and T. picturata and found individuals with fertile offspring, showing a low reproductive isolation between them.
The use of molecular and morphologic markers has generated important information about the phylogenetic and genealogical relationships between species of triatomine that present taxonomic problems. In the case of molecular markers, the Cyt B sequences of triatomine species present in Mexico have been analyzed, separating the members of the Phyllosoma complex from other species and defining the phylogenetic relationships of this and other complexes.17
The analysis of the antennal phenotype is a powerful quantitative tool for Triatominae study, allowing characterization of genera, species, and populations.25 Catalá and others26 showed high similarities among the antennal phenotypes of species of the Phyllosoma complex: T. pallidipennis, T. phyllosoma, and T. longipennis with a better differentiation of T. pallidipennis from Morelos.
This work analyzed individuals of the Phyllosoma complex collected in sympatric areas and determined possible crossbreeding using Cyt B sequence analysis. Additionally, the antennal phenotype of these individuals was also analyzed. Natural crossbreeding between sympatric species of the Phyllosoma complex with fertile progeny would be a determining factor to understand the taxonomic and phylogenetics problems of one of the more important triatomine complexes in Mexico.
The study took place in three areas of Mexico: Talpa de Allende (20°22′ N, 104°54′ W) at an altitude of 1,134 meters above sea level (MASL), Autlán de Navarro (19° 46′ N, 104° 22′ W) at an altitude of 900 MASL, and finally the Teocuitatlán de Corona (20° 1′ N, 103° 1′ W) at an altitude of 1,375 MASL. All these localities belong to the Jalisco State in the central west part of Mexico (Figure 1) where T. pallidipennis, T. picturata, and T. longipennis species have been reported.8,11,12,27 The average annual temperature ranges from 21.3 to 23.5°C and average annual precipitation ranges from 719.8 to 1,029.7 mm.
The triatomine used in this study were adult females, collected according to Martínez-Ibarra and others.24 All specimens were identified morphologically as T. longipennis, T. pallidipennis, and T. picturata with the Lent and Wygodzinsky16 key. Triatoma picturata have an overall dark brown corium with one large, subtriangular basal coloration, and yellow or orange-red subapical coloration with short, slightly decumbent setae, and are not more than 0.2 mm long. They have pronotum with an extensively orange-yellow posterior lobe and a yellowish connexivum with a subrectangular black spot (Figure 2A). Triatoma pallidipennis have a mostly yellowish white corium and an extensively black connexivum with subrectangular orange spots (Figure 2B). Triatoma longipennis have a largely black corium, a black connexivun with yellow or orange-red markings basally and subapically with short, slightly decumbent setae, and are not more than 0.3 mm long. They have pronotum with an entirely black posterior lobe (Figure 2C). Other female specimens presented morphological characteristics with an intermediate phenotype between T. picturata and T. pallidipennis: a largely yellowish white corium, pronotum with extensively orange-yellow rounded humeral angles, a length of more than 25 mm, strongly widened abdomen, abundant pilosity, the first antennal segment attaining or surpassing clypeus apex, and spongy fossulae absent in both sexes. These specimens were designated Triatoma sp. (Figure 2D). They were similar to those named “hybrids” described by Mazzotti and Osorio14 and Martinez-Ibarra and others.15,24 Other individuals of the species T. longipennis, T. pallidipennis, and T. picturata were collected in areas where only one species has been reported. Triatoma phyllosoma (Phyllosoma complex), Triatoma dimidiata, and Triatoma barberi were also included as outgroups in the phylogenetic analysis. They were a denominated-type species and showed the characteristics reported by Lent and Wygodzinsky.16 The origin and number of the analyzed individuals are described in Table 1. All of these insects were identified by the same specialist (Dr. Martínez-Ibarra).
Genomic DNA was extracted from one leg of each insect. The extraction was carried out according to Martínez and others.17 The oligonucleotides used for the amplification of Cyt B sequences were previously described.17 Approximately 200 ng of genomic DNA was amplified by polymerase chain reaction (PCR).
The fragments obtained by amplification were subcloned in the cloning vector pCR 2.1 (Invitrogen) and sequenced using the vector primers (T7 promoter and M13 reverse). Sequencing was performed for both chains in the ABI prism sequencer (Model Perkin-Elmer 310).
In addition to the 28 sequences amplified in this work, several accessed Cyt B sequences were analyzed (see paragraph below). Multiple alignments were performed with the CLUSTAL W program, version 1.8.28 The Kimura two parameters distances were calculated using MEGA program, version 1.02.29,30 The program Modeltest 3.731 was used to determine the appropriate molecular evolution model. The general time-reversible model with gamma distribution and invariant sites (GTR + G + I)30 was used for Cyt B sequences. The phylogenetic reconstruction using Bayesian inference was performed with the MrBayes 3.1.2 program.32–34 The analysis was run for 10 million generations, sampling trees every 100 generations. The Cyt B data were treated as three separate partitions based on codon positions. Trees with scores lower than those at stationery (burn-in) were discarded from the analysis. The sampled trees that reached the stationary phase were collected and used to build majority consensus trees. The Cyt B sequences used are shown in Table 1.
Antennae were excised from each specimen at the stem level, treated with 4% KOH, and neutralized with 5% acetic acid. The individual preparations were mounted on microscope slides with glycerin to 50% (diluted with phosphate buffered saline [PBS]) and observed with a light microscope at 400×. The sensilla on the ventral side of the three distal segments were drawn using a drawing chamber. Each type of sensillum was counted separately. The receptors were classified in accordance with Catalá and Schofield35 and counted along the pedicel (P) and flagellum segments (F1 and F2). Four types of receptors were used for this analysis: Bristles (BR, a mechanoreceptor), Basiconica (BA), thick-walled trichoid (TK), and thin-walled trichoid (TH) (chemo receptors with different functions).35
Fifty-one individuals with 12 variables were analyzed. Averages and standard deviations of sensilla number were calculated by type and antennal segment. The data set were used to produce multivariate analysis: Principal components (PCA), cluster analysis, and discriminant analysis (DA) using STATISTICA version 8 (Statsoft).25,26,35 For comparative purposes, the antennal data from 7 T. dimidiata females from Veracruz (Mexico) were added. These data belong to the ECLAT database for antennal phenotypes in CRILAR-CONICET, Argentina. The data set of sympatric individuals was analyzed using PADWIN version 6 (http://www.mpl.ird.fr/morphometrics) to estimate functions that identify the studied groups.
Twenty-eight individuals were sequenced for Cyt B, and all data were deposited in GenBank (Table 1). The size of the amplified Cyt B gene fragment was 313 bp with a number of variables (44%) and informative sites (38.7%) and an average A + T nucleotide composition percentage of 64%.
The proteins were determined for each nucleotide sequence according to the specific mitochondrial invertebrate code. No deletions, no insertions, and no stop codons were present in this amino acid sequence. Analysis based on the third codon position and use of the preferential codon supported the fact that the sequences represent mitochondrial DNA (mtDNA) and not nuclear pseudogenes.
The genetic distances were calculated according to Kimura 2 parameters. Species belonging to the Phyllosoma complex showed small distances between 0 and 0.16 (Table 2). No differences (genetic distance = 0) were observed among the following individuals: EU790619/EU790626/EU790632/EU790636/EU790637 (T. pallidipennis/T. picturata/T. pallidipennis/Triatoma sp./T. sp.), EU790624/EU790628/EU790633/EU790634/EU790635 (T. longipennis/T. sp./T. sp./T. sp./T. sp.), EU790616/EU790618/EU790627/EU790638 (T. longipennis/T. longipennis/T. picturata/T. sp.), and EU790617/EU790622 (T. longipennis/T. pallidipennis) (Table 2). The sequence DQ198817 taken from the GenBank, identified as T. picturata by Pfeiles and others,36 showed a close genetic distance with T. pallidipennis (AY859419, AF045724, and DQ198814), suggesting a mistake in its classification or the possibility that this sequence belongs to an individual result of natural crossbreeding.
For the phylogenetic analysis, the Bayesian and the Parsimony methods (data not shown) were used, both with similar topology and support. Sequences of the species denominated type (see Materials and Methods, specimens previously reported in the GenBank with genetic and morphologic analysis)6,17 were used as controls for these analyses. These were species of the Phyllosoma complex present in localities in Mexico without sympatry with other species (Figure 3).
The phylogenetic trees of the sympatric species showed genetic inconsistencies. Specimens morphologically classified as T. pallidipennis (individuals with keys EU790619, EU790621, EU790631, and EU790632) and T. longipennis (EU790624) were grouped with type specimens of T. picturata, which are represented by the AY859413 sequences. This clade also grouped the majority of the individuals with mixed characteristics from both species as Triatoma sp. (individuals EU790628, EU790633, EU790634, EU790635, EU790636, and EU790637). In the clade of the type species T. pallidipennis/T. bassolsae, represented by the individuals AY859419, AY859420, AF045724, DQ198814, and AY859410, individuals morphologically classified T. longipennis (EU790616, EU790617, EU790618, EU790625, and EU790640), T. picturata (EU790627 and DQ198817), and T. sp. (EU790638) were also found. The specimen EU790630 morphologically designated as T. sp. was localized in the T. phyllosoma clade. Individual EU790629, morphologically classified as T. pallidipennis, was found in the T. mazzottii clade. Finally, individual EU790620 did not have clear concordance with any clade (Figure 3). These data indicated the possible process of crossbreeding between species of the Phyllosoma complex living in sympatry. The majority of the branches showed above 70% posterior probability values.
To study some morphological parameters of these sympatric individuals, antennal analysis was used. The four mentioned types of sensilla (BR, BA, TK, and TH) are present in the three segments of the antenna (pedicel and Flagellum 1 and 2). Table 3 shows means and standard deviations for all sensilla types in the studied populations.
Considering that specimens used in this work were preliminarily identified by traditional (qualitative) morphologic characteristics, we decided to check the antennal phenotype of the three species not found in sympatry: T. pallidipennis from Morellos, T. picturata from Nayarit, and T. longipennis from Teocuitatlan de Corona. Triatoma dimidiata from Veracruz was added to the analysis as an outer group. First, a principal PCA was carried out. The PCA compares the individuals by similitude without previous grouping. Figure 4 shows that the first function explains 71% of the variance and the second function explains 19%, which adds up to 90% of the variance. Two major groups were clearly identified: 1) T. dimidiata specimens (dmVe; Figure 4) and 2) the Phyllosoma complex (paMo, piNa, and loTe; Figure 4). However, within this last group T. pallidipennis is better separated, whereas T. longipennis and T. picturata showed mixed phenotypes.
Because the numbers of specimens for each group were insufficient for a DA with the 12 antennal variables, we used the first three factors of the PCA as variables. The analysis was significant for the two first discriminant functions (P < 0.001, F = 16.05, Wilks 0.038). All T. dimidiata (100%), 80% of T. pallidipennis, 72% of T. picturata, and 62% of T. longipennis were correctly classified. The specimens for T. picturata and T. longipennis shared almost the same phenotype and did not differ significantly. These results indicate low differentiation within the populations (“species”) not in sympatry and prevent new analysis using them as true species.
The PCA for the populations (“species”) living in sympatry in Jalisco (Autlan de Navarro and Talpa de Allende) was carried out. Only T. dimidiata (the outer group) was well differentiated; the Phyllosoma complex populations living in sympatry showed distribution with no significant Mahalanobis distances (data not show).
The DA using the two first components indicated that only 33–50% of specimens from the Phyllosoma complex obtain a correct classification as different phenotypes. A tree cluster with Euclidean distances (complete linkage) using two first principal components illustrates very well the position of all individuals from populations living in sympatry (Figure 5). Triatoma dimidiata specimens congregate on a first branch and all Phyllosoma complex specimens appear mixed.
The DA carried out with the two principal components for all populations together (living or not in sympatry) produces similar results. The distances between the populations were not significant with the exception of the population of T. pallidipennis from Morelos State (data not shown).
In this study, taxonomic analysis of sympatric populations of the Phyllosoma complex species from Talpa de Allende, Autlan de Navarro, and Teocuitatlan de Corona in the state of Jalisco, Mexico were performed, allowing a better understanding of the taxonomic and phylogenetic relationships between these important North American triatomines.
The collected individuals in the present study are an example of the morphological complexity of the Phyllosoma complex and show the genetic and morphologic classification problems that several studies have reported. For example, some species such as Triatoma brailovsky, Triatoma bolivari, T. dimidiata, Triatoma hegnery, T. mexicana, Triatoma gerstaeckeri, Triatoma sanguisuga, Triatoma ryckmani, and Triatoma recurva have been tentatively included in this complex. Nevertheless, these data remain controversial, and more studies are needed to clarify their status.13,16,17
The results presented in this work agree with previous molecular analyses where small genetic distances with the Cyt B were found. Additionally, microsatellite data in internal transcribed spacer 2 (ITS 2) showed the same microsatellite sequence (AT5 TTT AT6), which indicates that the Phyllosoma complex species have low interspecific variation as a result of a recent divergences.17,18
Using Cyt B gene sequences, it was possible to genetically identify introgressions related to crossbreeding in each specimen, regardless of the fact that these species show morphologic differences, thereby showing the importance of this marker. These introgressions suggest closely related species or subspecies of recent origins that could promote adaptive evolution and speciation, which is in contrast with production of sterile descendent, that bring to an evolutionary dead-end.37
The phylogenetic tree constructed with the sequences of the Cyt B clearly reveals the introgression process, which means the organisms have a morphologic feature not corresponding exactly with their genetics status. Their wrong position in the tree correlates with possible mistakes in the morphological identification, which could be caused by the extensive numbers of phenotypes that resulted from natural crossbreeding. For example, in the Phyllosoma complex the DQ198817 sequence taken from GenBank, identified as T. picturata, showed genetic distance near T. pallidipennis (AY859419, AF045724, and DQ198814). In the case of Triatoma brasiliensis, 11 different phenotypes were identified in hybrid zones.38
The antennal phenotype analysis showed that these quantitative morphological characteristics are closely linked to adaptive genetic characteristics. Even when comparing populations not living in sympatry, the specimens show similar antennal phenotypes, which indicates close relationships. In coincidence with Catalá and others,26 only T. pallidipennis from Morelos exhibits a higher morphological distance and could be identified as a separated population. All other populations, living or not in sympatry, do not show morphological distances usually found between species. These results confirm that the chromatic and qualitative characters that are traditionally used are not appropriate to identify populations from this complex.
The analysis with the Cyt B marker and the antennal phenotypes has been shown to be a reliable tool for species, genera, and population classification. In this study, these markers showed that the sympatric individuals in the localities in the state of Jalisco are mixed populations and are probably products of crossbreeding between different individuals living in close proximity in this natural area, suggesting that the Phyllosoma complex species are morphotypes with chromatic and genetics varieties, which preserve the possibility of natural breeding and probable fertile descent.14 Importantly, this possibility is also supported by laboratory crossbreeding experiments with the same species used in this work. A high degree of crossbreeding with fertile offspring was found between T. longipennis females and T. picturata males.15 Our study suggests that natural crossbreeding played a crucial role in the origin and diversification of wild triatomine species as has been suggested for T. brasiliensis.39
It will be important to carefully analyze the collected individuals from the state of Jalisco and from other sympatric areas in Mexico where there are individuals of the Phyllosoma complex because these populations might be the result of crossbreeding, and it will be difficult to classify them with traditional morphological criteria. This study strongly suggests the need for quantitative morphologic and genetic analysis before classifying these individuals.
The consequences of the existence of individuals with mixed characteristics could be of epidemiological importance because defecation patterns, susceptibility to the T. cruzi infection, domiciliation, and nutritional preferences could be different to those of the parental individuals.40 It is not known if these individuals are more capable to resist ambiental stress and better adapted to their habitat. Studies that focus on these aspects are currently being conducted in our laboratories.
In conclusion, our results strongly support the hypothesis that T. pallidipennis, T. longipennis, and T. picturata are subspecies of the Phyllosoma complex. The same is true for genetic studies of T. mazzottii, T. phyllosoma, and T. bassolsae,17 but in this case, natural hybrid must be found and experimental studies that confirm the crossbreed should be made. On the basis of these results, we propose the modification of the taxonomic status of these species to subspecies and suggest that they are named T. phyllosoma pallidipennis, T. phyllosoma longipennis, T. phyllosoma picturata, T. phyllosoma mazzottii, T. phyllosoma phyllosoma, and T. phyllosoma bassolsae.
Financial support: This work was support by grant number IN 212806 from PAPIIT-DGAPA Universidad Nacional Autonoma de Mexico. Fernando Martínez-Hernandez would like to acknowledge Consejo Nacional de Ciencia y Tecnologia for the scholarship during his Ph.D. studies and the Programa de Apoyo a los Estudiantes de Posgrado from Universidad Nacional Autonoma de Mexico for the economical support to visit the Centro Regional de Investigacion Cientifica y Transferencia Tecnologica (CRILAR), Anillaco, La Rioja, Argentina.
Authors' addresses: Fernando Martínez-Hernandez, Guiehdani Villalobos, Patricia de la Torre, Juan P. Laclette, and Bertha Espinoza, Departamento de Immunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Distrito Federal, Mexico, E-mails: xm.moc.oohay@zyxrehf, xm.moc.oohay@adheiug, xm.manu.rodivres@errotldp, xm.manu.rodivres@ettelcal, and xm.manu.sacidemoib@ugseb. Jose A. Martínez-Ibarra, Centro Universitario del Sur, Universidad de Guadalajara, Ciudad Guzmán, Jalisco, México, E-mail: xm.gdu.rusuc@arrabia. Silvia Catalá, Centro Regional de Investigación Científica y Transferencia Tecnológica (CRILAR), Anillaco, La Rioja, Argentina and Departamento de Parasitología, Anillaco - La Rioja, Argentina, E-mail: ra.moc.ralirc@alatacs. Ricardo Alejandre-Aguilar, Departamento de Parasitología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, D.F., Mexico, E-mail: firstname.lastname@example.org.
Reprint requests: Bertha Espinoza, Departamento de Immunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, Distrito Federal, Circuito Escolar, Ciudad Universitaria, P.C. 04510, Mexico, D.F., Tel: (525) 56 22 89 43, Fax: (525) 56 22 91 98, E-mail: xm.manu.sacidemoib@ugseb.