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Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae)

Abstract

BACKGROUND

Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity.

METHODS

Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples.

OBJECTIVES

To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons.

FINDINGS

We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%).

MAIN CONCLUSIONS

Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

Key words:
Chagas disease vectors; cytochrome b gene; cytochrome C oxidase subunit I; internal transcribed spacer 2; karyotype evolution; morphometric analyses; sex chromosome fusion


The Triatominae subfamily (Hemiptera: Heteroptera: Reduviidae) includes more than 150 blood-sucking species grouped into 16 genera.11. Lent H, Wygodzinsky P. Revision of the Triatominae (Hemiptera: Reduviidae), and their significance as vectors of Chagas disease. Bull Am Mus Nat Hist. 1979; 163: 123-520.,22. Costa J, Dale C, Galvão C, Almeida CE, Dujardin JP. Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979-2021. Mem Inst Oswaldo Cruz. 2021; 116: e210015. These insects act as vectors of Trypanosoma cruzi, the etiological agent of Chagas disease, which is recognised as the most serious human parasitic disease of Latin America, affecting 5-6 million people.22. Costa J, Dale C, Galvão C, Almeida CE, Dujardin JP. Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979-2021. Mem Inst Oswaldo Cruz. 2021; 116: e210015.,33. Rojas de Arias A, Monroy C, Guhl F, Sosa-Estani S, Santos WS, Abad-Franch F. Chagas disease control-surveillance in the Americas: the multinational initiatives and the practical impossibility of interrupting vector-borne Trypanosoma cruzi transmission. Mem Inst Oswaldo Cruz. 2021; 116: e210130. In the absence of vaccines or adequate drugs for large-scale treatment, the reduction of disease incidence depends mainly on vector control of bug’s populations in human dwellings.22. Costa J, Dale C, Galvão C, Almeida CE, Dujardin JP. Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979-2021. Mem Inst Oswaldo Cruz. 2021; 116: e210015.,33. Rojas de Arias A, Monroy C, Guhl F, Sosa-Estani S, Santos WS, Abad-Franch F. Chagas disease control-surveillance in the Americas: the multinational initiatives and the practical impossibility of interrupting vector-borne Trypanosoma cruzi transmission. Mem Inst Oswaldo Cruz. 2021; 116: e210130. An accurate taxonomic identification and knowledge about the genetics of these insects are keys to ensure successful entomological surveillance after control campaigns.22. Costa J, Dale C, Galvão C, Almeida CE, Dujardin JP. Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979-2021. Mem Inst Oswaldo Cruz. 2021; 116: e210015.

The Panstrongylus genus belongs to the Triatomini tribe and is included within the North American lineage, constituting a paraphyletic group.44. Marcilla A, Bargues MD, Abad-Franch F, Panzera F, Carcavallo RU, Noireau F, et al. Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera, Reduviidae, Triatominae), vectors of Trypanosoma cruzi. Infect Genet Evol. 2002; 1(3): 225-35.,55. Justi SA, Russo CAM, Santos-Mallet JR, Obara MT, Galvão C. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasit Vectors. 2014; 7: 149.,66. Monteiro FA, Weirauch C, Felix M, Lazoski C, Abad-Franch F. Evolution, systematics, and biogeography of the Triatominae, vectors of Chagas disease. Adv Parasitol. 2018; 99: 265-344.Panstrongylus is the third most relevant genus within Triatominae in terms of species richness (one fossil and 14 living species) and epidemiological impact.77. Patterson JS, Barbosa SE, Feliciangeli MD. On the genus Panstrongylus Berg 1879: evolution, ecology and epidemiological significance. Acta Trop. 2009; 110(2-3): 187-99. Among these species, Panstrongylus megistus is the most significant Chagas disease vector, particularly in Brazil. Although the remaining Panstrongylus species are primarily sylvatic, some of them have gained attention, as they are involved in domiciliation processes and hence the transmission of Chagas disease to humans. Panstrongylus rufotuberculatus is one of these species, with a wide distribution area extending from Mexico to Argentina, from lowland rainforests to arid highlands of up to 2600m above sea level.11. Lent H, Wygodzinsky P. Revision of the Triatominae (Hemiptera: Reduviidae), and their significance as vectors of Chagas disease. Bull Am Mus Nat Hist. 1979; 163: 123-520.,77. Patterson JS, Barbosa SE, Feliciangeli MD. On the genus Panstrongylus Berg 1879: evolution, ecology and epidemiological significance. Acta Trop. 2009; 110(2-3): 187-99.,88. Noireau F, Bosseno MF, Vargas F, Brenière SF. Apparent trend to domesticity observed in Panstrongylus rufotuberculatus Champion, 1899 (Hemiptera, Reduviidae) in Bolivia. Res Rev Parasitol. 1994; 54(4): 263-4. Additionally it is adapted to dry as well as humid ecotopes, being found in a great variety of sylvatic hosts including armadillos, kinkajous, opossums, rodents, bats and birds burrows.11. Lent H, Wygodzinsky P. Revision of the Triatominae (Hemiptera: Reduviidae), and their significance as vectors of Chagas disease. Bull Am Mus Nat Hist. 1979; 163: 123-520. Furthermore, several authors reported breeding colonies of P. rufotuberculatus inside and around dwellings in Bolivia,88. Noireau F, Bosseno MF, Vargas F, Brenière SF. Apparent trend to domesticity observed in Panstrongylus rufotuberculatus Champion, 1899 (Hemiptera, Reduviidae) in Bolivia. Res Rev Parasitol. 1994; 54(4): 263-4.,99. Dujardin JP, Forgues G, Torrez M, Martinez E, Cordoba C, Gianella A. Morphometric of domestic Panstrongylus rufotuberculatus in Bolivia. Ann Trop Med Parasitol. 1998; 92(2): 219-28.,1010. Depickère S, Durán P, López R, Chávez T. Presence of intradomicile colonies of the triatomine bug Panstrongylus rufotuberculatus in Muñecas, La Paz, Bolivia. Acta Trop. 2011; 117(2): 97-100. Colombia,1111. Wolff M, Castillo D. Domiciliation trend of Panstrongylus rufotuberculatus in Colombia. Mem Inst Oswaldo Cruz. 2002; 97(3): 297-300. Ecuador,1212. Grijalva MJ, Villacís AG, Moncayo AL, Ocaña-Mayorga S, Yumiseva CA, Baus EG. Distribution of triatomine species in domestic and peridomestic environments in central coastal Ecuador. PLoS Negl Trop Dis. 2017; 11(10): e0005970.,1313. Barnabé C, Grijalva MJ, Santillán-Guayasamín S, Yumiseva CA, Waleckx E, Brenière SF, et al. Genetic data support speciation between Panstrongylus howardi and Panstrongylus chinai, vectors of Chagas disease in Ecuador. Infect Genet Evol. 2020; 78: 104103. Peru1414. Marín E, Santillán R, Cuba C, Jurberg J, Galvão C. Intra-domiciliary capture of Panstrongylus rufotuberculatus (Champion, 1899) (Hemiptera, Reduviidae, Triatominae) in Piura, Peru. Cad Saude Publica. 2007; 23(9): 2235-8. and Venezuela.1515. Traviezo-Valles LE, Berkefeld D, Aldana E. Infección natural de Panstrongylus rufotuberculatus (Hemiptera: Reduviidae) al sureste del Estado Lara, Venezuela. Bol Mal Salud Amb. 2008; 48(1): 99-101. Some studies highlight the high rates of house infestation1010. Depickère S, Durán P, López R, Chávez T. Presence of intradomicile colonies of the triatomine bug Panstrongylus rufotuberculatus in Muñecas, La Paz, Bolivia. Acta Trop. 2011; 117(2): 97-100.,1111. Wolff M, Castillo D. Domiciliation trend of Panstrongylus rufotuberculatus in Colombia. Mem Inst Oswaldo Cruz. 2002; 97(3): 297-300. and infection with Trypanosoma cruzi.1212. Grijalva MJ, Villacís AG, Moncayo AL, Ocaña-Mayorga S, Yumiseva CA, Baus EG. Distribution of triatomine species in domestic and peridomestic environments in central coastal Ecuador. PLoS Negl Trop Dis. 2017; 11(10): e0005970. The domiciliary presence of P. rufotuberculatus represents an important epidemiological risk factor for Chagas disease transmission in several Latin American countries, mainly in areas where the principal vector (e.g. Triatoma infestans) is absent due to vector control programs.1010. Depickère S, Durán P, López R, Chávez T. Presence of intradomicile colonies of the triatomine bug Panstrongylus rufotuberculatus in Muñecas, La Paz, Bolivia. Acta Trop. 2011; 117(2): 97-100.

Following the description of the male holotype from Panama by Champion (1899), several authors have emphasised the chromatic, morphologic and morphometric variation of P. rufotuberculatus along their geographical distribution.88. Noireau F, Bosseno MF, Vargas F, Brenière SF. Apparent trend to domesticity observed in Panstrongylus rufotuberculatus Champion, 1899 (Hemiptera, Reduviidae) in Bolivia. Res Rev Parasitol. 1994; 54(4): 263-4.,99. Dujardin JP, Forgues G, Torrez M, Martinez E, Cordoba C, Gianella A. Morphometric of domestic Panstrongylus rufotuberculatus in Bolivia. Ann Trop Med Parasitol. 1998; 92(2): 219-28.,1616. Hiwat H. Triatominae species of Suriname (Heteroptera: Reduviidae) and their role as vectors of Chagas disease. Mem Inst Oswaldo Cruz. 2014; 109(4): 452-8. For example, the carinae limiting the central depression of the scutellum is entirely black in specimens from Bolivia, Brazil, Costa Rica, Peru and Venezuela, while it is red or reddish in Colombia, Panama and Suriname insects.88. Noireau F, Bosseno MF, Vargas F, Brenière SF. Apparent trend to domesticity observed in Panstrongylus rufotuberculatus Champion, 1899 (Hemiptera, Reduviidae) in Bolivia. Res Rev Parasitol. 1994; 54(4): 263-4.,1616. Hiwat H. Triatominae species of Suriname (Heteroptera: Reduviidae) and their role as vectors of Chagas disease. Mem Inst Oswaldo Cruz. 2014; 109(4): 452-8. A traditional morphometric analysis of head capsules of individuals found in the domiciles from Bolivia (La Paz) versus sylvatic specimens deposited in the collection of the Natural History Museum of London (NHM), from Mexico, Ecuador and Panama (including the holotype of P. rufotuberculatus), showed a consistent variation of metric properties.99. Dujardin JP, Forgues G, Torrez M, Martinez E, Cordoba C, Gianella A. Morphometric of domestic Panstrongylus rufotuberculatus in Bolivia. Ann Trop Med Parasitol. 1998; 92(2): 219-28. Until now, no geometric morphometric approach had been applied.

As other hemipteran species, Panstrongylus genus presents holocentric chromosomes characterised by the absence of a primary constriction. Chromosomal analyses of eight out of the 14 Panstrongylus species reveal that this genus is variable in its diploid chromosome number (21, 23 or 24 chromosomes in males), including the number of autosomes (18 and 20), sex chromosome systems (X1X2Y and X1X2X3Y) and different amount of autosomal C-heterochromatin.1717. Panzera F, Pita S, Lorite P. Chromosome structure and evolution of Triatominae: a review. In: Guarneri AA, Lorenzo MG, editors. Triatominae - the biology of Chagas disease vectors. Entomology in Focus. Vol. 5. Switzerland: Springer; 2021. pp. 65-99. Previous cytogenetic studies on P. rufotuberculatus were restricted to individuals from Colombia (Antioquia and Santander) showing a male diploid chromosome number of 23 chromosomes (20 autosomes plus X1X2Y) and C-heterochromatic regions on most autosomes.1818. Crossa RP, Hernández M, Caraccio MN, Rose V, Valente SA, da Costa Valente V, et al. Chromosomal evolution trends of the genus Panstrongylus (Hemiptera, Reduviidae), vectors of Chagas disease. Infect Genet Evol. 2002; 2(1): 47-56.

We examined the cytogenetic characteristics of P. rufotuberculatus individuals collected in several localities from three countries (Bolivia, Colombia and Ecuador). This material was also employed to address wing morphometrics analyses, which included an additional sample from French Guiana not analysed by cytogenetics. We determined, for the first time, the chromosomal position of major ribosomal loci (rDNA clusters) by fluorescence in situ hybridisation (FISH). This chromosomal trait is considered as species specific and very useful to distinguish chromosomally undifferentiated species, such as observed in several species complexes.1919. Panzera Y, Pita S, Ferreiro MJ, Ferrandis I, Lages C, Pérez R, et al. High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera). Cytogenet Genome Res. 2012; 138(1): 56-67.,2020. Panzera F, Pita S, Nattero J, Panzera Y, Galvão C, Chavez T, et al. Cryptic speciation in the Triatoma sordida subcomplex (Hemiptera, Reduviidae) revealed by chromosomal markers. Parasit Vectors. 2015; 8: 495.,2121. Pita S, Lorite P, Cuadrado A, Panzera Y, de Oliveira J, Alevi KCC, et al. High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae). Med Vet Entomol. 2021; https://doi.org/10.1111/mve.12552.
https://doi.org/10.1111/mve.12552...
In order to check the phylogenetic relationships of these individuals, sequences analyses of one nuclear (internal transcribed spacer 2 - ITS-2) and two mitochondrial DNA fragments (cytochrome b - cyt b- and cytochrome C oxidase subunit I - coI) were performed, including sequences of P. rufotuberculatus from different countries and others Panstrongylus spp. available in GenBank.

MATERIALS AND METHODS

Materials - The specimens were identified according to morphological keys established in Lent and Wygodzinsky.11. Lent H, Wygodzinsky P. Revision of the Triatominae (Hemiptera: Reduviidae), and their significance as vectors of Chagas disease. Bull Am Mus Nat Hist. 1979; 163: 123-520. Table I and Fig. 1 show the geographic origin and number of P. rufotuberculatus individuals studied by morphometric, cytogenetics and molecular analyses (including its GenBank accession numbers). Previously published cytogenetic data are also incorporated.1818. Crossa RP, Hernández M, Caraccio MN, Rose V, Valente SA, da Costa Valente V, et al. Chromosomal evolution trends of the genus Panstrongylus (Hemiptera, Reduviidae), vectors of Chagas disease. Infect Genet Evol. 2002; 2(1): 47-56.

TABLE I
Geographic origin of Panstrongylus rufotuberculatus specimens used in molecular analyses (ITS-2, cyt b and coI), cytogenetics (CYT) and morphometrics of wings (MM)

Fig. 1:
Latin American map showing the geographic location of the 10 sampling sites of Panstrongylus rufotuberculatus individuals studied by genetic markers in this paper. Site names and other details are given in Table I.

In our phylogenetic analyses, we used all Panstrongylus sequences available in GenBank, in addition to sequences obtained by us. However, several ITS-2 and cyt b sequences identified as P. chinai, P. howardi and P. rufotuberculatus, submitted in GenBank in 2014 by Sempertegui-Sosa et al., were not included (Unpublished data). Barnabé et al.1313. Barnabé C, Grijalva MJ, Santillán-Guayasamín S, Yumiseva CA, Waleckx E, Brenière SF, et al. Genetic data support speciation between Panstrongylus howardi and Panstrongylus chinai, vectors of Chagas disease in Ecuador. Infect Genet Evol. 2020; 78: 104103. recognised, in several of these sequences, labeling errors in their species identification. Since Panstrongylus species belong to the North American Triatomini lineage, other species from this lineage were also included, as well as several species from the South American lineage as out-groups. Since there were few coI sequences available in GenBank for the fragment employed in the present paper, we have sequenced individuals of other Panstrongylus species: P. chinai (MZ643675); P. lignarius/herreri (MZ643676); P. geniculatus (MZ643678, MZ643679) and P. tupynambai (MZ643677).

Morphometric study - The wings pictures assembled for these morphometric studies were provided by different authors (TC, AV, JPD and two others cited in the acknowledgments). Since the pictures from Bolivia and Colombia did not harbor a size scale, our statistical analyses of the total sample were restricted to shape comparisons only, without including size analyses.

Six landmarks were selected on the wings in addition to 14 semilandmarks that were used to capture the curved lines of veins between landmarks (Fig. 2). All landmarks and semilandmarks were submitted to partial Procrustes superimposition (GPA),2222. Rohlf FJ. Rotational fit (Procrustes) methods. In: Rohlf F, Bookstein F, editors. Proceedings of the Michigan Morphometrics Workshop. University of Michigan Museums: Ann Arbor; 1990. p. 227-36. and semilandmarks then subjected to sliding procedure.2323. Dujardin JP. A template-dependent semilandmarks treatment and its use in medical entomology. Infect Genet Evol. 2019; 70: 197-207. The tangent space projections of 67 individuals were used as input for a principal component analysis (PCA). The discriminant analysis used the nine first PCA as input, and was illustrated by the factor map of first and second discriminant factor (Fig. 3). This analysis compared males, females and countries as separated groups. Because of the very small samples from Colombia, this country was mixed with the neighbor country Ecuador.

Fig. 2:
landmarks and semilandmarks of the wing venation in Panstrongylus rufotuberculatus. Numbers correspond to true landmarks type I (LM I) and letters to semilandmarks. The semilandmarks are partly capturing the shape of three veins: the anal vein (group “a”), the cubital vein (group “b”) and the median vein (group “c”). Their positions were computed depending on the position of two flanking landmarks.

Fig. 3:
factor map of the two first discriminant factors (DF 1, horizontal axis and DF 2, vertical axis) derived from first nine principal components which together represented 73% of the total shape variation in Panstrongylus rufotuberculatus. The contribution of each DF to the total discriminant space is indicated between brackets. Bolivia is completely distinguished on DF1, French Guiana is distinguished on DF2.

Cytogenetic studies - We analysed 33 P. rufotuberculatus bugs whose geographical origin is detailed in Table I. Gonads were removed from adult insects and fixed in ethanol-acetic acid (3:1). C-banding and fluorescent in situ hybridisation (FISH) using as a probe an 18S rDNA fragment (included within 45S rDNA clusters) were carried out on chromosome preparations obtained by the squash method.1818. Crossa RP, Hernández M, Caraccio MN, Rose V, Valente SA, da Costa Valente V, et al. Chromosomal evolution trends of the genus Panstrongylus (Hemiptera, Reduviidae), vectors of Chagas disease. Infect Genet Evol. 2002; 2(1): 47-56.,1919. Panzera Y, Pita S, Ferreiro MJ, Ferrandis I, Lages C, Pérez R, et al. High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera). Cytogenet Genome Res. 2012; 138(1): 56-67. Slides were examined under a Nikon Eclipse 80i microscope and the images were obtained with a DS-5Mc-U2 digital camera. For each specimen, at least 20 cells were analysed to determine the chromosome number, the C-banding pattern and the chromosomal location of the 45S rDNA clusters. The C-banding pattern of P. rufotuberculatus was previously described on individuals from Colombia (Antioquia and Santander).1818. Crossa RP, Hernández M, Caraccio MN, Rose V, Valente SA, da Costa Valente V, et al. Chromosomal evolution trends of the genus Panstrongylus (Hemiptera, Reduviidae), vectors of Chagas disease. Infect Genet Evol. 2002; 2(1): 47-56. In this paper, we included more individuals from the previously reported populations, as well as from other localities in Colombia (La Guajira and Yolombó), Ecuador (Loja and Santo Domingo) and Bolivia (La Paz). The chromosomal locations of 45S ribosomal clusters were determined by FISH in P. rufotuberculatus individuals and described here for the first time. To elucidate the possible origin of the sex chromosome polymorphism, we estimate the relative length (in percent) of the X chromosome(s) in relation to the chromosome complement total length. Images were analysed using the Nikon Nis Elements 3.1. Advanced Software. We analysed 100 metaphase I and II images from three individuals from each sex chromosome system.

DNA extraction and sequencing - Bugs legs, stored in 70% ethanol, were used for DNA extraction using standard phenol-chloroform technique. A 210 bp ITS-2 fragment was amplified by PCR using the 5.8T and 28T primers,44. Marcilla A, Bargues MD, Abad-Franch F, Panzera F, Carcavallo RU, Noireau F, et al. Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera, Reduviidae, Triatominae), vectors of Trypanosoma cruzi. Infect Genet Evol. 2002; 1(3): 225-35. a 600 bp fragment of coI was amplified with ACO and COIb primers,2424. Calleros L, Panzera F, Bargues MD, Monteiro FA, Klisiowicz DR, Zuriaga MA, et al. Systematics of Mepraia (Hemiptera-Reduviidae): cytogenetic and molecular variation. Infect Genet Evol. 2010; 10(2): 221-8. and a 419 bp fragment of cyt b with the primers CYTB7432F and CYTB7433R.2525. Monteiro FA, Barrett TV, Fitzpatrick S, Cordon-Rosales C, Feliciangeli D, Beard CB. Molecular phylogeography of the Amazonian Chagas disease vectors Rhodnius prolixus and R. robustus. Mol Ecol. 2003; 12(4): 997-1006. PCR products were Sanger-sequenced by Macrogen Inc. (Korea). Forward and reverse sequences were aligned using Bioedit v.7.0.9.

Genetic diversity and phylogenetic analyses - Considering that ITS-2 nuclear sequences have wide size differences among species, gaps (indels) were included as informative characters in sequence-based and alignment analyses2626. Simmons MP, Ochoterena H. Gaps as characters in sequence-based phylogenetic analyses. Syst Biol. 2000; 49(2): 369-81. and added as an additional block into the data file using FastGap v.1.2 software (http://www.aubot.dk/FastGap_home.htm).

For the three molecular markers, MAFFT v7.453 was employed to align sequences. Genetic distances for mitochondrial markers were calculated using APE package v5.4.12727. Paradis E, Schliep K. Ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019; 35(3): 526-8. in R v3.6.1, using the Kimura 2-parameter substitution model (K2-p). The best fitting substitution models for the mitochondrial genes were estimated using Smart Model Selection and jModelTest v.2.1.102828. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8): 772. for the nuclear ITS-2. Decisions were taken under Bayesian information criteria (BIC).

Maximum likelihood (ML) phylogenetic trees were obtained using PhyML v3.2929. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010; 59(3): 307-21. Bootstrap values were estimated from 1000 replicates. In order to include indels information for ITS-2 sequences, a Bayesian phylogenetic tree was inferred with MrBayes v.3.2.7a.3030. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61(3): 539-42. The posterior probability of the phylogenetic tree was estimated by Metropolis-coupled Markov chain Monte Carlo (MCMCMC). The MCMCMC settings consisted of two simultaneous, independent runs with four chains each (with default heating temperature 0.1), which were run for 10×6 generations and sampled every 1000 generations with a 25% burn-in. Convergence was assessed using the average standard deviation of split frequency values (< 0.01) and by the Effective Sample Size (ESS > 200).

RESULTS

Morphometric variation (Fig. 3) - On the discriminant space derived from shape variables, males and females were grouped together as expected. The first discriminant factor (DF) represented 77% of the total shape variation among samples (Fig. 3). On this first axis, males and females from Bolivia constituted a completely separated group. The second DF represented 18% of the total shape variation, separating the Guiana sample from the others, but not completely from the Colombia-Ecuador sample. The remaining 2 DF represented only 4% of the total variation and were not presented here.

C-banding and fluorescence in situ hybridisation (Fig. 4) - Chromosomal analyses of 33 P. rufotuberculatus individuals from Bolivia, Colombia and Ecuador identified two chromosomal groups, also called patterns or cytotypes, with differences in the number of sex chromosomes and in the chromosomal location of 45S ribosomal clusters. Both cytotypes presented autosomal C-heterochromatin located at one or both chromosomal ends in almost all autosomal pairs.

Fig. 4:
male meiosis in Panstrongylus rufotuberculatus. Top row (A-E): P. rufotuberculatus from Colombia and Ecuador named cytotype 1 (2n = 23 = 20A + X1X2Y) showing two X chromosomes and 45S ribosomal clusters on one autosome. Bottom row (F-J): P. rufotuberculatus from Bolivia named cytotype 2 (2n = 22 = 20A + XY) showing only one X chromosome and ribosomal clusters in X and Y chromosomes. C-banding: A, B, D, F, G and I. Fluorescent in situ hybridization with 18S ribosomal DNA probe: C, E, H and J. Chromosomes in blue (DAPI stain) and ribosomal signals (45S rDNA clusters) in red on one autosome (arrows in C and E) or on the X and Y chromosomes (H and J). (A and G): First meiotic prophases showing C-dots dispersed in the nuclei. First and second meiotic metaphases (B, C, G, H and D, E, I, J respectively). Bar = 10 μm.

Cytotype 1: Individuals from Colombian and Ecuadorian populations (Fig. 4A-E, top row,). Male individuals presented a diploid number (2n) of 23 chromosomes, with 10 autosomal pairs plus three sex chromosomes: X1X2Y. Females have 2n = 24 (20 autosomes plus X1X2X1X2). Early meiotic prophase was characterised by the presence of a largest heterochromatic chromocenter constituted by the association of the sex chromosomes (Fig. 4A, arrowhead). Furthermore, several heterochromatic dots were scattered throughout the nucleus (Fig. 4A, arrows), corresponding with terminal C-regions of autosomal pairs. Autosomal chromosomes showed scarce variation regarding their size (Fig. 4B-E). The Y sex chromosome was middle sized and totally C-heterochromatic, while X1 and X2 chromosomes were the smallest of the complement and had an intermediate staining (Fig. 4B,D). In metaphase II, the three sex chromosomes disposed as a pseudotrivalent, in the center of an autosomal ring-like shape. FISH results indicated that 45S rDNA clusters were located on the largest autosomal pair (Fig. 4C, E, arrows).

Cytotype 2: Individuals from Bolivian population (Fig. 4F-J, bottom row). All individuals presented a diploid number (2n) of 22 chromosomes, with 10 autosomal pairs plus two sex chromosomes (XY in males and XX in females). Autosomal C-heterochromatin distribution was similar to what was observed in individuals from Colombia and Ecuador (Fig. 4A, F). In addition, the Y sex chromosome was middle sized and totally heterochromatic. The X chromosome was similar in size to the Y chromosome (Fig. 4G-I) with an intermediate staining. During metaphase II, the X and Y chromosomes formed a pseudobivalent (Fig. 4I). The 45S rDNA clusters were situated on both sex chromosomes (X and Y) (Fig. 4H, J).

To clarify the origin of the sex chromosome polymorphism, we measured the average length of the X chromosomes over the total length of the chromosome complement. In Bolivian male individuals, presenting XY sex chromosomes, the mean length of the X chromosome represented 5.98 ± 0.17%. In the individuals from Colombian and Ecuadorian populations, the sum of the X1 and X2 chromosomes average lengths represented 5.87 ± 0.15%.

Nuclear ITS-2 (Fig. 5) - The best fitted model was the HYK + G and selected for the construction of the ITS-2 Bayesian tree. This phylogenetic tree included sequences of the five Panstrongylus species together with 19 sequences of P. rufotuberculatus available in GenBank, two of them sequenced in this study (Table I). Identical sequences were grouped in the same haplotype, so only one was represented in the tree [Supplementary data (Table)]. Since Panstrongylus species belong to the North American lineage, other triatomine species from this lineage were also included. South American lineage species are considered as out-groups. This analysis showed that the Panstrongylus genus is clearly paraphyletic. All P. rufotuberculatus sequences (four haplotypes representing 19 sequences) are grouped together in one clade with high statistical support, and also separated from the remaining five Panstrongylus species.

Fig. 5:
Bayesian phylogenetic tree obtained from ITS-2 fragments including the sequences of the six Panstrongylus species available in GenBank (P. lignarius and P. herreri counted as one species). The Panstrongylus genus is paraphyletic; with P. rufotuberculatus clade clearly separated from the other Panstrongylus species. The 19 individuals of P. rufotuberculatus, showed as four haplotypes, are clustered in one clade, including P. rufotuberculatus from Bolivia. Circles represent statistical support obtained through posterior probability, gray circles denote above 0.75 and black circles above 0.9. DNA sequence alignment is represented at the right panel, where each color represents a different DNA base. Accession numbers of P. rufotuberculatus from Bolivia and Colombia are MZ647516 and MZ647517 respectively.

Mitochondrial cyt b fragment (Fig. 6) - Maximum likelihood (HKY85+G+I) analysis using cyt b fragment sequences of seven Panstrongylus species plus P. rufotuberculatus is shown in Fig. 6 (collapsed tree). This phylogenetic analysis depicted the genus Panstrongylus as a paraphyletic clade. The 11 sequences identified as P. rufotuberculatus were grouped into two well supported clades. One of them includes the sequences from Bolivia and Colombia (La Guajira) and the other one the remaining sequences from Ecuador and Mexico. Uncollapsed tree could be seen in Supplementary data (Figure).

Fig. 6:
collapsed maximum likelihood phylogenetic tree obtained from cytochrome b (cyt b) fragment, including all sequences of Panstrongylus species available in GenBank. The paraphyly of the Panstrongylus genus is clearly shown. The 11 individuals recognized as P. rufotuberculatus were grouped into two well supported clades, clearly separated from the remaining seven Panstrongylus species. The individual from Bolivia (in blue) is closely related to P. rufotuberculatus from Colombia (La Guajira). Accession numbers of sequences presented herein are depicted in bold. Gray circles denote bootstraps support above 0.75 and black circles above 0.9. Uncollapsed tree could be seen in Supplementary data (Figure).

Mitochondrial coI fragment (Fig. 7) - The GTR+G+I substitution model was selected for the construction of the coI ML tree. This analysis included five Panstrongylus species plus sequences of P. rufotuberculatus. This genetic marker also supports the paraphyly of Panstrongylus. All individuals (eight) initially identified as P. rufotuberculatus are closely related to each other constituting a clearly separated clade from the remaining Panstrongylus species. Within this clade, it is possible to identify four well supported subclades: (a) Mexican individuals from Oaxaca and Veracruz, (b) Ecuadorian and Central Colombia (Antioquia) individuals, (c) North Colombia (La Guajira), and (d) Bolivian specimens. The specimens from Bolivia appear as a sister group of P. rufotuberculatus from Colombia (La Guajira).

Fig. 7:
maximum likelihood phylogenetic tree obtained from cytochrome C oxidase subunit I (coI) fragment from six Panstrongylus species, including all other triatomine species available in GenBank. The genus Panstrongylus is clearly paraphyletic. The eight individuals of P. rufotuberculatus are grouped in the same clade, clearly separated from the remaining five Panstrongylus species. Depicted in blue are P. rufotuberculatus from Bolivia. Gray circles denote bootstraps support above 0.75 and black circles above 0.9.

Average genetic distances (K2-p) among the four P. rufotuberculatus groups vary between 10.0% to 19.7% for cyt b and from 9.0% to 15.8% for coI fragments. Comparing P. rufotuberculatus from Bolivia with the other groups, the average genetic distances are close to the highest values, varying between 10.7% and 18.7% for cyt b and 10.6% and 15.8% for coI fragments. For both markers, the closest group to Bolivian individuals is the one from North Colombia (La Guajira): K2-p of 10.7% for cyt b and 10.6% for coI (Table II).

TABLE II
Mitochondrial DNA sequence differentiation among four Panstrongylus rufotuberculatus geographic groups represented in Table I. Mean Kimura 2-parameter (K2-p) distances (in percentages) computed with cyt b (above diagonal) and coI (below diagonal) partial sequences; mean within-group distances (cyt b / coI) are on the diagonal

DISCUSSION

Wing venation patterns - Due to the incomplete information about the wings size, no comparisons of size were possible, and its possible effect on the shape variables as extracted from our landmarks and semilandmarks remains unknown. Our work hypothesis considers that the possible allometric effect on shape variables did not bias the global comparison.

Shape as disclosed by the geometric morphometrics approach is a polygenic character3131. Klingenberg CP, Leamy LJ, Cheverud JM. Integration and modularity of quantitative trait locus effects on geometric shape in the mouse mandible. Genetics. 2004: 166(4); 1909-21. and most of its variation has been attributed to genetic drift rather than to environmental conditions.3232. Dujardin JP, Slice DE. Contributions of morphometrics to Medical Entomology. In: Tibayrenc M, editor. Encyclopedia of infectious diseases modern methodologies. Wiley & Sons, Inc.; 2006. p. 433-45. For this reason, the shape-based discriminant space is frequently reminiscent of the geographic map, as in our case here (Fig. 3).

The clear cut shape-based discrimination obtained between the Bolivian sample and the remaining ones parallels the separate position of the Bolivian origin in the morphospace of the head capsule.99. Dujardin JP, Forgues G, Torrez M, Martinez E, Cordoba C, Gianella A. Morphometric of domestic Panstrongylus rufotuberculatus in Bolivia. Ann Trop Med Parasitol. 1998; 92(2): 219-28. Considering the wide geographic sample of P. rufotuberculatus individuals, the observed morphometric variation pattern is likely to be the reflection of genetic drift. However, the morphometric approach by itself cannot tell us whether it is due to a spatial or biological isolation.

Sex chromosome polymorphism in Heteroptera - In Triatominae as well as in Reduviidae, it is generally agreed that XX/XY is the ancestral sex chromosome system. Sex mechanisms with multiple X or Y chromosomes are believed to be most probably originated through fragmentation(s) of the ancestral X and Y chromosome, respectively.3333. Ueshima N. Insecta 6. Hemiptera II: Heteroptera. In: John B, editor. Animal Cytogenetics. Vol. 3. Insecta 6 Gebrüder Bornträger, Berlin-Stuttgart; 1979. Intraspecific variation in the number of sex chromosomes (consider as polymorphisms) are very uncommon. It has been detected in only ten of the more than 1,000 heteropteran species analysed so far.3333. Ueshima N. Insecta 6. Hemiptera II: Heteroptera. In: John B, editor. Animal Cytogenetics. Vol. 3. Insecta 6 Gebrüder Bornträger, Berlin-Stuttgart; 1979.,3434. Papeschi AG. Sex chromosome polymorphism in species of Belostoma (Belostomatidae, Heteroptera). Hereditas. 1996; 124: 269-74.,3535. Poggio MG, Provecho YM, Papeschi AG, Bressa MJ. Possible origin of polymorphism for chromosome number in the assassin bug Zelurus femoralis longispinis (Reduviidae: Reduviinae). Biol J Linn Soc. 2013; 110(4): 757-64. Almost all of these variations were due to the fragmentation of the X chromosome, which was supported both by their meiotic chromosomal behavior and by the size comparison of the sex chromosomes between the individuals with simple system (XY) and those with multiple systems (XnY).3434. Papeschi AG. Sex chromosome polymorphism in species of Belostoma (Belostomatidae, Heteroptera). Hereditas. 1996; 124: 269-74.,3535. Poggio MG, Provecho YM, Papeschi AG, Bressa MJ. Possible origin of polymorphism for chromosome number in the assassin bug Zelurus femoralis longispinis (Reduviidae: Reduviinae). Biol J Linn Soc. 2013; 110(4): 757-64. Up to now, intraspecific variation in the number of sex chromosomes has not been reported in the Triatominae subfamily.

Almost all species from the North American Triatomini lineage (38 out of 41), which includes all Panstrongylus species, presented multiple X chromosomes. According to Panzera et al.,1717. Panzera F, Pita S, Lorite P. Chromosome structure and evolution of Triatominae: a review. In: Guarneri AA, Lorenzo MG, editors. Triatominae - the biology of Chagas disease vectors. Entomology in Focus. Vol. 5. Switzerland: Springer; 2021. pp. 65-99. the X1X2Y chromosome sex system is considered as the ancestral one in this lineage. Therefore, the XY mechanism observed in Bolivian individuals could represent a derived character originated by a fusion of the ancestral X1 and X2 chromosomes. The X chromosome relative length in Bolivian P. rufotuberculatus was significantly similar to the sum of chromosomes X1 and X2 of the individuals from Ecuador and Colombia. Hence, the fusion of the X1 and X2 chromosomes is the most likely explanation for the emergence of the XY system in P. rufotuberculatus from Bolivia.

Location of 45S ribosomal clusters - Another chromosome variation found within P. rufotuberculatus was the chromosomal position of the major ribosomal DNA loci. In the Bolivian individuals, the rDNA clusters were located on both sex chromosomes (Fig. 4F-J, bottom row), while in individuals from Colombia and Ecuador they were on one autosomal pair (Fig. 4A-E, top row).

The analysis of 92 triatomine species so far, revealed that the chromosomal position of the 45S ribosomal clusters was a species specific character.2121. Pita S, Lorite P, Cuadrado A, Panzera Y, de Oliveira J, Alevi KCC, et al. High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae). Med Vet Entomol. 2021; https://doi.org/10.1111/mve.12552.
https://doi.org/10.1111/mve.12552...
Intraspecific variation in the rDNA clusters location is an exceptional event within Hemiptera, reported only in one triatomine species: T. infestans.1919. Panzera Y, Pita S, Ferreiro MJ, Ferrandis I, Lages C, Pérez R, et al. High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera). Cytogenet Genome Res. 2012; 138(1): 56-67.,3636. Panzera F, Ferreiro MJ, Pita S, Calleros L, Pérez R, Basmadjián Y, et al. Evolutionary and dispersal history of Triatoma infestans, main vector of Chagas disease, by chromosomal markers. Infect Genet Evol. 2014; 27: 105-13. Another apparently intraspecific variation reported in Rhodnius ecuadoriensis from Peru and Ecuador by Pita et al.3737. Pita S, Panzera F, Ferrandis I, Galvão C, Gómez-Palacio A, Panzera Y. Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes. Mem Inst Oswaldo Cruz. 2013; 108(3): 376-82. turned out to be explained by the existence of different species.3838. Abad-Franch F, Monteiro FA, Pavan MG, Patterson JS, Bargues MD, Zuriaga MA, et al. Under pressure: phenotypic divergence and convergence associated with microhabitat adaptations in Triatominae. Parasit Vectors. 2021; 14(1): 195.

In the Triatomini tribe, the location of rDNA clusters on one autosomal pair is by far the most frequent pattern and is considered as the ancestral character for this group.1717. Panzera F, Pita S, Lorite P. Chromosome structure and evolution of Triatominae: a review. In: Guarneri AA, Lorenzo MG, editors. Triatominae - the biology of Chagas disease vectors. Entomology in Focus. Vol. 5. Switzerland: Springer; 2021. pp. 65-99.,2121. Pita S, Lorite P, Cuadrado A, Panzera Y, de Oliveira J, Alevi KCC, et al. High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae). Med Vet Entomol. 2021; https://doi.org/10.1111/mve.12552.
https://doi.org/10.1111/mve.12552...
This trend is more acute in the North American lineage, since the autosomal rDNA location is highly conserved including all Panstrongylus species analysed hitherto.2121. Pita S, Lorite P, Cuadrado A, Panzera Y, de Oliveira J, Alevi KCC, et al. High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae). Med Vet Entomol. 2021; https://doi.org/10.1111/mve.12552.
https://doi.org/10.1111/mve.12552...
Therefore, the sex chromosome location pattern observed in P. rufotuberculatus from Bolivia is a very atypical event and should be considered as an apomorphic character. The movement of the ribosomal clusters from autosomes to both sex chromosomes is probably due to transposition events or ectopic recombination, as observed in other triatomine species.1717. Panzera F, Pita S, Lorite P. Chromosome structure and evolution of Triatominae: a review. In: Guarneri AA, Lorenzo MG, editors. Triatominae - the biology of Chagas disease vectors. Entomology in Focus. Vol. 5. Switzerland: Springer; 2021. pp. 65-99.,1919. Panzera Y, Pita S, Ferreiro MJ, Ferrandis I, Lages C, Pérez R, et al. High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera). Cytogenet Genome Res. 2012; 138(1): 56-67.,2121. Pita S, Lorite P, Cuadrado A, Panzera Y, de Oliveira J, Alevi KCC, et al. High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae). Med Vet Entomol. 2021; https://doi.org/10.1111/mve.12552.
https://doi.org/10.1111/mve.12552...
The mobilisation of rDNA loci from autosomes to sex chromosomes alters the genetic recombination of the involved chromosome regions. The differences in genetic recombination rates among autosomes and sex chromosomes are even more extreme in triatomines considering the achiasmatic nature of sex chromosomes.1717. Panzera F, Pita S, Lorite P. Chromosome structure and evolution of Triatominae: a review. In: Guarneri AA, Lorenzo MG, editors. Triatominae - the biology of Chagas disease vectors. Entomology in Focus. Vol. 5. Switzerland: Springer; 2021. pp. 65-99.,1919. Panzera Y, Pita S, Ferreiro MJ, Ferrandis I, Lages C, Pérez R, et al. High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera). Cytogenet Genome Res. 2012; 138(1): 56-67.,2121. Pita S, Lorite P, Cuadrado A, Panzera Y, de Oliveira J, Alevi KCC, et al. High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae). Med Vet Entomol. 2021; https://doi.org/10.1111/mve.12552.
https://doi.org/10.1111/mve.12552...
,3939. Solari AJ. Autosomal synaptonemal complexes and sex chromosomes without axes in Triatoma infestans (Reduviidae, Hemiptera). Chromosoma. 1979; 72: 225-40. As a result of the reduced recombination, genetic barriers to gene flow could arise rapidly between populations with different sex chromosome systems and location of ribosomal clusters, leading to their divergence and speciation. This was suggested for other insect groups such as Coleoptera and Lepidoptera.4040. Galián J, Proença SJ, Vogler AP. Evolutionary dynamics of autosomal-heterosomal rearrangements in a multiple-X chromosome system of tiger beetles (Cicindelidae). BMC Evol Biol. 2007; 7: 158. 41. -íchová J, Nguyen P, Dalíková M, Marec F. Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features. PLoS One. 2013; 8(5): e64520.,4141. -íchová J, Nguyen P, Dalíková M, Marec F. Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features. PLoS One. 2013; 8(5): e64520. As gene flow is more restricted between sex chromosomes than autosomes, sex linked genes are particularly efficient to produce post-zygotic barriers4242. S-tre GP, Borge T, Lindroos K, Haavie J, Sheldon BC, Primmer C, et al. Sex chromosome evolution and speciation in Ficedula flycatchers. Proc R Soc London B. 2003; 270(1510): 53-9. and therefore rearrangements involving sex chromosomes can be particularly effective as isolation mechanisms.

Most likely, crosses between P. rufotuberculatus individuals with different cytotypes could give rise to an adult progeny, which will include individuals with different numbers of X chromosomes and chromosomes with ribosomal loci. As a consequence of chromosomal pairing and segregation during both meiotic divisions, this hybrid progeny will produce a number of unbalanced gametes, both in the number of X chromosomes and of chromosomes carrying ribosomal loci, resulting in reproductive disadvantages. This negative effect is exacerbated by the changes in the number of X chromosomes because they alter the sex determination control and the gene regulation. A similar effect was described in coleopteran species.4040. Galián J, Proença SJ, Vogler AP. Evolutionary dynamics of autosomal-heterosomal rearrangements in a multiple-X chromosome system of tiger beetles (Cicindelidae). BMC Evol Biol. 2007; 7: 158. 41. -íchová J, Nguyen P, Dalíková M, Marec F. Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features. PLoS One. 2013; 8(5): e64520.

We propose that the variability in the number of X chromosomes and rDNA position observed in P. rufotuberculatus populations reflects chromosomal rearrangements leading to reproductive incompatibility between both cytotypes. Hence, it is likely that they do not represent intraspecific variability.

Molecular markers - In concordance with previous phylogenetic analyses,44. Marcilla A, Bargues MD, Abad-Franch F, Panzera F, Carcavallo RU, Noireau F, et al. Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera, Reduviidae, Triatominae), vectors of Trypanosoma cruzi. Infect Genet Evol. 2002; 1(3): 225-35.,55. Justi SA, Russo CAM, Santos-Mallet JR, Obara MT, Galvão C. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasit Vectors. 2014; 7: 149.,66. Monteiro FA, Weirauch C, Felix M, Lazoski C, Abad-Franch F. Evolution, systematics, and biogeography of the Triatominae, vectors of Chagas disease. Adv Parasitol. 2018; 99: 265-344.,4343. Kieran TJ, Gordon ERL, Zaldívar-Riverón A, Ibarra-Cerdeña C, Glenn TC, Weirauch C. Ultraconserved elements reconstruct the evolution of Chagas disease-vectoring kissing bugs (Reduviidae: Triatominae). Syst Entomol. 2021; 46: 725-40. our results support the paraphyletic nature of the Panstrongylus genus and their inclusion within North American Triatomini lineage (Figs 5-7). Furthermore, previous reports and the here obtained results also showed that P. rufotuberculatus specimens constitute always a separate clade from the remaining Panstrongylus species. Despite the low number of individuals here analysed (38 for the three molecular markers), phylogenetic trees also showed that all individuals morphologically identified as P. rufotuberculatus, including those from Bolivia, are closely related. In conclusion, despite its important chromosomal differences, P. rufotuberculatus from Bolivia seems to be a sister group to the remaining P. rufotuberculatus individuals of other countries.

The average genetic distances (K2-p) of P. rufotuberculatus from Bolivia to others P. rufotuberculatus varies between 10.7% to 18.7% and 10.6% to 15.8% for cyt b and coI fragments respectively (Table II). The closest individuals to the Bolivian samples are those from La Guajira (North Colombia) with 10.7% and 10.6% K2-p distances respectively. The last ones present the regular chromosomal cytotype (Fig. 4). For cyt b gene, this distance was much greater than the cut-off value of 7.5% proposed by Monteiro et al.4444. Monteiro FA, Donnelly MJ, Beard CB, Costa J. Nested clade and phylogeographic analyses of the Chagas disease vector Triatoma brasiliensis in Northeast Brazil. Mol Phyl Evol. 2004; 32(1): 46-56. to validate distinct triatomine species. For example, within the closely related species of dimidiata complex (T. dimidiata and those later described as T. mopan4545. Dorn PL, Justi SA, Dale C, Stevens L, Galvão C, Lima-Cordón R, et al. Description of Triatoma mopan sp. n. from a cave in Belize (Hemiptera, Reduviidae, Triatominae). ZooKeys. 2018; 775: 69-95.and T. huehuetenanguensis4646. Lima-Cordón RA, Monroy MC, Stevens L, Rodas A, Rodas GA, Dorn PL, et al. Description of Triatoma huehuetenanguensis sp. n., a potential Chagas disease vector (Hemiptera, Reduviidae, Triatominae). ZooKeys. 2019; 820: 51-70.), the cyt b sequences diverged from 8.0% to 15.5%.4747. Monteiro FA, Peretolchina T, Lazoski C, Harris K, Dotson EM, Abad-Franch F, et al. Phylogeographic pattern and extensive mitochondrial DNA divergence disclose a species complex within the Chagas disease vector Triatoma dimidiata. PLoS One. 2013; 8(8): e70974. Furthermore, Panstrongylus sister species such as P. chinai and P. howardi presented a K2-p distance of 7.8%.4848. Villacís AG, Dujardin JP, Panzera F, Yumiseva CA, Pita S, Santillán-Guayasamín S, et al. Chagas vectors Panstrongylus chinai (Del Ponte, 1929) and Panstrongylus howardi (Neiva, 1911): chromatic forms or true species? Parasit Vectors. 2020; 13(1): 226. For coI sequences, the value of 10.6% observed between P. rufotuberculatus from Bolivia and La Guajira was also significantly higher than those reported for other closely related triatomine species, as 7.1% between Mepraia spinolai and M. gajardoi,2424. Calleros L, Panzera F, Bargues MD, Monteiro FA, Klisiowicz DR, Zuriaga MA, et al. Systematics of Mepraia (Hemiptera-Reduviidae): cytogenetic and molecular variation. Infect Genet Evol. 2010; 10(2): 221-8. or 5.3% between T. sordida and the recently described species T. rosai.4949. Alevi KCC, de Oliveira J, Garcia ACC, Cristal DC, Delgado LMG, Bittinelli IF, et al. Triatoma rosai sp. nov. (Hemiptera, Triatominae): a new species of Argentinian Chagas disease vector described based on integrative taxonomy. Insects. 2020; 11(12): 830. In summary, the great genetic differentiation observed between the two chromosomal cytotypes of P. rufotuberculatus, either by cyt b and coI fragments, suggests that both groups should be considered as distinct species.

Within P. rufotuberculatus, both mitochondrial markers identified four groups: Bolivia, Mexico, North Colombia, and Ecuador plus central Colombia (Table II, Figs 1, 5-7). Unexpectedly, the genetic K2-p distances observed between these four groups are similar to those observed between different species. This would suggest that P. rufotuberculatus is most likely a complex of several taxa (Table II). Therefore, it is essential to analyse a greater number of individuals to confirm these high genetic divergences. Unfortunately, chromosomal data from the Mexican group is unavailable at the moment.

Evolutionary origin of P. rufotuberculatus from Bolivia - The close relationship obtained in the phylogenetic analyses and the external morphology similarities suggest that P. rufotuberculatus from Bolivia is a sister group of the remaining P. rufotuberculatus from other countries (Figs 4-6). Variations in the number of X chromosomes and the rDNA loci chromosomal location of P. rufotuberculatus from Bolivia are most likely explained by at least two different chromosomal rearrangements. Our hypothesis implies an ancestor with a multiple sex system (X1X2Y) and ribosomal clusters in one autosomal pair, similar to most Panstrongylus species (including P. rufotuberculatus from Colombia and Ecuador). In this ancestor, the first rearrangement that should have occurred was the fusion of the X1 and X2 chromosomes, resulting in a single fused X chromosome. Subsequently, the complete mobilisation of the ribosomal loci from one autosome pair to the newly fused X chromosome may have occurred, followed by another transfer (partial) of rDNA clusters from the X chromosome to the Y chromosome. Considering the regular meiotic segregation of sex chromosomes in Bolivian individuals, and the fact that heterozygous individuals for each chromosomal marker were not detected, it is very likely that both rearrangements occurred a long time ago. This could be supported by the significant genetic distances observed among P. rufotuberculatus from Bolivia and its sister groups (Table II).

The present paper reports, for the first time, an extensive morphometric and genetic differentiation within Panstrongylus rufotuberculatus. Important chromosomal differences, that would prevent genetic exchange between both chromosomal groups, are here revealed. This evidence is supported by high values of genetic distances and consistent morphometric differences between the groups. Deeper and widely molecular analyses would help to confirm genetic differentiation observed among P. rufotuberculatus from different countries.

All the P. rufotuberculatus material from Bolivia here analysed, was collected in 2004 and 2006 and belongs to a single locality (La Paz, Muñecas, Camata), both from domiciliary and peridomiciliary environments. However, the geographical distribution of P. rufotuberculatus in Bolivia is wider, since it has already been described in other provinces of La Paz Department (Nor Yungas, Sud Yungas, Caranavi, Inquisivi), associated with anthropic structures, particularly intradomiciles.88. Noireau F, Bosseno MF, Vargas F, Brenière SF. Apparent trend to domesticity observed in Panstrongylus rufotuberculatus Champion, 1899 (Hemiptera, Reduviidae) in Bolivia. Res Rev Parasitol. 1994; 54(4): 263-4.,99. Dujardin JP, Forgues G, Torrez M, Martinez E, Cordoba C, Gianella A. Morphometric of domestic Panstrongylus rufotuberculatus in Bolivia. Ann Trop Med Parasitol. 1998; 92(2): 219-28.,1010. Depickère S, Durán P, López R, Chávez T. Presence of intradomicile colonies of the triatomine bug Panstrongylus rufotuberculatus in Muñecas, La Paz, Bolivia. Acta Trop. 2011; 117(2): 97-100.

A detailed morphological examination and other biological aspects of P. rufotuberculatus from Bolivia are now needed to determine external differences that allow its taxonomic recognition as well as for its formal description as a separated species.

ACKNOWLEDGEMENTS

To Cristina Aznar (AG University, Cayenne, French Guiana) for allowing us to dissect specimens of her collections from French Guiana. The few pictures from Colombia were kindly provided by Harling Caro-Riano (Universidad de Antioquia, Instituto de Biología, Medellín, Colombia). Ecuadorian collection permit (N° MAE-DNB-CM-2015-0030) and export permit (N°71-2018-EXP-CM-FAU-DNB/MA).

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  • Financial support: From Uruguay: MEC/DICYT/II/FVF/2019/054 and CSIC-Udelar (project grant No. 160); From Spain: Spanish Junta de Andalucía by AUIP and “Programa Operativo FEDER Andalucía 2014-2020”. UNICEF/UNPD/World Bank/WHO Special Programme for TDR [A20785], PAHO [A60655], and Pontificial Catholic University of Ecuador (L13254).

Publication Dates

  • Publication in this collection
    02 Feb 2022
  • Date of issue
    2021

History

  • Received
    02 Aug 2021
  • Accepted
    16 Nov 2021
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