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Trends in Taxonomy of Chagas Disease Vectors (Hemiptera, Reduviidae, Triatominae): From Linnaean to Integrative Taxonomy

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi and transmitted mainly by members of the subfamily Triatominae. There are currently 157 species, grouped into 18 genera and five tribes. Most descriptions of triatomine species are based on classical taxonomy. Facing evolutionary (cryptic speciation and phenotypic plasticity) and taxonomic (more than 190 synonymizations) problems, it is evident that integrative taxonomy studies are an important and necessary trend for this group of vectors. Almost two-and-a-half centuries after the description of the first species, we present for the first time the state-of-the-art taxonomy of the whole subfamily, covering from the initial classic studies to the use of integrative taxonomy.


Triatominae: The Vectors of Chagas Disease
Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) [1]. This disease is found mainly in 21 Latin American countries, where it is mostly vector-borne, more specifically by members of the subfamily Triatominae (Hemiptera, Reduviidae) [1]. Triatomines or kissing bugs are hematophagous insects that have a habit of defecating during or after the blood meal-if they are infected with T. cruzi, they release the parasite in the feces/urine [1]. An estimated 8 million people are infected worldwide, and more than 65 million people at risk of acquiring the disease, which causes more than 12,000 deaths per year, the vector control being the most useful method to prevent new infections [1,2].
There are currently 157 species (154 extant species and three fossils), grouped into 18 genera and five tribes (Table 1) [3][4][5][6][7], being all potential vectors of T. cruzi. Taxonomic studies of Triatominae started in the 18th century with the description of Triatoma rubrofasciata (De Geer, 1773) (as Cimex rubro-fasciatus) [8]. Almost two and a half centuries after the description of the first species, we presented for-the first time-a review of the state-of-the-art of taxonomy of the whole subfamily, covering from the initial classic studies to the use of integrative taxonomy, a term formally introduced only in 2005 to describe taxa by integrating information from different data and methodologies [9,10].

Applications and Limitations of Triatominae Taxonomic Studies
For 225 years (1773-1998), the descriptions of triatomine species have been based only on studies of classical taxonomy (using descriptive morphology, comparative morphology, and/or morphometry) ( Table 2). Although these analyses are imperative and are present in the description of all species of the subfamily Triatominae (Table 2), in the last decade, other approaches (such as biochemical [5,11], cytogenetic [5,12], phylogenetic [5,[13][14][15][16][17] and/or of reproductive barriers [5]) started to be combined with the characterization of morphology and/or morphometry, employing the integrative taxonomy in the study of these insect vectors (Table 2).
More than 190 synonymization acts occurred in the subfamily Triatominae [18,19], with the majority of synonymized taxa being described from classical taxonomy. The use of combined analyses for the characterization of a taxon greatly reduces the chances of synonymization (although it does not make it impossible [19,20]). Based on the synonymization events and the importance of multi-analyses for the characterization of a taxon, we will discuss the current issues, applications, and limitations of classical, molecular, and integrative taxonomy. 79 Triatoma dispar (Lent, 1950

Classical Taxonomy
Classical taxonomy underlies most taxonomic studies of species description in the subfamily Triatominae ( Table 2). The morphological and morphometric studies applied in the last described taxa are: morphological study of the head, thorax, abdomen, and male and female genitalia (with optical microscopy (OM) and/or scanning electronic microscopy (SEM)), and morphometric study of the head, thorax, abdomen and appendices (using OM) [5][6][7][15][16][17]132].
Although the use of morphological and morphometric characters is essential to describe a new taxon (since the diagnosis of the species needs to be made based on specimens that will be deposited, such as vouchers, in entomological collections), evolutionary events of cryptic speciation [14] and phenotypic plasticity [14] [15] represent two of the four paraphyletic strains of R. robustus Larrousse, 1927 [134,135] (the application of integrative taxonomy allowed description of the species from specimens initially characterized as R. robustus [136]). On the other hand, was demonstrated that R. taquarussiensis Rosa et al., 2017 (species described by integrative taxonomy [20]) represented only an intraspecific polymorphism of R. neglectus Lent, 1954 [19] (from studies of molecular taxonomy combined with experimental crosses it was possible to synonymize the species [19]).
Morphological convergence events can also hinder the classic taxonomy of these vectors [129]. The paraphyletic genus Triatoma Laporte, 1832 needs several studies from a taxonomic and systematic point of view [137]. Triatoma tibiamaculata , for example, is a species that has morphological characteristics that bring it together and groups it (until now) as a Triatoma [138]. However, the generic status of this vector has been questioned several times [134,137,138]-since it presents cytogenetic [139], structural [140] and phylogenetic [137,138] characteristics that bring it closer to Panstrongylus (which highlights the importance of studies with integrative taxonomy).

Molecular Taxonomy
The first phylogenetic trees with molecular markers were published only in 1998 [141], giving rise to the phylogenetic systematics and molecular taxonomy of these vectors. Although no species of triatomine has been described by molecular taxonomy (Table 2), the combination of phylogenetic analyses with morphological and morphometric studies in species description studies (integrative taxonomy) has been a trend in the last decade [5,[13][14][15][16][17] (Table 2), since it provides greater reliability of the specific status of the taxa and allows, above all, to understand the evolutionary history of the species.
In addition to the contributions mentioned above, molecular taxonomy and phylogenetic systematics allowed the evaluation and re-validation of the taxonomic status of some species: reinclusion of Linshcosteus Distant, 1904 genus in Triatomini tribe (extinguishing the Linshcosteini tribe) [30]; inclusion of Psammolestes Bergroth, 1911 species in the genus Rhodnius [30] (proposal not accepted by the scientific community due to the differences that support the generic status of Psammolestes [17]); inclusion of the species T. flavida , and N. obscura Maldonado & Farr, 1962 [4,143]); confirmation of the generic status of Mepraia [137]; and inclusion of T. dimidiata (Latreille, 1811) in the Meccus Stål, 1859 genus (genus that later was considered invalid and the Meccus species started to be considered as Triatoma [137,144,145]).
Although the International Code of Zoological Nomenclature does not consider groupings of triatomines to be complexes or subcomplexes [146], Justi et al. [137] suggests that these groupings should represent monophyletic groups. In the genus Triatoma, for example, studies based on phylogenetic systematics evaluated the position of several species that had been grouped mainly by geographic distribution and morphological similarities and proposed regrouping and/or the creation of new monophyletic groups [137,147,148]. Species well defined as natural groups (monophyletic) are currently the T. brasiliensis [149,150], T. sordida [151], T. rubrovaria [151], T. infestans [137], and T. vitticeps [148] subcomplexes.

Integrative Taxonomy
The data integration in the integrative taxonomy can be done by cumulation or congruence [152]. The use of combined tools to delimit a species of triatomine occurred for the first time in 1998 by Frias et al. [111] who combined morphological, morphometric, cytogenetic, and reproductive barriers data to describe M. gajardoi (Table 2). However, only in the last decade has the integrative taxonomy has been more applied in the study of these vectors ( Table 2).
This tendency to integrate different analyses to characterize a taxon, made it possible to resolve ancient taxonomic issues, such as the description by T. mopan Dorn et al. (2018) and T. huehuetenanguensis Lima-Cordón et al. (2019) from specimens initially characterized as T. dimidiata [16,17,153,154] and the recent description of T. rosai Alevi et al., 2020 from the allopatric population of T. sordida (Stål, 1859) from Argentina [5,155,156]. In addition, the specific status of T. bahiensis Sherlock & Serafim, 1967 (a species that for more than three decades has been synonymous with T. lenti Sherlock & Serafim, 1967 [101]) has been revalidated based on integrative taxonomy [149].
On the other hand, even if the integrative taxonomy provides more robustness in the characterization of the new taxa (decreasing the chance of synonymization), does not prevent this event can occur (as mentioned above for R. taquarussuensis which has been synonymous with R. neglectus Lent, 1954 [19]). Although morphological, morphometric, and cytogenetic intraspecific variation had been described in the genus Rhodnius [157,158], the description of R. taquarussuensis was based on these factors [20]. Thus, synonymization event occurred through phylogenetic analyses and experimental crosses [19]. We suggest that integrative taxonomy work should include molecular studies and, whenever possible, reproductive barriers to confirm the taxon specific status following the biological concept of species [159][160][161].
In general, most articles of description based on integrative taxonomy combine only morphological and morphometric data with molecular analyses (Table 2). However, it is worth mentioning that in 2020 the description of T. rosai was published based on morphometric, morphological, molecular data, and experimental crosses that have been combined with information from the literature about the species (cytogenetic data [155,156], electrophoresis pattern [155], cuticular hydrocarbons pattern [162], geometric morphometry [163], cycle, and average time of life [164][165][166] as well as geographic distribution [18,[42][43][44]50,51]), becoming the most complete article of species description of the subfamily Triatominae [5].

Overview of Tools Applied to Taxonomic Studies of Triatomines
In addition to species descriptions, several taxonomic studies have been carried out to assess the specific status of valid species and, above all, to assist in the correct classification of Chagas disease vectors. Based on this, we will specifically discuss the application of each taxonomic tool.

Morphometry
Like morphological studies, morphometric studies are also present in the description of all triatomines (at first, showing the size of specimens and structures and, later, by means of geometric morphometry [4]). These measurable data are very important from a taxonomic point of view, as a visual identification system was recently developed from morphometric data that has the potential to automate the identification of triatomines [173,174].

Chemotaxonomy
In 1964, Actis et al. [175] used, for the first time, biochemical studies with hemolymph protein electrophoresis to compare species of triatomines, giving rise to chemotaxonomy. Isoenzymes were applied to different species of Rhodnius [176], the T. brasiliensis subcomplex [177] and Mexican Triatoma [178]. However, recently, biochemical studies are rare from a taxonomic perspective; they contribute to the integrative taxonomy as shown by Jurberg et al. [11] and Alevi et al. [5] with the species descriptions of T. pintodiasi Jurberg et al., 2013 and T. rosai respectively.

MALDI-TOF MS
Laroche et al. [194] used, for the first time, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis to differentiate triatomine species. The researchers were able to differentiate species from French Guiana by MALDI-TOF. Subsequently, Souza et al. [195] used these analyses to differentiate 12 species of the genus Rhodnius. Furthermore, Souza et al. [196] also differentiated the species of Cavernicola Barber, 1937.

Omics
In 2017, omics tools (transcriptomics) were used for the first time in taxonomic studies of triatomines to confirm the specific status of R. montenegrensis [197]. In 2019, Brito et al. [198] also validated the specific status of R. montenegrensis and confirmed that this species refers to strain II of the paraphyletic group of R. robustus.

Concluding Remarks
Classical taxonomy, over the last few decades, has been revitalized by integrative taxonomy leading to success in the identification and delimitation of new species through the use of multiple and complementary approaches. Most descriptions of triatomine species are based on classical taxonomy. Facing evolutionary (cryptic speciation and phenotypic plasticity) and taxonomic (more than 190 synonymizations) problems has indicated that it is evident that integrative taxonomy studies are an important and necessary trend for this group of vectors. However, from the synonymization of R. taquarussuensis (which was described through integrative taxonomy [20] and was later synonymized with R. neglectus [19]), it is evident that phylogenetic studies (molecular taxonomy) should be considered among the analyses used for the description of new species from the integrative taxonomy (Figure 1).