Triatoma guazu Lent and Wygodzinsky Is a Junior Synonym of Triatoma williami Galvão, Souza and Lima

Simple Summary Triatomines are blood-sucking insects, potential vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Triatoma guazu and Triatoma williami are phylogenetically very close and occur in sympatry. Morphologic, morphometric, and genetic analyses were performed to discuss the taxonomic status of these species. Morphometric and molecular data do not show diagnostic characteristics between species, whereas their different patterns of connexival spots were considered a phenotypic polymorphism, common in triatomines. These results suggest T. guazu as a junior synonym of T. williami. Therefore, the synonym between these species is formally proposed here. Abstract Triatoma guazu Lent and Wygodzinsky and Triatoma williami Galvão, Souza, and Lima (Hemiptera: Triatominae) are found in human dwellings and are potential vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Triatoma guazu was described based solely on a single female specimen, from the municipality of Villarica, Guairá Department, Paraguay, and posteriorly, a male from Barra do Garças, Mato Grosso, Brazil was described and designated as the allotype of this species. Triatoma williami is found in the central-west of Brazil between Goiás, Mato Grosso, and Mato Grosso do Sul. However, the taxonomic “status” of these species is questioned. Previous studies indicate the lack of isoenzymatic diagnostic loci, morphometric similarity, low genetic divergence, and close evolutionary relationship of these species. In this study, we compared the morphology, morphometry, and mitochondrial DNA fragments of the populations of the two species. The morphological diagnostic characteristic among these species is the difference in the connexivum spots pattern, which has been recognized as a phenotypic variation that exists among populations resulting from ecological diversity. Furthermore, our analysis also revealed the morphometric similarity and low genetic divergence between these species. Therefore, in the present paper, we formally propose T. guazu as a junior synonym of T. williami.

Triatoma williami Galvão, Souza, and Lima, 1965 was described based on specimens from the municipality of Piranhas, Goiás state, in honor of Dr. William Barbosa for his efforts to create a research institute in the region (Instituto de Patologia Tropical) [9]. This species also occurs in other states of the Cerrado, the tropical savanna ecoregion located

Materials and Methods
Morphological analysis. We examined 44 individuals (12 Triatoma guazu and 32 T. williami) including the type specimens, deposited in Coleção Entomológica do Instituto Butantan (CEIB), São Paulo, Brazil, and Coleção de Triatomíneos do Instituto Oswaldo Cruz (CTIOC), Fiocruz, Rio de Janeiro, Brazil (see examined material in Supplemental Data S1, . Photographs and morphological analysis were taken with a Leica DMC 2900 camera attached to a Leica M205C stereomicroscope (Figures 1 and 4-7). Images were edited using Adobe Photoshop 7.0.1. General morphological terminology mainly follows original descriptions of the species [9,12] and previous studies on Triatominae (e.g., Lent and Wygodzinsky [13]). Images were edited using Adobe Photoshop 7.0.1. General morphological terminology mainly follows original descriptions of the species [9,12] and previous studies on Triatominae (e.g., Lent and Wygodzinsky [13]).    Images were edited using Adobe Photoshop 7.0.1. General morphological terminology mainly follows original descriptions of the species [9,12] and previous studies on Triatominae (e.g., Lent and Wygodzinsky [13]).    Images were edited using Adobe Photoshop 7.0.1. General morphological terminology mainly follows original descriptions of the species [9,12] and previous studies on Triatominae (e.g., Lent and Wygodzinsky [13]).                Geometric morphometrics. The adult head capsule was analyzed from 12 specimens of T. guazu (from Mato Grosso State) and 20 of T. williami (from the States of Goiás, Mato Grosso and Mato Grosso do Sul), in addition to the outgroup consisting of 20 specimens of T. matogrossensis (from Mato Grosso State) and three specimens of T. oliveirai (from the Rio Grande do Sul State) (see examined material in Supplemental Data S1, Figure 1). The head was photographed using a digital camera Nikon coolpix 990, and the landmark coordinates were recorded with TpsDig version v. 2.05 (New York, United States) [26] to achieve a better definition of the head capsule conformation of the specimens [26] ( Figure  6). Landmarks were superimposed to Generalized Procrustes Analysis in TPsRelw 1.53 [27][28][29][30]. This method allows the calculation of shape variables among taxa after alignment of the landmarks (to ensure homology). The multivariate differences were evaluated by the Lambda test of Wilks. The scores matrix was examined by Canonical Variate Analysis (CVA) to plot and observe the position of each specimen on the "Shape Discriminant Space". The observed average distances were used to analyze the relationship among the species through the reconstruction of an UPGMA dendrogram. All analyses were executed in JMP 3.2.6 (SAS Institute, Cary, NC, USA).
Analysis of male genitalia. The dissections of a male genitalia of a specimen identified as T. williami were made by first removing the pygophore from the abdomen with a pair of forceps and then clearing it in 20% NaOH solution for 24 h. The dissected structures Geometric morphometrics. The adult head capsule was analyzed from 12 specimens of T. guazu (from Mato Grosso State) and 20 of T. williami (from the States of Goiás, Mato Grosso and Mato Grosso do Sul), in addition to the outgroup consisting of 20 specimens of T. matogrossensis (from Mato Grosso State) and three specimens of T. oliveirai (from the Rio Grande do Sul State) (see examined material in Supplemental Data S1, Figure 1). The head was photographed using a digital camera Nikon coolpix 990, and the landmark coordinates were recorded with TpsDig version v. 2.05 (New York, NY, USA) [26] to achieve a better definition of the head capsule conformation of the specimens [26] (Figure 6). Landmarks were superimposed to Generalized Procrustes Analysis in TPsRelw 1.53 [27][28][29][30]. This method allows the calculation of shape variables among taxa after alignment of the landmarks (to ensure homology). The multivariate differences were evaluated by the Lambda test of Wilks. The scores matrix was examined by Canonical Variate Analysis (CVA) to plot and observe the position of each specimen on the "Shape Discriminant Space". The observed average distances were used to analyze the relationship among the species through the reconstruction of an UPGMA dendrogram. All analyses were executed in JMP 3.2.6 (SAS Institute, Cary, NC, USA).
Analysis of male genitalia. The dissections of a male genitalia of a specimen identified as T. williami were made by first removing the pygophore from the abdomen with a pair of forceps and then clearing it in 20% NaOH solution for 24 h. The dissected structures were studied and photographed in glycerol (see the label of the analyzed specimen at Data S1). The photographs were obtained using a digital camera (Sony DSC-W830, Budapest, Hungary). Images were edited using Adobe Photoshop CS6.
In relation to the male genitalia portions, the terminology used here follows mostly Lent and Wygodzinsky [13]. However, "vesica" as recognized by Lent and Wygodzinsky [13] and Lent et al. [14] has been considered to be absent in reduviids. The assumed equivalent structure in reduviids is a somewhat sclerotized appendage of the phallosoma or the endosoma [31], but not the homologous vesica that occurs in other heteropterans such as Pentatomomorpha [32]. Thus, this term is not used here for the median process of endosoma, which is named as such.
Scanning Electron Microscopy. The samples of T. guazu examined were obtained from colonies maintained by the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil (see the label of the analyzed specimens at Data S1). The females were washed with detergent and subsequently metallized. Then, cross-sections were performed between abdominal tergites II and III. The genitals were dehydrated in an alcoholic series on silica and fixed in aluminum stubs. The genitalia were plated on the Rudolf Barth Plataforma de Microscopia Eletrônica de Varredura (IOC) and were photographed using the microscopes JEOL JSM-6390LV.
Molecular analysis. A total of 64 DNA sequences of Triatoma brasiliensis Neiva, 1911 [33], T. guazu, Triatoma jatai Gonçalves, Teves-Neves, Santos-Mallet, Carbajal-dela-Fuente, and Lopes, 2013 [34], T. matogrossensis and T. williami were retrieved from Gen-Bank [35] for 16S, COI, COII and CytB. Triatoma oliveirai is a rare species and does not have any DNA sequence publicly available. DNA sequence data were aligned separately for each marker using ClustalW [36] implemented in the MEGA-X [37]. MEGA-X was used then to identify the number of variable sites. A pairwise divergence calculation was performed in the MEGA-X program, using the bootstrap method of resampling with 500 replications and the Kimura-2-parameters as the substitution model [38]. Species comparisons were performed by calculating the difference between inter and intraspecific genetic distances. Here, we considered as 'true' species those which interspecific divergences were above intraspecific divergences observed for Triatoma species.

Results
In the present study, we highlight new diagnostic characters among these species, in addition to those mentioned by Lent and Wygodzinsky [13]: length of the anteocular region in relation to the postocular region; eye position in relation to the ventral surface of the head; length of the submedian carina of the pronotum; and absence of spongy fossa (see Supplemental Table S2). The morphological data did not reveal diagnostic characters to differentiate between T. guazu and T. williami, except for the connexivum spots pattern (see Supplemental Data S2). Based on the original description, both species in the dorsal view have spots on the intersegmental sutures of the connexivum, which are dark and wide in T. guazu, and dark and narrow in T. williami. However, the present analysis evidenced a continuous variation, within the populations of the two species, from narrow to wide dark spots (Figure 7). The discriminant analysis of the head capsule shape showed that specimens originally designated as T. guazu are not distinct from T. williami specimens ( Figure 8A,B). The male genitalia of a specimen identified as T. williami ( Figure 9A-E) revealed the same characteristics as those described to T. guazu [14]. It is noteworthy that the mentioned uniformity was recorded in the structures which are usually more important to diagnosis or to distinguish species of Triatoma, such as the phallic portions (articulatory apparatus, pedicel, phallothecal plate, including the struts and the processes of the endosoma) ( Figure 9A-E), which presented the same shape, dimensions, and peculiarities in T. williami as described in T. guazu by Lent et al. [14]. The male genitalia of a specimen identified as T. williami ( Figure 9A-E) revealed the same characteristics as those described to T. guazu [14]. It is noteworthy that the mentioned uniformity was recorded in the structures which are usually more important to diagnosis or to distinguish species of Triatoma, such as the phallic portions (articulatory apparatus, pedicel, phallothecal plate, including the struts and the processes of the endosoma) ( Figure 9A-E), which presented the same shape, dimensions, and peculiarities in T. williami as described in T. guazu by Lent et al. [14]. In the same way, the character set of the female genitalia of T. guazu did not reveal differences when compared to T. williami, genitalia described by Teves et al. [17] ( Figure  10A-C). It was not possible to compare the genitalia in posterior view, because there is no description for T. williami. However, in posterior view, we observed the following characters for T. guazu: appendix not visible; gonocoxite VIII elongated and slightly wider; abdominal segments IX and X slightly downward and as wide as long; and tergite IX posterior margin clearly separated from tergite X. In the same way, the character set of the female genitalia of T. guazu did not reveal differences when compared to T. williami, genitalia described by Teves et al. [17] ( Figure 10A-C). It was not possible to compare the genitalia in posterior view, because there is no description for T. williami. However, in posterior view, we observed the following characters for T. guazu: appendix not visible; gonocoxite VIII elongated and slightly wider; abdominal segments IX and X slightly downward and as wide as long; and tergite IX posterior margin clearly separated from tergite X. A total of 56 sequences were selected and analyzed (Table 1): 16S gene fragments (20 sequences, 356 bp, 48 variable sites); COI (16 sequences, 201 bp, 52 variable sites); COII (eight sequences, 284 bp, and 79 variable sites); and CytB (12 sequences, 313 bp, and 91 variable sites). In general, those markers provide strong evidence that T. guazu and T. williami are the same species. Pairwise comparisons revealed that sequences of these two species diverge less than 2% for all markers analyzed (16S = 0.2%, COI = 1.5%, COII = 1.7%, CytB = 0.9%). These levels of divergence were below some intraspecific comparisons in T. matogrossensis (see Tables 2-5; see Supplemental Table S4 A-D) and T. brasiliensis (see Table 5; Table S4.A). A similar pattern was found for COII and CytB pairwise divergences, which T. guazu and T. williami diverged 1.7% and 0.9%, respectively, and intraspecific divergences within T. matogrossensis reached up to 3.2% in COII sequences, and 1.2% in intraspecific divergences within T. brasiliensis CytB sequences.  A total of 56 sequences were selected and analyzed (Table 1): 16S gene fragments (20 sequences, 356 bp, 48 variable sites); COI (16 sequences, 201 bp, 52 variable sites); COII (eight sequences, 284 bp, and 79 variable sites); and CytB (12 sequences, 313 bp, and 91 variable sites). In general, those markers provide strong evidence that T. guazu and T. williami are the same species. Pairwise comparisons revealed that sequences of these two species diverge less than 2% for all markers analyzed (16S = 0.2%, COI = 1.5%, COII = 1.7%, CytB = 0.9%). These levels of divergence were below some intraspecific comparisons in T. matogrossensis (see Tables 2-5; see Supplemental Table S2A-D) and T. brasiliensis (see Table 5; Table S2A). A similar pattern was found for COII and CytB pairwise divergences, which T. guazu and T. williami diverged 1.7% and 0.9%, respectively, and intraspecific divergences within T. matogrossensis reached up to 3.2% in COII sequences, and 1.2% in intraspecific divergences within T. brasiliensis CytB sequences.

Discussion
Lent and Wygodzinsky [13] considered T. oliveirai to be the closet taxon of T. guazu, because they have connexival spots in a similar pattern. In contrast, our morphological review confirms that T. guazu and T. oliveirai are not a closely related species.
In contrast, the low genetic divergence, lack of morphological diagnostic characters, and low morphometric distinction observed between T. guazu and T. williami corroborate previous studies which stated that they are very similar taxa [14][15][16][17][18][19][20][21][22]. Additionally, the variation in the spots pattern in the connexivum is compatible with phenotypic variations between populations of the same species, possibly a consequence of different ecological characteristics, not standing as a feature to consider them as a separate species [14,39].
The close phylogenetic relationship between T. guazu and T. williami had already been addressed in previous studies [15,[19][20][21][22], being confirmed in phylogenetic reconstructions with high node support. Almeida et al. [40] reported low genetic divergence for species from southern Brazil, which may be the result of recent speciation events. Previous studies used 16S, COI, COII, and CytB, with fragments between 200 and 300 bp, to separate species from Triatoma brasiliensis [41], Triatoma matogrossensis [19] and Triatoma rubrovaria subcomplexes [40], highlighting the effectiveness of our approach.
Populations of T. guazu and T. williami occur in the same municipality of Barra do Garças and their biology remains unknown. These sympatric populations show chromatic variations, but chromatic differences provide weak evidence to support distinct species. Triatoma infestans var. melanosoma Lent, Jurberg, Galvão, and Carcavallo, 1994 [42] and Triatoma infestans sensu stricto (Klug, 1834) [43] have sympatric populations, exhibit different color patterns and are the same species [39,44]. Conversely, individuals from different populations of Triatoma rubrovaria (Blanchard, 1843) [45] are polychromatic [13,46,47]. Indeed, Dale et al. [47] observed 16 different chromatic patterns in the collar, pronotum, and connexivum of this species, questioning the validity of these variations as diagnostic characters to designate new species.
Despite the chromatic patterns, which seems to be a continuous variation, the distinct number of heteropyknotic filaments in spermatogenesis is the only character to our knowledge that differentiate T. guazu from T. williami [25,48]. Perhaps both phenotypes are present in the two species and more research involving a better sampling strategy with sympatric and allopatric populations could shed light on this subject. We also emphasize the importance of population genetics studies to identify the presence (or the lack of) barriers to the gene flow of specimens with different chromatic patterns. So far, morphological traits, traditional and geometric morphometry, isozyme and DNA sequencing data prove that T. guazu is a junior synonym of T. williami.

Conclusions
Finally, we emphasize the importance of population genetics studies to identify the presence (or the lack of) barriers to the gene flow of specimens with different chromatic patterns. So far, morphological traits, traditional and geometric morphometry, comparison of genitalia in SEM, isozyme and DNA sequencing data prove that T. guazu is a junior synonym of T. williami. Taxonomy Table 1].