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. 2026 Jul 20;121(Suppl 1):e250293. doi: 10.1590/0074-02760250293

Cladistics of South American Triatoma (Hemiptera: Reduviidae): a morphological overview of T. infestans complex

João Paulo Sales Oliveira-Correia 1, Hélcio Reinaldo Gil-Santana 2, Cleber Galvão 1,+
PMCID: PMC13395083  PMID: 42484119

Abstract

BACKGROUND

Triatoma infestans complex comprises 39 species of South American triatomines, organised into different subcomplexes. These species act as vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. However, the evolutionary relationships among the subcomplexes remain unresolved.

OBJECTIVES

In this study, we present the most comprehensive morphological cladistic analysis of these species to date.

METHODS

Phylogenetic relationships were examined in a new analysis based on morphological data. We analysed a data matrix comprising 48 terminal taxa and 72 morphological characters, coded under implicit parsimony weights, with a range of concavities (k1-80).

FINDINGS

Our results indicate that the T. infestans and Triatoma rubrovaria subcomplexes constitute monophyletic groups, while Triatoma brasiliensis, Triatoma matogrossensis and Triatoma sordida subcomplexes are merophyletic. We also confirm that Triatoma maculata does not belong to the homonymous subcomplex.

MAIN CONCLUSIONS

The study presents the first cladistic analysis and provides a solid foundation for understanding phylogenetic relationships within of the T. infestans complex.

Key words: evolutionary relationship, subcomplexes, Triatominae, vectors, Chagas disease

Triatomines are insects of public health importance, acting as vectors of the protozoan Trypanosoma cruzi (Chagas, 1909), 1 the etiological agent of Chagas disease (CD). All triatomines, commonly known as kissing bugs, belong to the subfamily Triatominae Jeannel, 1919 (Hemiptera), which has 159 species (156 living and three fossils) distributed in five tribes and 19 genera. 2 3

The genus Triatoma Laporte, 1832 is composed of 76 species, divided into three groups, following their geographic distributions: (i) Triatoma rubrofasciata group, species from Central and North America, in addition to the Old World; (ii) Triatoma dispar group, species from the Andes Mountains; and (iii) Triatoma infestans group, species from South America. 4 5 6 7

Species of the T. infestans group are classified into specific complexes based on phenotypic similarity, geographic distribution, phylogeny and epidemiology. 7 Among these, the T. infestans complex encompasses a greater diversity of species, with 37 taxa, distributed into eight subcomplexes. 7 However, these groupings have undergone modifications, and the most recent proposal indicates the following subcomplexes: Triatoma brasiliensis, Triatoma costalimai, T. infestans, Triatoma maculata, Triatoma pseudomaculata, Triatoma rubrovaria, Triatoma sordida and Triatoma vitticeps. 8 9 10

Phylogenetic studies, with mitochondrial genes (with the markers 16S rDNA, 12S rDNA, cytochrome oxidase I, cytochrome oxidase II and cytochrome b), nuclear (with the markers 18S, 28S and Wingless) and ultraconserved elements, have indicated that the majority of subcomplexes do not represent a monophyletic group. 11 12 13 14 15

However, there is still no consensus regarding the characteristics that define these species groups, and further studies are needed to corroborate or refute their validity. 9 15 16 It is important to note that groups, complexes and subcomplexes are not taxonomic categories recognized by the International Code of Zoological Nomenclature; therefore, caution is recommended when proposing such groupings. 17

In the present study, we performed a cladistic analysis to test the monophyly of the current recognised subcomplexes within T. infestans complex and to investigate the evolutionary relationships.

MATERIALS AND METHODS

Selection of terminal taxa - The ingroup comprises all species of the T. infestans complex, which includes all South American species, except the five species belong to the T. dispar complex (Table). According to the previous hypotheses proposed by Hypša et al., 11 Gardim et al., 12 Justi et al., 13 14 Teves et al. 18 and Kieran et al., 15 and taking care to avoid the introduction of homoplasies associated with very distant taxa, the outgroup is composed by Dipetalogaster maxima (Uhler, 1894), Eratyrus mucronatus Stål, 1859, Nesotriatoma bruneri Usinger, 1944, Panstrongylus megistus (Burmeister, 1835), Panstrongylus tibiamaculatus (Pinto, 1926), Panstrongylus tupynambai Lent, 1942, Panstrongylus rufotuberculatus (Champion, 1899), Psammolestes tertius Lent & Jurberg, 1965, Rhodnius stali Lent, Jurberg & Galvão, 1993 and T. rubrofasciata (De Geer, 1773). These species are not closely related to the ingroup species and were used to polarise the characters, test the monophyly of the group, and root the resulting trees.

TABLE. List of material examined of the ingroup species, with respective deposit collections of types and non-type specimens.

Genus Species analysed (with status) Depository institutions
Triatoma arthurneivai Lent & Martins, 1940 (holotype) CTIOC
bahiensis Sherlock & Serafim, 1967 (syntype) CTIOC
baratai Carcavallo & Jurberg, 2000 (holotype) CTIOC
brasiliensis Neiva, 1911 (holotype) CEIOC
carcavalloi Jurberg, Rocha & Lent, 1998 (paratype) CTIOC
circummaculata (Stål, 1859) (syntype)* MfN
costalimai Verano & Galvão, 1958 (paratype) CTIOC
deaneorum Galvão, Souza & Lima, 1967 (holotype) CEIB
delpontei Romaña & Abalos, 1947 (paratype) CTIOC
garciabesi Carcavallo, Cichero, Martínez, Prosen & Ronderos, 1967 (holotype) CTIOC
guasayana Wygodzinsky & Abalos, 1949 (paratype) CTIOC
infestans (Klug, 1834) (non-type) CTIOC
jatai Gonçalves, Teves-Neves, Santos-Mallet, Fuente & Lopes, 2013 (paratype) CTIOC
juazeirensis Costa & Felix, 2007 (holotype) CEIOC
jurbergi Carcavallo, Galvão & Lent, 1998 (holotype) CTIOC
klugi Carcavallo, Jurberg, Lent & Galvão, 2001 (holotype) CTIOC
lenti Sherlock & Serafim, 1967 (holotype) CTIOC
limai Del Ponte, 1929 (holotype) CTIOC
maculata Erichson, 1848 (syntype)* MfN
matogrossensis (Leite & Barbosa, 1953) (non-type) CTIOC
melanica Argolo & Felix, 2006 (non-type) CEIOC
melanocephala Neiva & Pinto, 1923 (paratype) CTIOC
infestans var. melanosoma (Martínez, Olmedo & Carcavallo, 1987) (paratype) CTIOC
oliveirai (Neiva, Pinto & Lent, 1939) (holotype) CTIOC
patagonica Del Ponte, 1929 (non-type) CTIOC
petrocchiae Pinto & Barreto, 1925 (holotype) CTIOC
pintodiasi Jurberg, Cunha & Rocha, 2013 (holotype) CTIOC
platensis Neiva, 1913 (non-type) CTIOC
pseudomaculata Corrêa & Espínola, 1964 (holotype) CTIOC
rosai Alevi, Oliveira, Garcia, Cesaretto, Grzyb, Bittinelli, Resis, Ravazi, Bortolozo, Galvão, Azeredo-Oliveira, Tercilia & Madeira, 2020 (paratype) CTIOC
rubrofasciata (De Geer, 1773) (non-type) CTIOC
rubrovaria (Blanchard, 1843) (non-type) CTIOC
sherlocki Papa, Jurberg, Carcavallo, Cerqueira & Barata, 2002 (paratype) CTIOC
sordida (Stål, 1859) (non-type) CTIOC
vandae Carcavallho, Jurberg, Rocha, Galvão, Noireau & Lent, 2002 (holotype) CTIOC
vitticeps (Stål, 1859) (syntype)* MfN
williami Galvão, Souza & Lima, 1956 (paratype) CEIB and CTIOC
wygodzinskyi Lent, 1951 (paratype) CTIOC
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CTIOC: Coleção de Triatomíneos do Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; CEIB: Coleção Entomológica do Instituto Butantan, São Paulo, Brazil; CEIOC: Coleção Entomológica do Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; MfN: Hemimetabola Collection, Museum für Naturkunde, Berlin, Germany. *Analysed from photographs by Rodrigues et al. 48

Terminology, character and measurements - The adopted terminology mainly follows Lent & Wygodzinsky, 6 Weirauch 19 and Schuh & Weirauch. 20 The character survey was based on the original descriptions of each species and in publications by Galvão, 21 Lent et al., 22 23 24 25 Lent & Wygodzinsky, 6 Carcavallo et al., 26 27 Costa et al., 28 Weirauch, 19 Obara et al., 16 Jurberg et al., 29 Gonçalves et al., 30 Galvão & Dale, 31 Mendonça et al., 32 Rodrigues, 33 Alevi et al. 34 and Oliveira-Correia et al. 35

For measurements and photographs of the studied species, 213 specimens were used, of which 118 were females and 95 males [Supplementary data (Data 1)]. A Leica M205® stereoscopic microscope and a Leica DMC 2900 camera were used, with the aid of the version 4.9. All measurements are in millimetres (mm) [Supplementary data (Data 2, Fig. 1A-C)]. For the rostrum sensu Lent and Wygodzinsky, 6 we will use the term labium, as it has been used in more recent literature. 20 In almost all subfamilies of Reduviidae, including Triatominae, the first labial segment is lost or fused to the head capsule and is therefore not externally visible; the three visible segments correspond to segments II, III and IV. 29

The characters were coded by contingency, 36 following the standards of Sereno 37 and Brazeau. 38 The following characters have been encoded. The character matrix was created using the Mesquite 3.81 program 39 [Supplementary data (Table I)].

Phylogenetic analysis - We submitted the data set to a parsimony analysis under equal weights (EW) and implied weights (IW) in TNT 1.6. 40 The IW analysis carried the searches out under RAS+TBR, with a wide range of concavities (k3 to k80). The approach used here derives from Goloboff et al., 41 being the basic strategy to explore different parameters of the concavity constant (k) and group the searches when the Most-Parsimonious Trees (MPTs) are found, to investigate the effect of character weighting on hypotheses of relationships.

To make a robust and exhaustive analysis, the parameters used in all searches were 5000 TBR replications with 1000 trees saved per replication. To calculate the nodal support, we used TNT through bootstrap Poisson independent re-weighting and relative Bremer support. 42

RESULTS

The data matrix comprised 48 terminal taxa and 72 characters were encoded: 49 binary and 23 multistate [Supplementary data (Table II)]. The analysis recovered all Triatomini species studied, with 13 supported synapomorphies for both the EW and IW clades [Supplementary data (Fig. 2)]. EW analysis result in 21 MPTs (with L = 291, CI = 0.34, RI = 0.66) (Fig. 1).

Fig. 1: strict consensus of the 21 most parsimonious trees that resulted from the analysis under equal weights (EW) (L = 291, CI = 0.34, RI = 0.66). The classification follows. 7 49 Terminal taxa in colour: black = outgroup; red = Triatoma brasiliensis; green = Triatoma maculata; yellow = Triatoma matogrossensis; purple = Triatoma rubrovaria; grey = Triatoma infestans. (L = tree length, CI = consistency index, CR = retention index).

Fig. 1:

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For the IW analysis, we observed 12 strict consensus MPTs with the same topology. We chose the tree under k3 (Fig. 2, Fit = 2.18956, L = 250, CI = 0.41, RI = 0.59) for graphical representation, to map the Bootstrap and Bremer support values, to obtain character scores and basis for discussion. The disagreement between the EW and IW analyses was limited to the position of Triatoma oliveirai and the presence of polytomy in clades of the T. sordida, T. rubrovaria and T. brasiliensis subcomplexes (Fig. 2).

Fig. 2: strict consensus of the three most parsimonious trees that resulted from the analysis under implied weights (IW) with K = 3 (Fit = 2.18956, L = 250, CI = 0.41, RI = 0.59). Bremer support values (> 50) mapped above branches and bootstrap values over 50% are given below the nodes. Blue numbers on nodes (1-21) represent clades. The classification follows. 7 49 Terminal taxa in colour: black = outgroup; red = Triatoma brasiliensis; green= Triatoma maculata; yellow = Triatoma matogrossensis; purple = Triatoma rubrovaria; grey = Triatoma infestans. (L = tree length, CI = consistency index, CR = retention index).

Fig. 2:

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The Triatoma species recovered as a monophyletic group, except for Triatoma phyllosoma and T. rubrofasciata. However, P. tibiamaculatus is grouped close to the clade (T. vitticeps + Triatoma melanocephala) and distant from the other Panstrongylus species Berg, 1879 (Fig. 2).

We observed that the T. rubrovaria and T. infestans subcomplexes form monophyletic groups (Fig. 2). For the T. rubrovaria subcomplex, the clade (Triatoma pintodiasi + (Triatoma circummaculata + Triatoma limai) was supported by a synapomorphy: 32 (1), third visible segment of the labium as long as or longer than the second. It is worth noting that T. pintodiasi has the apomorphy (Fig. 3A); 9 (1), pronotum with predominantly orange or yellowish posterior lobe (Fig. 3B) [Supplementary data (Fig. 3.10)].

Fig. 3: characters highlighted in the analysis. (A) Triatoma pintodiasi, head, lateral view. (B) T. pintodiasi, dorsal view. (C) Triatoma deaneorum, posterior portion of the body, including its connexivum, dorsal view.

Fig. 3:

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Therefore, the T. brasiliensis subcomplex is not a monophyletic group, because T. vitticeps and T. melanocephala are distant from the other species of the subcomplex. Similarly, the T. maculata subcomplex is polyphyletic, with (Triatoma arthurneivai + Triatoma wygodzinskyi) being distant from T. maculata and T. pseudomaculata (Fig. 2).

The Triatoma matogrossensis and T. sordida subcomplexes are also not monophyletic groups (Fig. 2). Triatoma deaneorum is grouped in the same clade as most species of the T. sordida subcomplex, supported by a synapomorphy: 11 (1), pronotum, connexivum, in dorsal view, with dark spots in the shape of musical notes on the intersegmental suture (Fig. 3C). This clade is formed by all species of the T. sordida subcomplex, except Triatoma patagonica. On the other hand, the species of the T. matogrossensis subcomplex are close (Triatoma baratai + Triatoma jurbergi) and (Triatoma williami + (T. matogrossensis + Triatoma vandae), except T. costalimai and Triatoma jatai.

DISCUSSION

Among the characters used in the analysis, as well as previous taxonomic studies of triatomines, the connexivum spot pattern is a character frequently used in the comparison or diagnosis of species. However, recent studies have shown phenotypic variations in this character, which has resulted in the synonymies between some taxa (e.g., Triatoma melanosoma Lent, Jurberg, Galvão & Carcavallo, 1994 = T. infestans, currently T. infestans var. melanosoma) 24 and (Triatoma guazu Lent & Wygodzinsky, 1979 = T. williami). 35 In the present analysis, this character was used with caution and always in agreement with that presented in the type specimens examined, especially the primary types (name-bearing).

Our study presents the first evolutionary hypothesis based on morphological characters that encompasses most South American Triatoma species. The phylogenetic reconstructions included a larger number of taxa, including rare species that had been overlooked in previous studies. However, previous molecular analyses failed to clarify the deeper evolutionary relationships among clades. According to Justi et al., 13 state relationships within subcomplexes remain unresolved.

According to Schofield & Galvão, 7 T. melanocephala, Triatoma tibiamaculata (currently, P. tibiamaculatus nov. comb.) and T. vitticeps make up the T. brasiliensis subcomplex. Previous hypotheses have observed the close relationship between T. melanocephala and T. vitticeps. 15 However, Alevi et al., 9 based on morphological, phenotypic and genotype characteristics (cytogenetic and molecular), proposed the establishment of the T. vitticeps subcomplex (T. melanocephala, P. tibiamaculatus and T. vitticeps), since these species were distinct from all other subcomplexes of the T. infestans complex.

Phylogenetic analyses, based on molecular and cytogenetic data, have revealed a highly supported close relationship of P. tibiamaculatus with P. megistus. 13 15 43 According to Rodrigues, 33 these two species present the same characteristics in the terminal abdominal segments of the females (abdominal segments IX and X combined in the form of an elongated trapezoid) and (abdominal segments VII-X combined longer than wide), differentiating it from other South American Triatoma species. Our topology did not recover P. tibiamaculatus and P. megistus as sister species; however, we consider the morphological characteristics shared between P. tibiamaculatus and Triatoma spp. to represent homoplasies as suggested by Bitinelli et al. 43

The T. brasiliensis subcomplex was redefined as a monophyletic group composed of the species: T. brasiliensis, Triatoma juazeirensis, Triatoma melanica, Triatoma lenti, Triatoma petrocchiae and Triatoma sherlocki. 44 Here T. petrocchiae stands out as a sister species to the other species of the T. brasiliensis subcomplex, corroborating the hypotheses of Oliveira et al. 44 According to Kieran et al., 15 T. petrocchiae appears as a sister species to the clade [T. baratai + (T. williami + T. guazu) + (subcomplexo T. sordida)], as observed in the trees generated by data from Ultraconserved Elements + Ribosomal and morphological (based on chromatic pattern and presence of fossula spongiosa). In our morphological analysis, we verified the absence of fossula spongiosa in T. petrocchiae and T. sherlocki, which corroborates the hypothesis proposed by Kieran et al. 15 It is worth noting that the absence of fossula spongiosa is an ancestral characteristic, and their presence is a characteristic present in most Triatominae lineages.

There is no consensus regarding the T. maculata subcomplex, T. maculata consistently appears more distant from the other species within the subcomplex. 13 15 According to Galvão et al., 21 T. maculata and T. pseudomaculata can be distinguished by total body length, spot pattern and male genitalia. In our analysis, we highlighted the following characters for both species: presence of spots on the head; width of the eye in relation to the dorsal interocular space; size of the anterolateral angles; and presence of basal disc tubercles on the anterior lobe of the pronotum. According to Justi et al., 14 the separation of T. maculata from the other Triatoma species in the Southern Hemisphere, except for T. melanocephala and T. vitticeps, in agreement with our analyses, is associated with the uplift of the Northern Andes (23-10 Ma). Consequently, the ancestral population of T. maculata was restricted to the Guiana Shield.

Triatoma arthurneivai, T. pseudomaculata and T. wygodzinskyi are grouped in the T. maculata subcomplex. 7 Our morphological analysis recovered these three species at distant terminals, except for the clade (T. arthurneivai + T. wygodzinskyi). However, according to Lent & Wygodzinsky, 6 T. arthurneivai and T. wygodzinskyi are very similar morphologically, but can be distinguished by the male genitalia, spot pattern of the pronotum and corium of the hemelytron. Such characters were not used in the current analysis, as they presented many intraspecific variations. Thus, as suggested by Masarin et al., 10 the existence of the new T. pseudomaculata subcomplex was not corroborated in our morphological analysis.

Previous studies indicate that the T. matogrossensis subcomplex should be disregarded, as it represents a paraphyletic group, with its species relocated to the T. sordida and T. maculata subcomplexes. 8 12 13 15 Likewise, existing hypotheses concerning the T. sordida subcomplex suggest a close relationship with some of the species of the T. matogrossensis subcomplex (i. e., T. matogrossensis e T. vandae), 8 12 13 15 18 which was not supported by our analysis. We consider our study to provide a more comprehensive perspective, as it includes morphological data from all species of the T. sordida subcomplex and T. matogrossensis, encompassing rare species and those not previously included in phylogenetic analyses.

In the present analysis T. deaneorum, little-reported species not analysed in existing hypotheses, was included. 12 13 14 15 T. deaneorum proved to be a sister taxon of [Triatoma guasayana + (Triatoma rosai + Triatoma garciabesi + T. sordida)], forming a clade supported by one synapomorphy: 11(1), spot pattern of the dorsal connexivum plates in the form of musical notes, covering the intersegmental sutures. In contrast, Lent and Wygodzinsky 6 suggested this species was a hybrid of T. infestans and T. williami. However, our phylogenetic topologies do not refute a close relationship between this species and T. deaneorum.

The close relationship of T. garciabesi, T. rosai and T. sordida was the same for EW and IW. About two decades, T. garciabesi remained synonymous with T. sordida. 6 These two species were separated by morphological, morphometric, isoenzymatic, cytogenetic and phylogenetic studies, resulting in the revalidation of T. garciabesi. 45 46 47

Triatoma rosai was described by an integrative approach, using morphological, morphometric, molecular data and experimental crossings. 34 Our study confirms the delimitation of T. rosai (previously termed as intraspecific variation of T. sordida from Argentina) and T. sordida sensu stricto. 34 The following diagnostic character was highlighted: for T. sordida, the posterior process of the scutellum is as long as or longer than the main body of the scutellum, whereas in T. rosai it is shorter.

Triatoma costalimai and T. jatai are considered part of the T. matogrossensis subcomplex. 5 According to Pita et al., 8 these two species would be grouped into the new T. arthurneivai subcomplex (current T. pseudomaculata subcomplex proposed by Masarin et al. 10 ), which does not corroborate our hypothesis. However, Masarin et al., 10 based on experimental crosses and molecular data, proposed the creation of the new T. costalimai subcomplex composed of T. costalimai and T. jatai. These two species are morphologically similar and can be differentiated by the proportion and general coloration of the body, the pattern of connective tissue spots, hairiness on the labial segments, and the shape of the male genitalia. 30 In the present analysis, T. costalimai and T. jatai were recovered as sister species and highlighted a new diagnostic character for such species: for T. jatai, submedian carina of the pronotum, reaching the distal margin of the posterior lobe and, for T. costalimai, limited to the median region of the posterior lobe.

Currently, there is no consensus on which and how many species are part of the T. rubrovaria subcomplex. 8 13 14 15 According to Pita et al. 8 and Kieran et al., 18 T. patagonica and T. guasayana make up the T. rubrovaria subcomplex, which was not observed in the current analysis and previous studies. 8 12 15 47

Among the species of the T. rubrovaria subcomplex it was possible to observe the clade (T. pintodiasi + (T. circummaculata + T. limai)). According to Jurberg et al., 29 these three species present marked morphological similarity, since our analyses indicate the following diagnostic characters for T. circummaculata and T. limai: predominant coloration of the posterior lobe, pattern of spots on the dorsal plates of the connexivum, length of the third antennal segment in relation to the second and the shape of the posterior process of the scutellum.

All species of the T. infestans subcomplex were established in both EW and IW analyses. The EW reconstruction showed T. oliveirai in the same clade as the other species of the T. infestans subcomplex. Although additional evidence is required, T. oliveirai may belong to this a subcomplex, as the species occur sympatrically in southern Brazil. We can observe that the T. infestans subcomplex was monophyletic in all analyses, but we cannot clearly highlight the evolutionary relationship with the other species studied.

However, we highlight that: (i) the T. infestans and T. rubrovaria subcomplexes form monophyletic groups; (ii) the T. brasiliensis subcomplex proved to be polyphyletic: T. melanocephala and T. vitticeps form a distant clade and T. sherlocki and T. petrocchiae form a clade sister to most of the species in the subcomplex; (iii) we confirmed the inappropriateness of the T. matogrossensis subcomplex and the close relationship of T. deaneorum with the T. sordida subcomplex; (iv) T. maculata is not part of the subcomplex that bears its name; and (v) T. patagonica is not included in the T. sordida subcomplex.

In conclusion, this study presents the first comprehensive cladistic analysis of the T. infestans complex, including all species, except for the recently described Triatoma chiarii. 3 This work provides a solid foundation for understanding the phylogenetic relationships within this complex.

SUPPLEMENTARY MATERIALS

Supplementary material

ACKNOWLEDGEMENTS

To Flávia Virgino, curator of the Entomological Collection/Butantan; Márcio Felix, curator of the Entomological Collection/Fiocruz; and Hugo Lopes Guimarães, curator of the Triatominae Collection, for support and unrestricted access to the collection. We also thank Jader de Oliveira for the photographs of the Triatoma williami and Triatoma deaneorum specimens from the Entomological Collection/Butantan.

DATA AVAILABILITY

The present study examined adult specimens of both sexes [Supplementary data]. All specimens are deposited in the following entomological collections: CTIOC - Coleção de Triatomíneos, Instituto Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil; CEIOC - Coleção Entomológica, Instituto Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil; CEIB - Coleção Entomológica, Instituto Butantan, São Paulo, Brazil; MfN - Hemimetabola Collection, Museum für Naturkunde, Berlin, Germany.

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Mem Inst Oswaldo Cruz.

FIRST REVIEW ROUND - REVIEWERS COMMENTS

Anonymous

REVIEWER #1

Dear authors,

The manuscript presents an important study on the Triatoma infestans complex. Therefore, several aspects of this work deserve attention before publication.

There are already robust doctoral theses on the web that have not been published due to a lack of support in the morphological cladistics of these species.

Many groups, when working with cladistics, use representatives from more distant groups to better root the trees; I believe you are aware of this. Aspects such as the lack of supplementary table 5 with morphological characters are difficult to evaluate. I have made other serious suggestions in the attached file that should be considered. Some references are already outdated (as in the second paragraph of the introduction). We need to know the reason for the choice of species as "outgroup".

I believe that this manuscript, in its current form, should not be published in Memórias.

I await news.

All the best.

REVIEWER #2

This manuscript provides the most comprehensive morphological cladistic analysis to date of South American Triatoma, particularly the T. infestans complex. The study is methodologically sound and based on extensive examination of type and reference specimens. The use of both equal and implied weighting, coupled with bootstrap and Bremer support, is appropriate and strengthens the morphological framework presented.

Nevertheless, the paper would benefit from a more critical discussion of the continued use of traditional subcomplexes. The results clearly demonstrate that several of these assemblages are not monophyletic, consistent with extensive molecular and cytogenetic evidence (e.g., for T. guasayana, T. patagonica, T. maculata). Maintaining these outdated categories risks obscuring, rather than clarifying, the true evolutionary relationships within Triatoma. I strongly encourage the authors to address whether the subcomplex framework remains conceptually valid or should be replaced by clades supported by integrative data.

Additionally, Figures 1 and 2 visually group taxa under color codes corresponding to these non-monophyletic subcomplexes. This graphical choice may mislead readers by implying monophyly where it is not supported. I recommend revising the figures so that colors or labels only represent recovered monophyletic clades, or alternatively, indicate non-monophyletic assemblages with neutral tones or dashed outlines.

Overall, this study is a valuable contribution to Triatominae systematics, and its impact would be enhanced by aligning the interpretation and visual presentation with the phylogenetic results and with modern integrative frameworks.

AUTHORS' RESPONSE TO THE REVIEWERS

Dear Dr. Adeilton Brandão,

Please find below a detailed document containing our responses to the reviewers' comments.

We sincerely appreciate the time and valuable suggestions provided by the reviewers, which have significantly contributed to improving the quality and clarity of our work. All comments were carefully addressed in the revised manuscript, and the corresponding changes are clearly indicated.

Thank you for your consideration.

Reviewer 1

Dear authors,

The manuscript presents an important study on the Triatoma infestans complex. Therefore, several aspects of this work deserve attention before publication.

There are already robust doctoral theses on the web that have not been published due to a lack of support in the morphological cladistics of these species.

Many groups, when working with cladistics, use representatives from more distant groups to better root the trees; I believe you are aware of this. Aspects such as the lack of supplementary table 5 with morphological characters are difficult to evaluate. I have made other serious suggestions in the attached file that should be considered. Some references are already outdated (as in the second paragraph of the introduction). We need to know the reason for the choice of species as "outgroup".

I believe that this manuscript, in its current form, should not be published in Memórias.

I await news.

All the best.

A: Dear Reviewer,

We appreciate your careful reading and detailed considerations regarding our manuscript, as well as your recognition of the relevance of the study on the Triatoma infestans complex. Your observations were fundamental to the improvement of the manuscript.

Regarding the observation about the existence of unpublished theses due to the fragility of morphological cladistic support, we emphasize that the present study was conducted with rigorous criteria for taxon selection, encompassing most species of the complex and a greater number of morphological characters compared to previous studies. The character set was thoroughly reviewed and subjected to parsimony analyses with different search strategies, aiming for greater robustness and support for the recovered clades.

The choice of the outgroup is justified in the manuscript, considering that our study seeks to observe the phylogenetic relationships of some Triatoma species, and excessively distant taxa can compromise character polarization, correct rooting of the tree, and increase homoplasies.

Regarding the absence of Supplementary Table 5, we acknowledge the error in the submission. The table, containing the complete and detailed list of morphological characters and their states, has now been submitted. The other suggestions were included. The modifications made were substantial, increasing the clarity of the text and the analyses.

Reviewer: Why not use the paper that described the nineteenth genera?

A: The suggestion was incorporated. In addition, the species number was updated, as well as the citation of the most recently described species, Triatoma chiarii.

Reviewer: The references are very outdated; we have new groups being proposed now.

A: Since Schofield & Galvão (2009), Triatoma has traditionally been divided into three major groups: rubrofasciata, dispar, and infestans. To date, this general classification has been maintained. However, several modifications have been proposed within these groups, including the establishment of new subcomplexes, the suppression or redefinition of others, as well as changes in their composition. As discussed further below in the text.

Additionally, the number of existing species in Triatoma was modified from 75 to 76 species due to the description of Triatoma chiarii.

Reviewer: I was only able to view 47 references.

A: Typo. Corrected. Instead of 76, it was changed to 7.

Reviewer: Add reference Schofield and Galvão 2009?

A: Done.

Reviewer: (7-9)

A: Done. With reference to the description of Triatoma chiarii. Thus, it became (8-10).

Reviewer: In fact, the authors need to better explain why they chose these species, since only triatomines were selected.

A: The text has been modified for better understanding.

Reviewer: When we talk about cladistics, your selected species are all representative of Triatominae, and we needed representatives of Reduviidae, as in the works you referenced.

A: The use of excessively distant taxa as an outgroup is not recommended, since large phylogenetic distances can compromise the proper polarization of characters and the correct rooting of the tree. Very distant outgroups tend to introduce homoplasies, long-branch attraction effects, and ambiguities in the interpretation of character states, reducing the reliability of phylogenetic inferences. Therefore, the outgroup should be phylogenetically close enough to the ingroup to share homologous characters, but clearly external to it, ensuring more robust and consistent analyses. In this study, the ingroup consisted exclusively of South American species of Triatoma; for this reason, species that were not so distant, as well as the Old-World species Triatoma rubrofasciata and representatives of other genera from the tribes Triatomini and Rhodniini, were used as outgroups. Therefore, it is plausible to restrict the ingroup to triatomines.

Reviewer: Common museum abbreviation: MfN.

A: Done.

Reviewer: Why weren't the specimens from the outgroup listed here?

A: The outgroup is listed in the supplementary material.

Reviewer: When using specimens that are not from the type series, a bias is created with the phenotypic characteristics.

A: This is a typographical error. The type specimen of Triatoma brasiliensis has been analyzed and is deposited in the Coleção Entomológica de Instituto Oswaldo Cruz (CEIOC), as indicated in the supplementary material. The text has been corrected.

Reviewer: Several species were analyzed through photography; how can we be sure of the information?

A: The use of photographs as a data source was carried out carefully and did not replace, when available, the direct analysis of specimens. The images used correspond to high-resolution photographs from the study by Rodrigues et al (2020). List of the type specimens of Triatominae (Hemiptera: Heteroptera: Reduviidae) in the Hemimetabola Collection of the Museum für Naturkunde, Berlin. Zootaxa. 2020; 4809 (2): 329-348. These images allowed for the reliable observation of diagnostic morphological characters widely used in the taxonomy of Triatominae. Whenever possible, the information obtained through photographs was compared with original descriptions, taxonomic revisions, and directly examined material, ensuring the consistency and reliability of the data used.

Reviewer: I believe it is necessary to compile a list of the localities of each specimen used that are not "types". Especially for these cosmopolitan species.

A: The list can be found in the supplementary material.

Reviewer: We don't know how many specimens or the sex of each species was used.

A: The list is in the supplementary material. However, the quantity has been included in the text.

Reviewer: Using TNT nowadays is obsolete. I believe that for the dataset, 5000 replications is not an exhaustive analysis.

A: We recognize that there are currently several approaches and software widely used in phylogenetic inference. However, we opted for the use of TNT due to its recognized suitability for the analysis of morphological data and its proven efficiency in parsimony-based inferences. To ensure the robustness of the results, we performed 5,000 replications, employing equal and implicit weighting, as well as advanced heuristic search algorithms (e.g., New Technology Search, including sectoral searches, ratchet, and tree drifting). Additionally, independent searches were conducted, all converging to consistent topologies, indicating adequate exploration.

Recently, Goloboff & Morales (2023) presented an updated version of TNT, demonstrating its effectiveness in inferring trees with groups of different quantities of taxa and in extensive datasets, in addition to highlighting the program's efficiency in parsimony analyses, especially when combined with advanced heuristic strategies applied to morphological data. Recent studies corroborate the continued use of TNT as a robust tool for parsimony inference in insect morphological matrices. For example, analyses with beetles of the genus Tiaronthophagus employed TNT for the simultaneous evaluation of discrete and continuous characters, using multiple heuristic search methods; Molina & Gil-Azevedo (2021) proposed the phylogeny of neotropical and afrotropical Simulium based on parsimonious analyses; Gutérrez-Tejo et al. (2024) used TNT v1.6 with new technology search strategies and bootstrap support to infer phylogenetic relationships among Tetraopes species; and Li et al. (2025) applied TNT in analyses of Neuroptera (Mantispoidea), combining traditional and advanced searches and reporting node support by bootstrap and Bremer indices. Taken together, these examples demonstrate that TNT remains widely used in recent literature, particularly in phylogenetic studies of insects based on morphological characters or integrated datasets.

References

Roggero, A.; Moretto, P.; Barbero, E.; Palestrini, C. The Phylogenetic Relationships of Tiaronthophagus n.gen. (Coleoptera, Scarabaeidae, Onthophagini) Evaluated by Phenotypic Characters. Insects 2019, 10, 64. https://doi.org/10.3390/insects10030064

Molina Ó S; Gil-Azevedo, Leonardo H. Simulium (Trichodagmia) (Diptera, Simuliidae) phylogeny revisited: the Neotropical and Afrotropical connection. European Journal of Taxonomy, v. 773, p. 80-119, 2021. https://doi.org/10.5852/ejt.2021.773.1517

Gutiérrez-Trejo, N., Van Dam, M.H., Lam, A.W. et al. Phylogenomics of Tetraopes longhorn beetles unravels their evolutionary history and biogeographic origins. Sci Rep 14, 7285 (2024). https://doi.org/10.1038/s41598-024-57827-z

Goloboff, P. A., & Morales, J. C. (2023). TNT version 1.6, with a graphical interface for MacOS and Linux, including parallelization. Cladistics, 39(2), 144–153. https://doi.org/10.1111/cla.12524

Hongyu Li, De Zhuo, Bo Wang, Hiroshi Nakamine, Shûhei Yamamoto, Weiwei Zhang, James E Jepson, Michael Ohl, Ulrike Aspöck, Horst Aspöck, Thet Tin Nyunt, Horst Aspöck, Thet Tin Nyunt, Michael S Engel, Michael J Benton, Philip Donoghue, Xingyue Liu, A Double-Edged Sword: Evolutionary Novelty Along Deep-Time Diversity Oscillation in an Iconic Group of Predatory Insects (Neuroptera: Mantispoidea), Systematic Biology, Volume 74, Issue 3, May 2025, Pages 395–420, https://doi.org/10.1093/sysbio/syae068

Reviewer: Here we can see an inconsistency that the limited data provides; Triatoma rubrofasciata could not be Triatoma, according to your inference.

A: Considering that the objective of the analysis was exclusively to investigate the phylogenetic relationships between the species of the Triatoma infestans complex and its respective subcomplexes, it was not intended to evaluate whether Triatoma rubrofasciata belongs to the genus Triatoma or not. A proper definition of the taxonomic position of T. rubrofasciata would require a more comprehensive phylogenetic analysis, including all species of the genus, with the incorporation of taxa from North America, the Old World and Linshcosteus, to which this species is most closely related (according to Schofield & Galvão, 2009; Justi et al., 2014, 2016; Kieran et al., 2021), in addition to species from the Andes, other regions of South America, and the Spinolai complex.

Reference:

Schofield CJ, Galvão C. Classification, evolution, and species groups within the Triatominae. Acta Tropica. 2009; 110: 88-100.

Justi SA, Russo CA, Mallet JR, Obara MT, Galvão C. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasites & Vectors. 2014; 7: 137-149.

Justi SA, Galvão C, Schrago CG. Geological changes of the Americas and their influence on the diversification of the Neotropical kissing bugs (Hemiptera: Reduviidae: Triatominae). Plos Neglected Tropical Diseases. 2016; 10 (4): 1-22.

Kieran TJ, Gordon ERL, Zaldívar-Riverón A, Ibara-Cerdeña CN, Glenn TC, Weirauch C. Ultraconserved elements reconstruct the evolution of Chagas disease vectoring kissing bugs (Reduviidae: Triatominae). Systematic Entomology, 2021; 1-16.

Reviewer: We can see some inconsistencies here. This needs to be explained in the discussion since it contradicts more recent work.

A: The suggestions were included in the text.

Reviewer 2

This manuscript provides the most comprehensive morphological cladistic analysis to date of South American Triatoma, particularly the T. infestans complex. The study is methodologically sound and based on extensive examination of type and reference specimens. The use of both equal and implied weighting, coupled with bootstrap and Bremer support, is appropriate and strengthens the morphological framework presented.

Nevertheless, the paper would benefit from a more critical discussion of the continued use of traditional subcomplexes. The results clearly demonstrate that several of these assemblages are not monophyletic, consistent with extensive molecular and cytogenetic evidence (e.g., for T. guasayana, T. patagonica, T. maculata). Maintaining these outdated categories risks obscuring, rather than clarifying, the true evolutionary relationships within Triatoma. I strongly encourage the authors to address whether the subcomplex framework remains conceptually valid or should be replaced by clades supported by integrative data.

Additionally, Figures 1 and 2 visually group taxa under color codes corresponding to these non-monophyletic subcomplexes. This graphical choice may mislead readers by implying monophyly where it is not supported. I recommend revising the figures so that colors or labels only represent recovered monophyletic clades, or alternatively, indicate non-monophyletic assemblages with neutral tones or dashed outlines.

Overall, this study is a valuable contribution to Triatominae systematics, and its impact would be enhanced by aligning the interpretation and visual presentation with the phylogenetic results and with modern integrative frameworks.

A: We appreciate the constructive comments, as well as the positive assessment regarding the comprehensiveness and analytical rigor of the manuscript. The discussion was implemented to critically address these points, as well as the other suggestions.

The combined use of equal and implicit weighting, together with supporting metrics, is especially appreciated, as it reinforces the consistency of the presented morphological structure reinforces the consistency of the presented morphological structure. The results obtained show that several subcomplexes recognized within Triatoma are not monophyletic groups, consistent with independent evidence from molecular and cytogenetic data. This issue remains central and there is no consensus.

We opted to maintain the use of colors in Figures 1 and 2, since this scheme follows the pattern employed in previous studies on the subcomplexes and species of the Triatoma infestans complex and related groups, facilitating the comparison and discussion of the results in the context of previous literature. We emphasize that, throughout the manuscript, we explicitly stated that these chromatic groupings correspond to historically proposed and admittedly non-monophyletic subcomplexes, being used only as a visual and comparative resource. Even so, we revised the legends and the text to make this caveat even clearer, minimizing any possibility of misinterpretation.

Below are other issues highlighted by the reviewers.

Reviewer: Why not use the paper that described the nineteenth genera?

A: The suggestion was incorporated. In addition, the species number was updated, as well as the citation of the most recently described species, Triatoma chiarii.

Reviewer: The references are very outdated; we have new groups being proposed now.

A: Since Schofield & Galvão (2009), Triatoma has traditionally been divided into three major groups: rubrofasciata, dispar, and infestans. To date, this general classification has been maintained. However, several modifications have been proposed within these groups, including the establishment of new subcomplexes, the suppression or redefinition of others, as well as changes in their composition. As discussed further below in the text.

Additionally, the number of existing species in Triatoma was modified from 75 to 76 species due to the description of Triatoma chiarii.

Reviewer: I was only able to view 47 references.

A: Typo. Corrected. Instead of 76, it was changed to 7.

Reviewer: Add reference Schofield and Galvão 2009?

A: Done.

Reviewer: (7-9)

A: Done. With reference to the description of Triatoma chiarii. Thus, it became (8-10).

Reviewer: In fact, the authors need to better explain why they chose these species, since only triatomines were selected.

A: The text has been modified for better understanding.

Reviewer: When we talk about cladistics, your selected species are all representative of Triatominae, and we needed representatives of Reduviidae, as in the works you referenced.

A: The use of excessively distant taxa as an outgroup is not recommended, since large phylogenetic distances can compromise the proper polarization of characters and the correct rooting of the tree. Very distant outgroups tend to introduce homoplasies, long-branch attraction effects, and ambiguities in the interpretation of character states, reducing the reliability of phylogenetic inferences. Therefore, the outgroup should be phylogenetically close enough to the ingroup to share homologous characters, but clearly external to it, ensuring more robust and consistent analyses. In this study, the ingroup consisted exclusively of South American species of Triatoma; for this reason, species that were not so distant, as well as the Old-World species Triatoma rubrofasciata and representatives of other genera from the tribes Triatomini and Rhodniini, were used as outgroups. Therefore, it is plausible to restrict the ingroup to triatomines.

Reviewer: Common museum abbreviation: MfN.

A: Done.

Reviewer: Why weren't the specimens from the outgroup listed here?

A: The outgroup is listed in the supplementary material.

Reviewer: When using specimens that are not from the type series, a bias is created with the phenotypic characteristics.

A: This is a typographical error. The type specimen of Triatoma brasiliensis has been analyzed and is deposited in the Coleção Entomológica de Instituto Oswaldo Cruz (CEIOC), as indicated in the supplementary material. The text has been corrected.

Reviewer: Several species were analyzed through photography; how can we be sure of the information?

A: The use of photographs as a data source was carried out carefully and did not replace, when available, the direct analysis of specimens. The images used correspond to high-resolution photographs from the study by Rodrigues et al (2020). List of the type specimens of Triatominae (Hemiptera: Heteroptera: Reduviidae) in the Hemimetabola Collection of the Museum für Naturkunde, Berlin. Zootaxa. 2020; 4809 (2): 329-348. These images allowed for the reliable observation of diagnostic morphological characters widely used in the taxonomy of Triatominae. Whenever possible, the information obtained through photographs was compared with original descriptions, taxonomic revisions, and directly examined material, ensuring the consistency and reliability of the data used.

Reviewer: I believe it is necessary to compile a list of the localities of each specimen used that are not "types". Especially for these cosmopolitan species.

A: The list can be found in the supplementary material.

Reviewer: We don't know how many specimens or the sex of each species was used.

A: The list is in the supplementary material. However, the quantity has been included in the text.

Reviewer: Using TNT nowadays is obsolete. I believe that for the dataset, 5000 replications is not an exhaustive analysis.

A: We recognize that there are currently several approaches and software widely used in phylogenetic inference. However, we opted for the use of TNT due to its recognized suitability for the analysis of morphological data and its proven efficiency in parsimony-based inferences. To ensure the robustness of the results, we performed 5,000 replications, employing equal and implicit weighting, as well as advanced heuristic search algorithms (e.g., New Technology Search, including sectoral searches, ratchet, and tree drifting). Additionally, independent searches were conducted, all converging to consistent topologies, indicating adequate exploration.

Recently, Goloboff & Morales (2023) presented an updated version of TNT, demonstrating its effectiveness in inferring trees with groups of different quantities of taxa and in extensive datasets, in addition to highlighting the program's efficiency in parsimony analyses, especially when combined with advanced heuristic strategies applied to morphological data. Recent studies corroborate the continued use of TNT as a robust tool for parsimony inference in insect morphological matrices. For example, analyses with beetles of the genus Tiaronthophagus employed TNT for the simultaneous evaluation of discrete and continuous characters, using multiple heuristic search methods; Molina & Gil-Azevedo (2021) proposed the phylogeny of neotropical and afrotropical Simulium based on parsimonious analyses; Gutérrez-Tejo et al. (2024) used TNT v1.6 with new technology search strategies and bootstrap support to infer phylogenetic relationships among Tetraopes species; and Li et al. (2025) applied TNT in analyses of Neuroptera (Mantispoidea), combining traditional and advanced searches and reporting node support by bootstrap and Bremer indices. Taken together, these examples demonstrate that TNT remains widely used in recent literature, particularly in phylogenetic studies of insects based on morphological characters or integrated datasets.

References

Roggero, A.; Moretto, P.; Barbero, E.; Palestrini, C. The Phylogenetic Relationships of Tiaronthophagus n.gen. (Coleoptera, Scarabaeidae, Onthophagini) Evaluated by Phenotypic Characters. Insects 2019, 10, 64. https://doi.org/10.3390/insects10030064

Molina Ó S; Gil-Azevedo, Leonardo H. Simulium (Trichodagmia) (Diptera, Simuliidae) phylogeny revisited: the Neotropical and Afrotropical connection. European Journal of Taxonomy, v. 773, p. 80-119, 2021. https://doi.org/10.5852/ejt.2021.773.1517

Gutiérrez-Trejo, N., Van Dam, M.H., Lam, A.W. et al. Phylogenomics of Tetraopes longhorn beetles unravels their evolutionary history and biogeographic origins. Sci Rep 14, 7285 (2024). https://doi.org/10.1038/s41598-024-57827-z

Goloboff, P. A., & Morales, J. C. (2023). TNT version 1.6, with a graphical interface for MacOS and Linux, including parallelization. Cladistics, 39(2), 144–153. https://doi.org/10.1111/cla.12524

Hongyu Li, De Zhuo, Bo Wang, Hiroshi Nakamine, Shûhei Yamamoto, Weiwei Zhang, James E Jepson, Michael Ohl, Ulrike Aspöck, Horst Aspöck, Thet Tin Nyunt, Horst Aspöck, Thet Tin Nyunt, Michael S Engel, Michael J Benton, Philip Donoghue, Xingyue Liu, A Double-Edged Sword: Evolutionary Novelty Along Deep-Time Diversity Oscillation in an Iconic Group of Predatory Insects (Neuroptera: Mantispoidea), Systematic Biology, Volume 74, Issue 3, May 2025, Pages 395–420, https://doi.org/10.1093/sysbio/syae068

Reviewer: Here we can see an inconsistency that the limited data provides; Triatoma rubrofasciata could not be Triatoma, according to your inference.

A: Considering that the objective of the analysis was exclusively to investigate the phylogenetic relationships between the species of the Triatoma infestans complex and its respective subcomplexes, it was not intended to evaluate whether Triatoma rubrofasciata belongs to the genus Triatoma or not. A proper definition of the taxonomic position of T. rubrofasciata would require a more comprehensive phylogenetic analysis, including all species of the genus, with the incorporation of taxa from North America, the Old World and Linshcosteus, to which this species is most closely related (according to Schofield & Galvão, 2009; Justi et al., 2014, 2016; Kieran et al., 2021), in addition to species from the Andes, other regions of South America, and the Spinolai complex.

Reference:

Schofield CJ, Galvão C. Classification, evolution, and species groups within the Triatominae. Acta Tropica. 2009; 110: 88-100.

Justi SA, Russo CA, Mallet JR, Obara MT, Galvão C. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasites & Vectors. 2014; 7: 137-149.

Justi SA, Galvão C, Schrago CG. Geological changes of the Americas and their influence on the diversification of the Neotropical kissing bugs (Hemiptera: Reduviidae: Triatominae). Plos Neglected Tropical Diseases. 2016; 10 (4): 1-22.

Kieran TJ, Gordon ERL, Zaldívar-Riverón A, Ibara-Cerdeña CN, Glenn TC, Weirauch C. Ultraconserved elements reconstruct the evolution of Chagas disease vectoring kissing bugs (Reduviidae: Triatominae). Systematic Entomology, 2021; 1-16.

Reviewer: We can see some inconsistencies here. This needs to be explained in the discussion since it contradicts more recent work.

A: The suggestions were included in the text.

Mem Inst Oswaldo Cruz.

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Associated Data

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    Supplementary Materials

    Supplementary material
    1678-8060-mioc-121-s1-e250293-s1.pdf (6.3MB, pdf)

    Data Availability Statement

    The present study examined adult specimens of both sexes [Supplementary data]. All specimens are deposited in the following entomological collections: CTIOC - Coleção de Triatomíneos, Instituto Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil; CEIOC - Coleção Entomológica, Instituto Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil; CEIB - Coleção Entomológica, Instituto Butantan, São Paulo, Brazil; MfN - Hemimetabola Collection, Museum für Naturkunde, Berlin, Germany.

    Articles from Memórias do Instituto Oswaldo Cruz are provided here courtesy of Instituto Oswaldo Cruz

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