New Evidence of the Monophyletic Relationship of the Genus Psammolestes Bergroth, 1911 (Hemiptera, Reduviidae, Triatominae)

Abstract.

The genus Psammolestes within the subfamily Triatominae and tribe Rhodniini comprises the species Psammolestes arthuri, Psammolestes coreodes, and Psammolestes tertius, all potential vectors of Chagas disease. A feature of Psammolestes is their close association with birds, which makes them an interesting model for evolutionary studies. We analyzed cytogenetically Psammolestes spp., with the aim of contributing to the genetic and evolutionary knowledge of these vectors. All species of the Psammolestes showed the same chromosomal characteristics: chromocenter formed only by sex chromosomes X and Y, karyotype 2n = 22 and constitutive heterochromatin, and AT base pairs restricted to the sex chromosome Y. These results corroborate the monophyly of the genus and lead to the hypothesis that during the derivation of P. tertius, P. coreodes, and P. arthuri from their common ancestor, there was no reorganization in the number or structure of chromosomes.

Chagas disease is caused by the protozoan Trypanosoma cruzi (Chagas, 1909) and transmitted mainly by triatomines.¹ Presently, the subfamily Triatominae (Hemiptera, Reduviidae) consists of 152 species (150 living species and two fossil ones) distributed in 18 genera and five tribes (Alberproseniini, Bolboderini, Cavernicolini, Rhodniini, and Triatomini),² all the species being potential vectors of T. cruzi.

The tribe Rhodniini is a monophyletic group³ consisting of two genera with different phenotypes: one with long thin legs and a long head, living mainly in palm trees (genus Rhodnius Stål, 1859), and the other having a short head, strong legs, wide femora, and a very wide rostrum (the widest in all the subfamily), living in nests of birds of the family Furnariidae (genus Psammolestes Bergroth, 1911).⁴ The genera include 21 species of Rhodnius (divided into the groups pallescens, pictipes, and prolixus) and three species of Psammolestes.^2,5

Psammolestes coreodes Bergroth, 1911 is distributed in Argentina (Catamarca, Corrientes, Chaco, Entre Rios, Formosa, Santa Fe, Santiago del Estero, Salta, Jujuy, and Tucumán), Bolivia (Santa Cruz), Brazil (Mato Grosso), and Paraguay (Central); Psammolestes tertius Lent and Jurberg, 1965 is distributed in Brazil (Bahia, Ceará, Goiás, Mato Grosso, Maranhão, Minas Gerais, Para, Paraíba, Pernambuco, and São Paulo) and Peru (San Martin); and Psammolestes arthuri (Pinto, 1926) is distributed in Colombia (Meta) and Venezuela (Aragua, Cojedes, Miranda, Guárico, Portuguesa, Yaracuy, Anzoátegui, Apure, Lara, Táchira, Barinas, and Monagas).^6,7

Phylogenetic analyses of P. tertius and P. coreodes suggest that this genus is monophyletic³ (there are no phylogenetic studies of P. arthuri in the literature) and presents a phylogenetic relationship with the species of the prolixus group,³ which led to suggest the inclusion of the genus Psammolestes in the genus Rhodnius.⁸ Monteiro et al.³ suggest that perhaps Psammolestes should be regarded as a specialized lineage from the prolixus group of Rhodnius because the genus Psammolestes and species of the prolixus group share a common ancestral origin, which highlights the paraphyly of the genus Rhodnius.⁵

Based on mitochondrial DNA data presented by Monteiro et al.,³ Soares et al.⁹ suggest that Psammolestes has derived from a form similar to Rhodnius robustus Larrousse, 1927. In addition, the authors suggest that these triatomines spread from the Amazon region northward into the llanos of Venezuela, where P. arthuri is now abundant in furnariidae nests, and southeastward into the caatinga–cerrado path of Central Brazil. Furthermore, as predicted by Schofield and Dujardin,¹⁰ the authors suggest subsequent differentiation of P. tertius along a north–south cline, from the larger specimens of the northeastern caatinga region to the smaller individuals of the central cerrado. According to them, the third species of the genus, P. coreodes, from the Chaco region of Argentina and Paraguay, may represent the southernmost differentiation of these descending populations.

Cytogenetic studies on the genus Psammolestes started in 1950 with the description of the karyotype of P. coreodes.¹¹ After 48 years, the karyotype of P. tertius was described,¹² and in 2012, the constitutive heterochromatin pattern of the species was characterized by Panzera et al.¹³ (Table 1). In addition, a more recent cytogenetic study comparing P. tertius of different Brazilian states (Bahia and Ceará) was performed and showed the absence of intraspecific chromosome variation.¹⁴ The present work seeks to characterize the karyotype evolution and the chromatin composition of the species of the genus Psammolestes, with the aim of contributing to the genetic and evolutionary knowledge of these potential vectors.

Table 1.

Cytogenetic characteristics of species of the genus Psammolestes

Species	Karyotype	Meiosis	C-banding			CMA3/DAPI
		Chromocenter	A	X	Y	A	X	Y
Psammolestes arthuri	2n = 22 (20A + XY)	XY	–	–	+	–	CMA+	DAPI−
Psammolestes coreodes	2n = 22 (20A + XY)11	XY	–	–	+	–	CMA+	DAPI−
Psammolestes tertius	2n = 22 (20A + XY)12,14	XY13	–	–	+13,14	–	CMA+	DAPI−

Five adult males of each species were used for cytogenetic analysis. The wild species considered herein were P. tertius (Castro Alves, Bahia, Brazil), P. coreodes (Corumbá, Mato Grosso do Sul, Brazil), and P. arthuri (Maracay, Aragua, Venezuela), all of them being from the field. The seminiferous tubules were torn apart, crushed, and fixed on slides in liquid nitrogen. The cytogenetic techniques Lacto-Aceto Orcein^15,16 and C-banding¹⁷ were applied for the description of karyotype, characterization of meiosis, and description of heterochromatin pattern, respectively. Then the cytogenomic technique of CMA₃/DAPI banding was applied according to Schimid¹⁸ with the modifications provided by Severi-Aguiar et al.¹⁹ to differentiate the heterochromatin regions rich in AT and CG. The biological material was analyzed using a Jenaval light microscope (Zeiss, Jena, Germany) and Olympus BX-FLA fluorescence microscope.

All species of the genus Psammolestes presented the same chromosomal characteristics, namely, chromocenter formed only by sex chromosomes X and Y during the prophase (Figure 1A–C), euchromatic autosomes and sex chromosome X and heterochromatic sex chromosome Y (Figure 1D–F), sex chromosomes X rich in CG (Figure 1G–I) and Y rich in CG (Figure 1J–L), and karyotype 2n = 22 (Figure 1M–O). These characteristics confirm the data already described in the literature (Table 1) and corroborate the monophyly of this genus.

Figure 1.

Cytogenetic analyses of Psammolestes spp. Note the X and Y sex chromosomes forming the chromocenter of Psammolestes tertius (A, arrow), Psammolestes coreodes (B, arrow), and Psammolestes arthuri (C, arrow). Note that the autosomes and X chromosome are euchromatic and Y is heterochromatic in P. tertius (D), P. coreodes (E), and P. arthuri (F). Note that the X chromosome is rich in CG (G–I) and the Y chromosome is rich in AT (J–L) and the karyotype is 2n = 22 (20A + XY) (M–O) for P. tertius (G, J, and M), P. coreodes (H, K, and N), and P. arthuri (I, L, and O). X: X sex chromosome, Y: Y sex chromosome. Scale bar: 10 μm. This figure appears in color at www.ajtmh.org.

The karyotype 2n = 22 is present in all species of the tribe Rhodniini.^20,21 This karyotype is the same number of chromosomes as the ancestor of Triatominae,¹⁷ which indicates that the genomic reorganization events that occurred during the evolution of the tribe Rhodniini did not lead to numerical alterations in the chromosomes, unlike what happened to the tribe Triatomini, which presents karyotypes ranging from 2n = 21 to 25.^22,23

The chromocenter formed only by sex chromosomes X and Y is also shared with the species of Rhodnius,^13,24 which demonstrates that this meiotic behavior is present in all species of the tribe Rhodniini. In the tribe Triatomini, there are species with the pattern described for Psammolestes and species that present union of autosomes with sexual chromosomes in the formation of the chromocenter.²⁵ This meiotic behavior can be used as a taxonomic tool to group related species. For example, all species of the Triatoma brasiliensis complex present a chromocenter formed by the sex chromosomes plus a pair of autosomes (characteristics that make it possible to differentiate seven species in this complex from all other triatomine complexes).²⁶

With the exception of Rhodnius colombiensis Mejia, Galvão and Jurberg, 1999; Rhodnius nasutus Stål, 1859; Rhodnius pallescens Barber, 1932; R. pictipes Stål, 1872; and R. taquarussuensis Rosa et al. 2017, ^21,27 all Rhodnius species also have constitutive heterochromatin restricted to the sex chromosome Y, as well as observed in Psammolestes spp. The distribution pattern of constitutive heterochromatin is extremely important for the taxonomy of Triatominae, being one of the main tools used in the description of the last species of the tribe Rhodniini, namely, R. taquarussuensis.²¹ In addition, the three species of the genus Psammolestes showed the same DNA composition rich in AT and CG.

Heterochromatin loss/reorganization in AT and CG composition could have occurred during the speciation of Psammolestes spp, as observed for the species of the group pallescens.²⁸ However, considering that the ancestor of P. tertius, P. coreodes, and P. arthuri was similar to R. robustus (which does not present heterochromatin in the autosomes either, only in the sexual chromosome Y²⁷) and especially the degree of specialization of these species during their evolution (they inhabit only bird nests), it can be stated that the species maintained the genetic material without chromosomal changes.

The cytogenetic characteristics analyzed indicate chromosomal homogeneity in the genus Psammolestes, which corroborates the monophyletic feature of the genus and suggests that during the derivation of P. tertius, P. coreodes, and P. arthuri from the common ancestor, there was no reorganization in the number or structure of chromosomes.