Cryptic species, also called sibling or isomorphic species, are identical in their external appearance, or differ in apparently minor and not easily visible traits. In Triatominae, the use of different phenotypic and genetic markers has shown that cryptic speciation is a widespread phenomenon in this subfamily. The existence of sibling species has been described in different Triatoma groups such as the brasiliensis, dimidiata, phyllosoma and sordida subcomplexes, as well as in several species of Rhodnius and Panstrongylus (for review see ).
Sordida subcomplex species except T. patagonica are morphologically very similar, with overlapping geographical distribution and even sympatric in many regions with putative hybrids, which significantly increase the taxonomic confusion. Although there are phenotypic and genetic markers able to differentiate the species, its recognition requires high expertise which greatly hinders their application by vector control staff– leading to mistaken determination even within insectary material (see Tables 1–4). Below, we discuss our results for each chromosomal taxon here identified, in comparison with previously published determinations.
T. guasayana: The morphological differentiation of T. guasayana with T. sordida is very difficult, especially in the nymphal stages. The geographic distribution of both species overlaps in northern Argentina and in the Chaco region from Bolivia and Paraguay. T. guasayana is mainly sylvatic, occupying a great variety of habitats including bromeliads, similar to T. sordida. Peridomiciliary colonies are frequent, side by side with T. infestans and T. sordida, especially in chicken houses . Isoenzymes studies in Bolivia show the absence of hybrid forms confirming the reproductive isolation in nature of both species .
Genetic markers (isoenzymes, chromosomal and mitochondrial sequences) clearly differentiated T. guasayana from the other species of sordida subcomplex [7, 9, 10, 23–25]. However, probably due to their morphological similarity, COI sequences deposited in GenBank reveal at least one mistaken identification: a specimen from Santa Cruz, Bolivia (Chaco, Tita) (KC249342) originally identified as T. guasayana, should be recognized as T. sordida (Fig. 3). At phylogenetic level, genetic distances between T. guasayana and the other sordida taxa are the most extreme, between 14 and 14.9 % (Table 7). In fact T. guasayana seems more related to T. rubrovaria (10.3 %) (Table 7), as suggested by other authors using different mitochondrial genes [23–25]. Chromosomal similarities between these two species (lack of autosomal heterochromatin and ribosomal clusters on one autosomal pair) would also support the inclusion of T. guasayana in the rubrovaria subcomplex.
T. garciabesi: Until now, this species can be only described in Central and Northern Argentina [8, 12, 13]. Our results extend the T. garciabesi geographical distribution to other Chaco regions previously not described: Bolivian Chaco (Santa Cruz), western Paraguay (Departments of Boquerón and Presidente Hayes) and the Argentine provinces of Tucumán and Santiago del Estero (Table 2). This geographical distribution closely matches the predicted distribution for T. garciabesi based on ecological niche modeling . Most of them are sylvatic, but occasionally occupy peridomestic environments.
Different approaches support the taxonomic validity of this species: head and genitalia morphology , head and wing morphometrics , cuticular hydrocarbons , isoenzymatic and cytogenetic traits [7, 17], and molecular analyses  (Fig. 3). This paper establishes the chromosomal identity between T. garciabesi and the T. sordida group 2 from Bolivia defined by isoenzymes [9, 10].
Despite the genetic and phenotypic differentiation, taxonomic problems still persist in terms of distinguishing T. garciabesi from other species of the subcomplex. Individuals from established laboratory colonies (Salta, La Rioja, Santiago del Estero, Formosa and Chaco) which were originally identified as T. sordida, presented the chromosomal characteristics of T. garciabesi (Table 2). Hence, incorrect identification of this species is also seen in the sequences deposited in GenBank. The two individuals from the Bolivian Chaco (Romerillo) probably belong to T. garciabesi rather than T. sordida (Fig. 3). Recently, comparative analyses of T. sordida populations from Western (Chaco) and Eastern regions of Paraguay reveal striking differences in the feeding patterns, random amplified polymorphic DNA profiles (RAPD), and head and wing morphometrics . These authors suggested that this differentiation is associated to eco-geographical isolation by distance. However, our chromosomal studies suggest that T. sordida populations in Paraguay involve at least three taxa. In the Chaco region (Western Paraguay) we only found T. garciabesi, while in the Eastern region it appears that T. sordida Argentina and T. sordida sensu stricto coexist in sympatry. The ecological differentiation and distinct feeding patterns described by  support our results. We suspect that besides T. garciabesi and T. guasayana , also other cryptic sordida species may be present in Paraguay.
T. sordida sensu stricto: This species is the most geographically widespread species of the sordida subcomplex, found in large parts of Argentina, Brazil, Bolivia (Santa Cruz) and Paraguay (Central and Eastern) . Surprisingly according to our results, this species was not detected in Argentina despite the large number of populations and individuals analyzed (Tables 2–4). Individuals recognized as T. sordida group 1 in Bolivia by isoenzymes [9–11] belong to this chromosomal group (Table 3). All these populations occupy domestic and peridomestic habitats, although they are also found in sylvatic habitats such as birdnests, tree holes and under dry tree bark [9–11]. Historically, T. sordida is forming abundant colonies in peridomestic habitats (particularly chicken coops), with the ability to invade and colonize human habitations in Brazil [28, 29], Bolivia [9–11, 30] and Paraguay . Considering the sibling taxa here analyzed, T. sordida sensu stricto would be the most significant in terms of Chagas disease transmission , being the most common synanthropic species captured in Brazil .
T. sordida Argentina: For this chromosomal taxon, we considered as topotypes the individuals from San Luis del Palmar (Corrientes, Argentina). Chromosomal characteristics (without autosomal C-heterochromatin and ribosomal clusters in both sex chromosomes) were determined in most individuals from Argentina and some from Bolivia and Paraguay, all of them initially identified as T. sordida (Table 4). Ribosomal genes located on both sex chromosomes (Fig. 1g, h) is an uncommon feature in the genus Triatoma, only previously observed in four unrelated species of the 27 so far analyzed . The mtDNA sequence divergence between T. sordida Argentina with sympatric T. garciabesi is 7.0 % (Table 7), similar to that used to support specific denominations in the brasiliensis subcomplex  or Mepraia species . Sequence divergence between T. sordida Argentina and T. sordida sensu stricto is 5.3 %, similar to that observed between T. sanguisuga subspecies . However, isoenzymatic analyses involving 19 loci revealed a striking differentiation between T. sordida Argentina and T. sordida sensu stricto from Brazil, showing 3 different fixed alleles and 4 polymorphic loci . Considering this isoenzymatic diversity and the extreme chromosomal distinction (C-heterochromatin amount and ribosomal clusters location) between these taxa we suggest there may be complete genetic isolation between them. We therefore propose that T. sordida Argentina presents characteristics consistent with its designation as a new species. At the epidemiological level, T. sordida Argentina were found in various sylvatic ecotopes and peridomestic habitats, but in very low frequency in domestic environments compared to, say, T. sordida from Brazil [35, 36].
T. sordida La Paz: This chromosomal taxon was identified in domestic specimens collected from La Paz (Bolivia) (Table 5). These individuals exhibit heterochromatic autosomes similar to what is observed in T. sordida sensu stricto, but they differ in the position of the ribosomal clusters (Fig. 1). T. sordida La Paz showed rDNA clusters in one autosomal pair, while T. sordida sensu stricto has them on the X chromosome. Considering almost 100 heteropteran species analyzed to date, including 40 triatomine species, the chromosomal position of ribosomal genes appears to be a species-specific character, although variation in ribosomal gene location was reported in T. infestans [16, 17]. For this reason we cannot rule out that T. sordida sensu stricto and T. sordida La Paz are conspecific populations with different ribosomal gene locations. Furthermore, some hybrid individuals detected in Bolivia (Fig. 2) could plausibly have originated from crosses between these two chromosomal taxa, thereby strengthening the idea of an intraspecific variation in ribosomal genes. Unfortunately we do not have information about COI sequences of individuals from this chromosomal taxon.
Putative hybrids by chromosomal markers
Experimental crosses between T. sordida populations and with T. guasayana have been made by many researchers, showing either fertility or F1 sterility [8, 20, 37–39]. These results, apparently contradictory, can now be explained by the fact that the crossings involved distinct chromosomal taxa, as suggested in this paper. Currently, there is only one report that demonstrates the existence of natural hybrids in sordida subcomplex species. By isoenzymes, putative hybrids in low frequency (3 %) were recorded in two localities from Santa Cruz in Bolivia (Izozog and Tita) . According to these authors, in these regions at least three sordida subcomplex species (T. guasayana and T. sordida group 1 and group 2) coexist in sympatry, together with the putative hybrids. Here, in Izozog we also identified three species (T. guasayana, T. sordida sensu stricto and T. garciabesi, respectively) and putative hybrids, similar to isoenzymes . Since both heterochromatin and the ribosomal genes are inherited in Mendelian fashion [17, 21], the occurrence of heterozygous chromosomes for C-heterochromatin and the FISH patterns observed in the ribosomal cluster location (Fig. 2) suggests that these individuals are hybrids resulting from crosses among different taxa.