Abstract

The grasshopper Rhammatocerus brasiliensis shows polymorphism of B chromosomes, but the magnitude of B-chromosome occurrence and the factors that may contribute to their dispersion in the species remain unknown thus far. The present study analyzed the occurrence and dispersion of B chromosomes in R. brasiliensis individuals from 21 populations widely distributed in the Brazilian Northeast. The genetic connectivity between 10 populations was verified through analysis of ISSR markers from 200 individuals. Of the 21 populations, 19 presented individuals with one B chromosome, three with two, and one with three B chromosomes. The B chromosome is of medium size and constitutive heterochromatin (CH) located in the pericentromeric region. A variant B chromosome was observed in three populations, similar in size to that of chromosome X, gap and CH, and located in the terminal region. B chromosome frequencies in different populations varied from 0% to 18,8%, mean 8,5%. The wide distribution of the B chromosome is likely a consequence of the positive gene flow among the analyzed populations. B-chromosome occurrence in populations of R. brasiliensis possibly follows the population genetic structure of the species and, owing to the existence of a variant, its origin may not be recent.

Keywords:
Grasshopper; supernumerary chromosome; chromosome polymorphism; ISSR; genetic connectivity

Introduction

B chromosomes, also known as supernumerary chromosomes, are extra elements of the karyotype. These chromosomes do not obey the Mendelian laws of inheritance and do not present homology with the other chromosomes of the A complement (Camacho et al., 2000Camacho JPM, Sharbel TF and Beukeboom LW (2000) B-chromosome evolution. Philos Trans R Soc Lond B Bio Sci 355:163–178.). Moreover, they generally differ by the accumulation of repetitive DNA sequences, such as satellite DNA (Ruiz-Ruano et al., 2017Ruiz-Ruano FJ, Cabrero J, López-León MD and Camacho JPM (2017) Satellite DNA content illuminates the ancestry of a supernumerary (B) chromosome. Chromosoma 126:487–500.; Milani et al., 2017aMilani D, Ramos É, Loreto V, Martí DA, Cardoso AL, Moraes KCM, Martins C and Cabral-de-Mello DC (2017a) The satellite DNA AflaSAT-1 in the A and B chromosomes of the grasshopper Abracris flavolineata. BMC Genet 18:81.), ribosomal DNA and histone H3 (Oliveira et al., 2011Oliveira NL, Cabral-de-mello DC, Rocha MF, Loreto V, Martins C and Moura RC (2011) Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper Rhammatocerus brasiliensis (Acrididae, Gomphocerinae): Extensive chromosomal dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement and B chromosome. Mol Cytogenet 4:24.), U2 small nuclear RNA (Bueno et al., 2013Bueno D, Palacios-Gimenez OM and Cabral-de-Mello DC (2013) Chromosomal mapping of repetitive DNAs in the grasshopper Abracris flavolineata reveal possible ancestry of the B chromosome and H3 histone spreading. PLoS One 8:e66532.), and transposable elements (Palacios-Gimenez et al., 2014Palacios-Gimenez OM, Bueno D and Cabral-de-Mello DC (2014) Chromosomal mapping of two Mariner-like elements in the grasshopper Abracris flavolineata (Orthoptera: Acrididae) reveals enrichment in euchromatin. Eur J Entomol 111:329–334.). Although repetitive sequences are common in the composition of B chromosomes, genes conferring resistance to antibiotics and pathogens have also been identified (Coleman et al., 2009Coleman JJ, Rounsley SD, Rodriguez-Carres M, Kuo A, Wasmann CC, Grimwood J, Schmutz J, Taga M, White GJ, Zhou S et al. (2009) The genome of Nectria haematococca: Contribution of supernumerary chromosomes to gene expansion. PLoS Genet 5: e1000618.), besides others that participate in the control of the cell cycle (Valente et al., 2014Valente GT, Conte MA, Fantinatti BEA, Cabral-de-Mello DC, Carvalho RF, Vicari MR, Kocher TD and Martins C (2014) Origin and evolution of B chromosomes in the cichlid Fish Astatotilapia latifasciata based on integrated genomic analyses. Mol Biol Evol 31:2061–2072.; Ramos et al., 2016Ramos É, Cardoso AL, Brown J, Marques DF, Neill RJO and Martins C (2016) The repetitive DNA element BncDNA, enriched in the B chromosome of the cichlid fish Astatotilapia latifasciata, transcribes a potentially noncoding RNA. Chromosoma 126:313–323.).

The occurrence of B chromosomes is almost universal, being found in approximately 15% of the analyzed eukaryotes (Camacho, 2005Camacho M (2005) B Chromosomes. In: Gregory TR (ed) The Evolution of the Genome. Elsevier, San Diego, pp 223-286.). Among the animals, the insects of the order Orthoptera stand out for the great frequency of B chromosomes, being hotspots in the superfamilies Acridoidea, Grylloidea, Pyrgomorphoidea and Tetrigoidea (Palestis et al., 2010Palestis BG, Cabrero J, Robert T and Camacho JPM (2010) Prevalence of B chromosomes in Orthoptera is associated with shape and number of A chromosomes. Genetica 138:1181–1189.). In Acridoidea, the Acrididae family is considered a hotspot representing 17,1% (Palestis et al., 2010Palestis BG, Cabrero J, Robert T and Camacho JPM (2010) Prevalence of B chromosomes in Orthoptera is associated with shape and number of A chromosomes. Genetica 138:1181–1189.). Locusta migratoria (Linnaeus, 1758) and Eyprepocnemis plorans (Charpentier, 1825) of this family are used as models to better understand the occurrence, origin and evolution of the B chromosomes (Bueno et al., 2013Bueno D, Palacios-Gimenez OM and Cabral-de-Mello DC (2013) Chromosomal mapping of repetitive DNAs in the grasshopper Abracris flavolineata reveal possible ancestry of the B chromosome and H3 histone spreading. PLoS One 8:e66532.).

When the frequency of B chromosomes in a population is equal to or higher than 25%, it is suggested that their origin is recent (Araújo et al., 2001Araújo S, Pompolo S, Perfectti F and Camacho J (2001) Integration of a B chromosome into the A genome of a wasp. Proc Biol Sci 268:1127-1131.). Contrarily, in populations where the frequency is relatively low (below 25%), the B chromosome is considered as stable or in process of elimination (Riera et al., 2004Riera L, Petitpierre E, Juan C, Cabrero J and Camacho JPM (2004) Evolutionary dynamics of a B chromosome invasion in island populations of the grasshopper Eyprepocnemis plorans. J Evol Biol 17:716–719.). According to some authors (Camacho et al., 1997Camacho JPM, Shaw MW, López-Léon MD, Pardo MC and Cabrero J (1997) Population dynamics of a selfish B chromosome neutralized by the standard genome in the grasshopper Eyprepocnemis plorans. Am Nat 149:1030–1050., 2015Camacho JPM, Shaw MW, Cabrero J, Bakkali M, Ruíz-Estévez M, Ruíz-Ruano FJ, Martín-Blázquez R and López-León MD (2015) Transient microgeographic clines during B chromosome invasion. Am Nat 186:675–681.; Zurita et al., 1998Zurita S, Cabrero J, López-León MD and Camacho JPM (1998) Polymorphism regeneration for a neutralized selfish B chromosome. Evolution 52:274–277.), B chromosomes present four main life-cycle stages: 1) invasion, where a B chromosome arrives or emerges in a natural population and its frequency rises rapidly within only a few tens of generations; 2) resistance, when a B chromosome remains in the host genome through drive mechanisms; 3) near neutrality, when the frequency of B chromosomes varies randomly by genetic drift, or decreases due to negative selection in individuals with increased number of B chromosomes (this constitutes the longest stage, which can last for thousands of generations); and 4) regeneration, where the B chromosome depends on the accumulation of mutations for the emergence of novel variants that may be able to remain in the genome and substitute the former, neutralized B chromosome, reinitiating the B chromosome cycle and prolonging its evolutionary life.

For Neotropical grasshoppers, particularly those occurring in Brazil, most works performed so far have focused on the analysis of the origin and composition of B chromosomes through the mapping of different repetitive sequences, as observed for the species Abracris flavolineata (2n = 24, X0 + B and 2n = 25, X0 + 2Bs) (Bueno et al., 2013Bueno D, Palacios-Gimenez OM and Cabral-de-Mello DC (2013) Chromosomal mapping of repetitive DNAs in the grasshopper Abracris flavolineata reveal possible ancestry of the B chromosome and H3 histone spreading. PLoS One 8:e66532.; Milani and Cabral-de-Mello, 2014Milani D and Cabral-de-Mello DC (2014) Microsatellite organization in the grasshopper Abracris flavolineata (Orthoptera: Acrididae) revealed by FISH mapping: Remarkable spreading in the A and B chromosomes. PLoS One 9:e97956.; Palacios-Gimenez et al., 2014Palacios-Gimenez OM, Bueno D and Cabral-de-Mello DC (2014) Chromosomal mapping of two Mariner-like elements in the grasshopper Abracris flavolineata (Orthoptera: Acrididae) reveals enrichment in euchromatin. Eur J Entomol 111:329–334.; Menezes-de-Carvalho et al., 2015Menezes-de-Carvalho NZ, Palacios-Gimenez OM, Milani D and Cabral-de-Mello DC (2015) High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata. Mol Genet Genomics 290:1787–1792.; Milani et al., 2017aMilani D, Ramos É, Loreto V, Martí DA, Cardoso AL, Moraes KCM, Martins C and Cabral-de-Mello DC (2017a) The satellite DNA AflaSAT-1 in the A and B chromosomes of the grasshopper Abracris flavolineata. BMC Genet 18:81.,bMilani D, Palacios-Gimenez OM and Cabral-de-Mello DC (2017b) The U2 snDNA is a useful marker for B chromosome detection and frequency estimation in the grasshopper Abracris flavolineata. Cytogenet Genome Res 151:36–40.) and Xyleus discoideus angulatus (2n = 24, X0 + B and 2n = 25, X0 + 2Bs) (Loreto et al., 2008aLoreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241.; Machado et al., 2014Machado CB, Neto LCS, Loreto V and Souza MJ (2014) B chromosome prevalence and physical mapping of 18S rDNA and H4 histone sites in the grasshopper Xyleus discoideus angulatus (Romaleidae). Genet Mol Res 13:7052–7060.; Bernardino et al., 2017Bernardino ACS, Cabral-De-Mello DC, Machado CB, Palacios-Gimenez OM, Santos N and Loreto V (2017) B Chromosome variants of the grasshopper Xyleus discoideus angulatus are potentially derived from pericentromeric DNA. Cytogenet Genome Res 152:213–221.). For Rhammatocerus brasiliensis, the frequency of B chromosomes has also been analyzed in eight populations from Pernambuco and three from Bahia, states of the Brazilian Northeast, showing 2n = 24, X0 + B and 2n = 25, X0 + 2Bs (Loreto et al., 2008aLoreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241.,bLoreto V, Cabrero J, López-León MD, Camacho JPM and Souza MJ (2008b) Comparative analysis of rDNA location in five Neotropical gomphocerine grasshopper species. Genetica 132:95–101.; Oliveira et al., 2011Oliveira NL, Cabral-de-mello DC, Rocha MF, Loreto V, Martins C and Moura RC (2011) Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper Rhammatocerus brasiliensis (Acrididae, Gomphocerinae): Extensive chromosomal dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement and B chromosome. Mol Cytogenet 4:24.). Regarding Neotropical species, the distribution of B chromosomes in natural grasshopper populations has not been analyzed under consideration of the genetic structure of the populations, and such studies have only been performed in Palearctic grasshoppers (Keller et al., 2013Keller D, van Strien MJ, Herrmann M, Bolliger J, Edwards PJ, Ghazoul J and Holderegger R (2013) Is functional connectivity in common grasshopper species affected by fragmentation in an agricultural landscape? Agric Ecosyst Environ 175:39–46.; Manrique-Poyato et al., 2013aManrique-Poyato MI, López-León MD, Gómez R, Perfectti F and Camacho JPM (2013a) Population Genetic Structure of the Grasshopper Eyprepocnemis plorans in the South and East of the Iberian Peninsula. PLoS One 8:e59041.,bManrique-Poyato MI, López-León MD, Cabrero J, Perfectti F and Camacho JPM (2013b) Spread of a new parasitic B chromosome variant is facilitated by high gene flow. PLoS One 8:e83712., 2015Manrique-Poyato MI, López-León MD, Cabrero J, Gómez R, Perfectti F and Camacho JPM (2015) Geographical barriers impeded the spread of a parasitic chromosome. PLoS One 10:e0131277.). This kind of approach allows estimating the gene flow among individuals within and between populations, which may be a parameter directly related to the dispersion or isolation of B chromosomes (Manrique-Poyato et al., 2015Manrique-Poyato MI, López-León MD, Cabrero J, Gómez R, Perfectti F and Camacho JPM (2015) Geographical barriers impeded the spread of a parasitic chromosome. PLoS One 10:e0131277.).

R. brasiliensis is widely distributed in the Northeast of Brazil, with records in the states of Rio Grande do Norte, Paraíba, Pernambuco (Assis-Pujol, 1998Assis-Pujol C (1998) Aspectos morfológicos, taxonômicos e distribuição geográfica de cinco espécies de Rhammatocerus, Saussure, 1861 (Acrididae, Gomphocerinae, Scyllinini). Bol Mus Nac Rio Janeiro 387:1–27.), and Bahia (Loreto et al., 2008aLoreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241.). In Pernambuco, the species is found across the entire state, having been reported in 11 localities (Carbonell, 1995Carbonell CS (1995) Revision of the tribe Scyllinini, Nov. (Acrididae: Gomphocerinae), with descriptions of new genera and species. Trans Amer Entomol Soc 121:87–152.; Assis-Pujol, 1998Assis-Pujol C (1998) Aspectos morfológicos, taxonômicos e distribuição geográfica de cinco espécies de Rhammatocerus, Saussure, 1861 (Acrididae, Gomphocerinae, Scyllinini). Bol Mus Nac Rio Janeiro 387:1–27.; Loreto et al., 2008aLoreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241.). Because of its wide distribution (Carbonell, 1988Carbonell CS (1988) Rhammatocerus schistocercoides (Rehn, 1906), especie prejudicial para la agricultura en la región centro oeste de Brasil (Orthoptera, Acrididae, Gomphocerinae). Bol Mus Nac Rio de Janeiro 318:1-17.), R. brasiliensis is an interesting species for understanding the life-cycle stages of B chromosomes and how they may disperse. In this study, the following hypotheses were tested: 1) B chromosomes are dispersed among R. brasiliensis populations according to gene flow existing among them; 2) based on the relatively low frequency of B chromosomes in R. brasiliensis, as verified in different populations by Loreto et al. (2008b)Loreto V, Cabrero J, López-León MD, Camacho JPM and Souza MJ (2008b) Comparative analysis of rDNA location in five Neotropical gomphocerine grasshopper species. Genetica 132:95–101., it is expected that the B chromosome is currently in the near-neutrality stage.

With this in mind, we analyzed the occurrence, frequency and distribution of B chromosomes in R. brasiliensis individuals from 21 populations from the Brazilian Northeast, of which 10 were also analyzed regarding population genetic structure. In addition, the position of the constitutive heterochromatin (CH) was established in the B chromosomes of the species.

Material and Methods

Sampling

Specimens of R. brasiliensis (Bruner 1904) were collected from 21 populations in the Brazilian Northeast, of which 13 were located in the state of Pernambuco (PE), one in Sergipe (SE), one in Alagoas (AL), one in Paraíba (PB), one in Ceará (CE), one in Piauí (PI), and three in Bahia (BA) (Figure 1). Of these populations, 11 have been previously sampled and analyzed with regard to the occurrence and prevalence of B chromosomes by Loreto et al. (2008a)Loreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241. and Oliveira et al. (2011)Oliveira NL, Cabral-de-mello DC, Rocha MF, Loreto V, Martins C and Moura RC (2011) Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper Rhammatocerus brasiliensis (Acrididae, Gomphocerinae): Extensive chromosomal dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement and B chromosome. Mol Cytogenet 4:24..

The grasshoppers were collected with the help of an entomological net and transported to the Laboratório de Biodiversidade e Genética de Insetos (LBGI), Instituto de Ciências Biológicas da Universidade de Pernambuco. The collections were permitted by ICMBio/SISBIO under the license number 16278-1.

Conventional staining and C-banding

The karyotypes of 590 adult male specimens of R. brasiliensis were analyzed by conventional staining. First, the classic technique of testicular follicle squashing was performed, consisting in the maceration of the gonads in one drop of 45% acetic acid. Subsequently, the slides were subjected to heat of 65 °C for approximately 3 min for fixation of the chromosome preparation onto the slide, then stained with 5% Giemsa. The C-banding technique was performed according to Sumner (1972)Sumner A (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75:304–306., with duration of exposure to the basic solution [Ba(OH)₂] altered to 2 min, in individuals carrying B chromosomes from the populations of VIT and LC (Pernambuco – PE).

Analysis of occurrence and frequency of B chromosomes and statistical test

The occurrence, frequency, and distribution of B chromosomes were analyzed in a total of 1.274 specimens. Of these, 394 were individuals from 10 populations being analyzed for the first time (GOI–PE, LC–PE, SAL–PE, BUI–PE, OUR–PE, ARA–SE, MAC–AL, JP–PE, JN–PE and PIC–PI) and 196 were individuals from four populations with samplings being expanded (ITA–PE, VIT–PE, BEZ–PE and ST–PE). The data for the remaining specimens were obtained from the literature (Loreto et al., 2008bLoreto V, Cabrero J, López-León MD, Camacho JPM and Souza MJ (2008b) Comparative analysis of rDNA location in five Neotropical gomphocerine grasshopper species. Genetica 132:95–101.; Oliveira et al., 2011Oliveira NL, Cabral-de-mello DC, Rocha MF, Loreto V, Martins C and Moura RC (2011) Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper Rhammatocerus brasiliensis (Acrididae, Gomphocerinae): Extensive chromosomal dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement and B chromosome. Mol Cytogenet 4:24.), and included to provide a wider sampling for this species.

Using the software OpenEpi 3.01, a statistical significance test was carried out based on Fisher's exact test in order to verify whether the frequency of B chromosomes differs significantly among the populations. Furthermore, using the same software, analysis of confidence intervals (CI) was performed to estimate the maximum and minimum number that the frequency of B chromosomes may reach in an analysis with a population size of 1.000.000 specimens.

DNA extraction and ISSR marker amplification

The genomic DNA of R. brasiliensis specimens was extracted from the leg muscle according to protocol described by Sambrook and Russell (2001)Sambrook J and Russell D (2001) Molecular cloning, a laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press, New York, vol. 2.. Subsequently, the DNA was purified through enzymatic digestion with RNase (10 mg/mL) for 1 h at 37 °C. Next, the concentration and quality of the extracted DNA were verified via electrophoresis in 1% agarose gel by comparison with DNA from phage Lambda DNA/Hind III marker (Fermentas Life Sciences).

Polymerase chain reaction (PCR) amplification was performed from a total volume of 10 μL containing 5 ng of DNA, 1X PCR buffer, 5 mM of MgCl₂, 0,2 mM of dNTPs (2,5 mM), 1 pmol of primer (0,2 mM) and 1 U Taq polymerase (Invitrogen). The reactions were carried out in a thermocycler (Biosystems) as follows: Standard program at 94 °C for 4 min; 35 cycles at 94 °C for 30 s; annealing step (with temperature varying for each primer) for 45 s; extension at 72 °C for 2 min; and final extension at 72 °C for 7 min. The PCR products were visualized in 1,8% agarose gel using a transilluminator (Gel Logic/Carestream MI SE).

A total of 49 ISSR primers (Biotechnology Laboratory, University of British Columbia, Canada) were tested in one R. brasiliensis individual, with 33 presenting high numbers of amplified DNA fragments with good resolution. Selection of the most informative primers was accomplished by applying the 33 pre-selected primers to a sample of nine specimens from three populations (PIC–PI, JN–CE and JP–PB). Based on the genotyping, the data were grouped into a binary matrix of presence and absence. The analysis of the most informative primers was carried out according to the parameters number of total, monomorphic and polymorphic DNA fragments, and polymorphism indices: Polymorphism Information Content (PIC) (Roldán-Ruiz et al., 2000Roldán-Ruiz I, Loose M and Van Bockstaele E (2000) AFLP markers reveal high polymorphic rates in ryegrass (Lolium spp.). Mol Breed 6:125–134.), Marker Index (MI) (Prevost and Wilkinson, 1999Prevost A and Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98:107–112.) and Resolving Power (RP) (Varshney et al., 2007Varshney RK, Chabane K, Hendre PS, Aggarwal RK and Graner A (2007) Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated and elite barleys. Plant Sci 173:638–649.). Furthermore, the Pearson correlation was calculated among the PIC, MI, and RP indices. The seven most-informative primers (807, 835, 844, 845, 846, 857, 866) were applied to 200 individuals from 10 populations, with 20 obtained from each population, thus representing the wide distribution of the species in the Northeast of Brazil (Figure 1, Table 1).

Reproducibility test

The reproducibility of the ISSR markers was verified through analyses based on two independent PCR amplifications for all seven primers that were applied to all individuals of the studied populations. Non-reproducible loci were excluded from the genetic analysis.

Genetic diversity and population structure

The genetic diversity index (GD), mean expected heterozygosity (H_E), Wright's fixation index (F_ST) and molecular variance (AMOVA) were calculated with the software Arlequin 3.5 (Excoffier and Lischer, 2010Excoffier L and Lischer HEL (2010) Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:564–567.). The number of migrants (Nm) and coefficient of genetic differentiation (G_ST) were calculated in the software Popgen 1.31 (Yeh et al., 1999Yeh F, Yang R and Boyle T (1999) Microsoft Windows-based free ware for population genetic analysis (Release 1.31). University of Alberta, Edmonton.). The level of isolation by distance (IBD) was analyzed from the correlation between genetic and geographical distances (Mantel test, Sokal and Rohlf, 1995Sokal RR and Rohlf FJ (1995) Biometry. 3rd edition. Freeman, New York, pp 813–819.) using the software Arlequin 3.5 (Excoffier and Lischer, 2010Excoffier L and Lischer HEL (2010) Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:564–567.), based on 10.000 permutations. At last, the population model most adequate to the data was established with the software Hickory 1.1 (Holsinger et al., 2002Holsinger K, Lewis P and Dey D (2002) A Bayesian method for analysis of genetic population structure with dominant marker data. Mol Ecol 11:1157-1164.) considering four possible models: i) full model, where the values of population differentiation (theta, θ, an analog of Wright's parameter F_ST) and inbreeding (, an analog of F_IS) differ from zero; ii) model =0, which assumes absence of inbreeding within the populations; iii) model theta=0, based on the absence of population differentiation; and iv) the free model, where the values are chosen randomly, independent of a priori information. The choice of the most suitable model was based on the parameters deviance information criterion (DIC), an analog of the Akaike information criterion (AIC) in the selection via Bayesian models, and Dbar, a measure of the level of adjustment with which the model adapts to the analyzed data (the lower the value of both, the better).

Number of genetic clusters

The genetic clusters were analyzed with the software Structure 2.3.1 (Pritchard et al., 2000Pritchard JK, Stephens M and Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959.) using the Bayesian algorithm, where the K-value was identified and analyzed to determine the level of genetic mixture among the populations. For each value of K (from 1 to 10), 10 independent rounds were performed with 600.000 replications and burn-in of 60.000 interactions. Finally, we used the website Structure Harvester (Earl, 2012Earl DA and Vonholdt BM (2012) Structure Harvester: A website and program for visualizing structure output and implementing the Evanno method. Conserv Genet Resour 4:359–361.), which implements the method of Evanno et al. (2005)Evanno G, Regnaut S and Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study. Mol Ecol 14:2611–2620., to estimate the number of genetic groups (K) which best adjust the data.

Results

Cytogenetic data

The karyotype 2n = 23, X0, described for the species R. brasiliensis, was observed in 1.162 specimens. All chromosomes of the complement are acrocentric and were categorized, by size, into large (G1 – G3), medium (M4 – M8) and small pairs (P9 – P11). In addition, 2n = 24, X0 + B was seen in 107 individuals, with the B chromosome being acrocentric and of medium size (Figures 2a,b,d) in all populations exhibiting this polymorphism (ITA–PE, GOI–PE, VIT–PE, LC–PE, GRA–PE, SAL–PE, BON–PE, BUI–PE, SUR–PE, BEZ–PE, TOR–PE, ST–PE, OUR–PE, MAC–AL, JN–CE, PIC–PI, SOB–BA, RC–BA, and ITAB–BA); 2n = 25, X0 + 2Bs (Figure 2b) was observed in four specimens from ITA–PE, BEZ–PE, and OUR–PE; and 2n = 26, X0 + 3Bs in one individual from ITA–PE (Figure 2c, insert) (Table 2). Two distinct B chromosomes were observed: one verified in 19 of the 21 analyzed populations, of medium size and smaller than X. Another, here denoted as variant, was identified in the populations from ITA, BEZ and LC, with size similar to that of X and presence of a gap in the long arm (Figure 2c). In specimens with a single B chromosome, association with the X chromosome was observed (Figure 2a), whereas in the case of two B chromosomes, the association occurred with autosomes instead (Figure 2b).

The presence of B chromosomes was verified in almost all populations, with the exception of ARA–SE and JP–PB. Among populations with B chromosomes, ITAB–BA presented the highest frequency (18.8%) and SAL–PE the lowest (2.32%). The values for total frequency of males with B chromosomes in the populations varied from 0% to 18.8%, with a mean of 8.5% (Table 2). A significant difference existed in this regard between some populations (p-values > 0.05) identified by Fisher's pairwise exact test, namely: ITA–JP, 0.032; GRA–SUR, 0.031; GRA–ST, 0.012; GRA–OUR, 0.045; GRA–MAC, 0.008; GRA–JP, 0.007; GRA–JN, 0.031; GRA–PIC, 0.045; and BEZ–JP, 0.038 (Table 3).

The C-banding procedure revealed positive C bands in the pericentromeric region of all chromosomes of the A complement, as well as in the B chromosomes (Figure 2d). An additional block was observed in the terminal region of the variant B chromosome (Figure 2e).

Genetic diversity and population structure data

The seven ISSR primers used in the 10 analyzed populations generated a total of 95 reproducible fragments that varied in size between 300 and 2.500 base pairs (bp). Among the 95 analyzed loci, 71.05% were polymorphic, with the populations OUR–PE and ARA–SE presenting the highest and lowest percentages of polymorphism (90.5% and 13.6%), respectively (Table 4). In addition, the mean value for expected heterozygosity (H_E) in the populations was 0.311, with OUR–PE and MAC–AL presenting the highest and lowest values, corresponding to 0.36 and 0.28, respectively (Table 4).

The analysis of population models indicated the full model and =0 as the most adequate according to the parameters DIC and Dbar (Spiegelhalter et al., 2002Spiegelhalter DJ, Best NG, Carlin BP and van der Linde A (2002) Bayesian measures of model complexity and fit. J R Stat Soc Ser B Statistical Methodol 64:583–639.). However, the model =0, which assumes absence of inbreeding within the populations, is the most probable, owing to the gene flow evidenced by other analyses (F_ST, AMOVA) and the dispersion capacity of R. brasiliensis (Table 5).

Molecular variance (AMOVA) reached 15.32% among the populations and 64.68% within them. Moreover, low genetic differentiation and high gene flow were observed upon analysis of the fixation index, F_ST =0.15; the coefficient of genetic differentiation, G_ST = 0.17; and the number of migrants, Nm=2.3869. Most of the pairwise F_ST values were low, though significant values were obtained for the populations JN–CE and MAC–AL, ST–PE and ITA–PE, ST–PE and BUI–PE, and JN–CE and ST–PE (Table 6). The Mantel test revealed lack of correlation between the genetic and geographical distances (r: 0.023121; P: 0.828000), which indicates the absence of IBD.

The genetic clustering analysis performed with the software Structure presented K=2 as highest value, according to the method described by Evanno et al. (2005)Evanno G, Regnaut S and Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study. Mol Ecol 14:2611–2620. (Figure 3), with two genetic groups being observed in all analyzed populations (Figure 4).

Discussion

B chromosomes in R. brasiliensis

The polymorphism of the B chromosome in R. brasiliensis is well represented in Acrididae grasshoppers (Palestis et al., 2010Palestis BG, Cabrero J, Robert T and Camacho JPM (2010) Prevalence of B chromosomes in Orthoptera is associated with shape and number of A chromosomes. Genetica 138:1181–1189.). The broad distribution of R. brasiliensis individuals carrying B chromosomes suggests that this polymorphism is widespread among the populations, apparently there is a relation between the higher frequency of Bs and the geographical distribution, for example in GRA-PE, BEZ-PE, SUR-PE and VIT-PE. This tendency is likely associated with positive gene flow among those populations (better discussed below). Moreover, studies in other grasshopper species such as Eyprepocnemis plorans (Camacho et al., 1980Camacho JPM, Carballo AR and Cabrero J (1980) The B-chromosome system of the grasshopper Eyprepocnemis plorans subsp. plorans (Charpentier). Chromosoma 80:163–166.), Trimerotropis pallidipennis (Confalonieri, 1992Confalonieri V (1992) B-chromosomes of Trimerotropis pallidipennis (Oedipodinae, Acrididae): new effects on chiasma conditions. Caryologia 45:145–153.) and Dichroplus elongatus (Remis, 1989Remis MI (1989) Effects of supernumerary heterochromatin on chiasma condition in two species of Acrididae (Orthoptera). Genetica 79:53–61.) reported an increase in the frequency of recombination and chiasmata in host species, resulting in an advantageous increase in genetic variability (Camacho et al., 1980Camacho JPM, Carballo AR and Cabrero J (1980) The B-chromosome system of the grasshopper Eyprepocnemis plorans subsp. plorans (Charpentier). Chromosoma 80:163–166., 2002Camacho JPM, Bakkali M, Corral JM, Cabrero J, López-León MD, Aranda I, Martín-Alganza A and Perfectti F (2002) Host recombination is dependent on the degree of parasitism. Proc R Soc B Biol Sci 269:2173–2177.; Riera et al., 2004Riera L, Petitpierre E, Juan C, Cabrero J and Camacho JPM (2004) Evolutionary dynamics of a B chromosome invasion in island populations of the grasshopper Eyprepocnemis plorans. J Evol Biol 17:716–719.), which may be one of the reasons for the wide distribution of B chromosomes in R. brasiliensis.

The frequency of B chromosomes in R. brasiliensis was considered as high when above 11%, and as low when below 5%. However, all populations had values below 25%, which is indicative of the phase in which the B chromosome is in the life cycle (Camacho et al., 1997Camacho JPM, Shaw MW, López-Léon MD, Pardo MC and Cabrero J (1997) Population dynamics of a selfish B chromosome neutralized by the standard genome in the grasshopper Eyprepocnemis plorans. Am Nat 149:1030–1050.; Araújo et al., 2001Araújo S, Pompolo S, Perfectti F and Camacho J (2001) Integration of a B chromosome into the A genome of a wasp. Proc Biol Sci 268:1127-1131.). Given the values of the observed frequencies, the B of R. brasiliensis populations is probably in the stage of near neutrality of its life cycle. This is the longest phase, with successive generations being necessary to disperse the B chromosome, which has strictly vertical transmission. These frequencies may arise from stability, considering that these chromosomes tend to reach a balanced frequency over the years due to the efforts of the host genome to extinguish the B chromosome (Camacho et al., 1997Camacho JPM, Shaw MW, López-Léon MD, Pardo MC and Cabrero J (1997) Population dynamics of a selfish B chromosome neutralized by the standard genome in the grasshopper Eyprepocnemis plorans. Am Nat 149:1030–1050.; Riera et al., 2004Riera L, Petitpierre E, Juan C, Cabrero J and Camacho JPM (2004) Evolutionary dynamics of a B chromosome invasion in island populations of the grasshopper Eyprepocnemis plorans. J Evol Biol 17:716–719.).

The non-detection of B chromosomes in JP–PB and ARA–SE may be related either to elimination of the polymorphism or to low frequency in these populations as a manner of defense of the host genome, which attempts to get rid of parasite chromosomes (Cabrero et al., 2017Cabrero J, Martín-Peciña M, Ruiz-Ruano FJ, Gómez R and Camacho JPM (2017) Post-meiotic B chromosome expulsion, during spermiogenesis, in two grasshopper species. Chromosoma 126:633–644.). Most of the analyzed populations did not show a significant difference with regard to the occurrence and frequency of B chromosomes. This may be associated to the low frequency of B chromosomes in several populations and/or a relatively low number of sampled populations lacking these chromosomes. The sampling number of these groups was possibly insufficient to detect the occurrence of B chromosomes, a fact that was corroborated by the confidence interval calculation, which indicated maximum frequency of 8.2% in JP–PB and 26% in ARA–SE. In R. brasiliensis, the different frequencies may be associated with the sum of several other factors, such as the number of generations since the origination or emergence of the B chromosome; population differences in the accumulation of the B chromosome, in case this polymorphism brings an advantage to the host in a population; and action of genetic drift (in particular for ARA–SE, based on its low genetic diversity; Table 4), which may be eliminating individuals carrying B chromosomes.

The occurrence of R. brasiliensis individuals with two B chromosomes in the populations of BEZ–PE, OUR–PE and ITA–PE, as well as three B chromosomes in ITA–PE, is also observed in some Acrididae, for instance Aeropus sibiricus (Linnaeus, 1767) with four acrocentric B chromosomes (Jetybayev et al., 2018Jetybayev IY, Bugrov AG, Vladimirovna V and Rubtsov NB (2018) B chromosomes in grasshoppers: Different origins and pathways to the modern Bs. Genes 9:509–527.) and Eumastusia koebelei koebelei (Rehn, 1909), with two acrocentric B chromosomes (Anjos et al., 2016Anjos A, Loreto V and Cabral-de-Mello DC (2016) Cytogenetics organization of some repetitive DNAs and B chromosomes in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) (Orthoptera, Acrididae, Leptysminae). Comp Cytogen 10:219–228.). The presence of more than one B chromosome in R. brasiliensis is probably due to the non-disjunction of its sister chromatids during meiosis, as suggested by Camacho (2005)Camacho M (2005) B Chromosomes. In: Gregory TR (ed) The Evolution of the Genome. Elsevier, San Diego, pp 223-286.. Some mechanisms may be acting in this phenomenon, such as a control region of non-disjunction located in the B chromosome itself (in the terminal or pericentromeric region), as already observed in rye (Endo et al., 2008Endo TR, Nasuda S, Jones N, Dou Q, Akahori A, Wakimoto M, Tanaka H, Niwa K and Tsujimoto H (2008) Dissection of rye B chromosomes, and nondisjunction properties of the dissected segments in a common wheat background. Genes Genet Syst 83:23–30.; Banaei-Moghaddam et al., 2012Banaei-Moghaddam AM, Schubert V, Kumke K, Wei O, Klemme S, Nagaki K, Macas J, González-Sánchez M, Heredia V, Gómez-Revilla D et al. (2012) Nondisjunction in favor of a chromosome: The mechanism of rye B chromosome drive during pollen mitosis. Plant Cell 24:4124–4134.). Moreover, non-disjunction of B chromatids could involve B-specific products (proteins or ncRNAs) that could associate with the centromeric regions and retard the separation during anaphase, resulting in both B chromatids ending up in the gamete (Benetta et al., 2019Benetta ED, Akbari OS and Ferree PM (2019) Sequence expression of supernumerary B chromosomes: Function or fluff? Genes 10:123-135.). Various B-chromosome transcripts related to genes encoding proteins or pseudo-genes in rye (Banaei-Moghaddam et al., 2013Banaei-Moghaddam AM, Meier K, Karimi-Ashtiyani R and Houben A (2013) Formation and expression of pseudogenes on the B chromosome of rye. Plant Cell 25:2536–2544.), fish (Valente et al., 2014Valente GT, Conte MA, Fantinatti BEA, Cabral-de-Mello DC, Carvalho RF, Vicari MR, Kocher TD and Martins C (2014) Origin and evolution of B chromosomes in the cichlid Fish Astatotilapia latifasciata based on integrated genomic analyses. Mol Biol Evol 31:2061–2072.; Ramos et al., 2016Ramos É, Cardoso AL, Brown J, Marques DF, Neill RJO and Martins C (2016) The repetitive DNA element BncDNA, enriched in the B chromosome of the cichlid fish Astatotilapia latifasciata, transcribes a potentially noncoding RNA. Chromosoma 126:313–323.), fly (Bauerly et al., 2014Bauerly E, Hughes SE, Vietti DR, Miller DE, McDowell W and Hawley RS (2014) Discovery of supernumerary B chromosomes in Drosophila melanogaster. Genetics 196:1007–1016.) and cervids (Makunin et al., 2016Makunin AI, Kichigin IG, Larkin DM, O’Brien PCM, Ferguson-Smith MA, Yang F, Proskuryakova AA, Vorobieva NV, Chernyaeva EN and O’Brien SJ (2016) Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing. BMC Genomics 17:1–14.) provide the basis to hypothesize about the involvement of protein-coding genes or pseudogenes in non-disjunction control. The constitutive heterochromatin (CH) may also act on the cohesion of the sister chromatids of the B chromosome, leading to non-disjunction (Banaei-Moghaddam et al., 2012Banaei-Moghaddam AM, Schubert V, Kumke K, Wei O, Klemme S, Nagaki K, Macas J, González-Sánchez M, Heredia V, Gómez-Revilla D et al. (2012) Nondisjunction in favor of a chromosome: The mechanism of rye B chromosome drive during pollen mitosis. Plant Cell 24:4124–4134.). However, the CH of B chromosomes from R. brasiliensis observed in this study, as well as by Loreto et al. (2008a)Loreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241. and Milani et al. (2018)Milani D, Bardella V, Ferretti A, Palacios-Gimenez O, Melo A, Moura R, Loreto V, Song H and Cabral-de-Mello D (2018) Satellite DNAs unveil clues about the ancestry and composition of B chromosomes in three grasshopper species. Genes 9:523-537., is restricted to the pericentromeric and/or terminal region, indicating that other of the mechanisms listed above may be acting on the non-disjunction of the B chromosomes.

The small amount of CH restricted to the pericentromeric region of the B chromosomes from R. brasiliensis is different from that observed in other grasshopper species that possess B chromosomes with CH amplification rich in satellite DNA (Loreto et al., 2008aLoreto V, Cabrero J, López-Léon MD, Camacho JPM and Souza MJ (2008a) Possible autosomal origin of macro B chromosomes in two grasshopper species. Chromosome Res 16:233–241.; Bernardino et al., 2017Bernardino ACS, Cabral-De-Mello DC, Machado CB, Palacios-Gimenez OM, Santos N and Loreto V (2017) B Chromosome variants of the grasshopper Xyleus discoideus angulatus are potentially derived from pericentromeric DNA. Cytogenet Genome Res 152:213–221.; Milani et al., 2018Milani D, Bardella V, Ferretti A, Palacios-Gimenez O, Melo A, Moura R, Loreto V, Song H and Cabral-de-Mello D (2018) Satellite DNAs unveil clues about the ancestry and composition of B chromosomes in three grasshopper species. Genes 9:523-537.). However, the hypothesis cannot be discarded that other repetitive DNA segments may be dispersed in euchromatic regions, such as transposable elements (TEs), which can be present in both observed types of B chromosomes (common and variant). TEs may have accumulated in the B chromosome and promoted the amplification of sequences (Marques et al., 2018Marques A, Klemme S and Houben A (2018) Evolution of plant B chromosome enriched sequences. Genes 9:515.), contributing to the generation of the variant B chromosome.

B chromosomes can be differentiated with the C-banding technique based on the patterns of CH distribution, as observed in different types of B chromosomes in the grasshopper E. plorans (Henriques-Gil et al., 1984Henriques-Gil N, Santos J and Arana P (1984) Evolution of a complex polymorphism in the grasshopper Eyprepocnemis plorans. Chromosoma 89:290–293.). In addition, the terminal region of the variant B chromosome found in the population from LC–PE was also C-positive, which further contributes to the characterization of the variant B chromosome. This variant is probably undergoing the regeneration stage of the cycle, where it has accumulated modifications in order to escape neutralization by the host genome (Henriques-Gil and Arana, 1990Henriques-Gil N and Arana P (1990) Origin and substitution of B chromosomes in the grasshopper Eyprepocnemis plorans. Evolution 44:747–753.; Camacho et al., 1997Camacho JPM, Shaw MW, López-Léon MD, Pardo MC and Cabrero J (1997) Population dynamics of a selfish B chromosome neutralized by the standard genome in the grasshopper Eyprepocnemis plorans. Am Nat 149:1030–1050.). This way, the hypothesis of non-recent origin of B chromosomes is reinforced, since successive generations are necessary for the B chromosome to be nearly neutralized, accumulate modifications and scatter in the populations (Henriques-Gil and Arana, 1990Henriques-Gil N and Arana P (1990) Origin and substitution of B chromosomes in the grasshopper Eyprepocnemis plorans. Evolution 44:747–753.; Camacho et al., 1997Camacho JPM, Shaw MW, López-Léon MD, Pardo MC and Cabrero J (1997) Population dynamics of a selfish B chromosome neutralized by the standard genome in the grasshopper Eyprepocnemis plorans. Am Nat 149:1030–1050.).

Genetic connectivity between populations of R. brasiliensis

As previously observed in other organisms (Taylor et al., 2011Taylor SJ, Downie DA and Paterson ID (2011) Genetic diversity of introduced populations of the water hyacinth biological control agent Eccritotarsus catarinensis (Hemiptera: Miridae). Biol Control 58:330–336.; Pinheiro et al., 2012Pinheiro LR, Rabbani ARC, da Silva AVC, da Silva Lédo A, Pereira KLG and Diniz LEC (2012) Genetic diversity and population structure in the Brazilian Cattleya labiata (Orchidaceae) using RAPD and ISSR markers. Plant Syst Evol 298:1815–1825.; Izzatullayeva et al., 2014Izzatullayeva V, Akparov Z, Babayeva S, Ojaghi J and Abbasov M (2014) Efficiency of using RAPD and ISSR markers in evaluation of genetic diversity in sugar beet. Turkish J Biol 38:429–438.), the ISSR marker was polymorphic in R. brasiliensis, with 71% of the loci presenting variation in the ten analyzed populations. The expected heterozygosity (H_E) presented variation between 0.285 in MAC–AL and 0.363 OUR–PE. These values are similar to those described for other insects, such as the grasshoppers Pezotettix giornae (Rossi, 1794) (Gauffre et al., 2015Gauffre B, Mallez S, Chapuis MP, Leblois R, Litrico I, Delaunay S and Badenhausser I (2015) Spatial heterogeneity in landscape structure influences dispersal and genetic structure: empirical evidence from a grasshopper in an agricultural landscape. Mol Ecol 24:1713–1728.) and E. plorans (Manrique-Poyato et al., 2013aManrique-Poyato MI, López-León MD, Gómez R, Perfectti F and Camacho JPM (2013a) Population Genetic Structure of the Grasshopper Eyprepocnemis plorans in the South and East of the Iberian Peninsula. PLoS One 8:e59041.); the honey bee Apis mellifera meda (Skorikow, 1829) (Rahimi et al., 2016Rahimi A, Mirmoayedi A, Kahrizi D, Zarei L and Jamali S (2016) Genetic diversity of Iranian honey bee (Apis mellifera meda Skorikow, 1829) populations based on ISSR markers. Q Rev Biol 83:117–117.); as well as the low value in the beetle Canthon staigi (Pereira, 1953) (Ferreira-Neto et al., 2017Ferreira-Neto CA, Cruz GA dos S, Amorim IC, Balbino VQ and Moura RC (2017) Effects of fragmentation and anthropic pressure on the genetic structure of Canthon (Peltecanthon) staigi (Coleoptera: Scarabaeidae) populations in the Atlantic Forest domain. J Insect Conserv 21:267–276.) and the high value in Lucanus cervus (Linnaeus, 1758) (Snegin, 2014Snegin EA (2014) Analysis of gene flow between generations of various years in population of stag beetle (Lucanus cervus L.) based on RAPD and ISSR DNA markers. Adv Environ Biol 13:9-12,). Although it has already been evinced that in grasshoppers the presence of B chromosomes may contribute to the increase in genetic variability, this relationship was not observed for R. brasiliensis, in which some populations present low H_E, such as ST–PE (0,291) and JN–CE (0.290). Moreover, the total expected heterozygosity (H_T= 0.298) was greater than the subpopulation heterozygosity (H_S= 0.270), indicating that there may be a deficit of heterozygotes.

The data generated in this study suggest that the occurrence of B chromosomes in R. brasiliensis is related to its population genetic structure, given the wide dispersion of the B chromosome verified in the karyotyped individuals, positive gene flow (Nm = 2.3869), and low genetic differentiation F_ST (0.15) and G_ST (0.17) among the analyzed populations. Owing to the migratory behavior of R. brasiliensis (Carbonell, 1988Carbonell CS (1988) Rhammatocerus schistocercoides (Rehn, 1906), especie prejudicial para la agricultura en la región centro oeste de Brasil (Orthoptera, Acrididae, Gomphocerinae). Bol Mus Nac Rio de Janeiro 318:1-17.), this is an expected pattern, considering that the probable means of dispersion of the B chromosome in different geographic ranges, according to Camacho et al. (2015)Camacho JPM, Shaw MW, Cabrero J, Bakkali M, Ruíz-Estévez M, Ruíz-Ruano FJ, Martín-Blázquez R and López-León MD (2015) Transient microgeographic clines during B chromosome invasion. Am Nat 186:675–681., would be through gene flow between hosts and individuals from populations that do not have the B chromosome. The Mantel test suggested a lack of correlation between genetic and geographical distance, even between populations more than 700 km apart (ITA–PE and JN–CE). This may be contributing to the dispersion of the B chromosomes, as their transmission is strictly vertical (Munoz et al., 1998Munoz E, Perfectti F, Martin-Alganza A and Camacho JPM (1998) Parallel effects of a B chromosome and a mite that decrease female fitness in the grasshopper Eyprepocnemis plorans. Proc R Soc B Biol Sci 265:1903–1909.). Pairwise F_ST indicated an overall low genetic differentiation; however, some populations presented F_ST with significantly high values (JN–CE and MAC–AL, JN–CE and ST–PE, ST–PE and ITA–PE, ST–PE and BUI–PE; Table 6).

The data from molecular variance (AMOVA), which was greater within the populations (84.68%), when associated to Structure data, corroborate the low indices of genetic differentiation, where the presence of a mixture of two genetic profiles is related to migratory habit, wide mobility, and flight capacity of R. brasiliensis (Carbonell, 1988Carbonell CS (1988) Rhammatocerus schistocercoides (Rehn, 1906), especie prejudicial para la agricultura en la región centro oeste de Brasil (Orthoptera, Acrididae, Gomphocerinae). Bol Mus Nac Rio de Janeiro 318:1-17., 1995Carbonell CS (1995) Revision of the tribe Scyllinini, Nov. (Acrididae: Gomphocerinae), with descriptions of new genera and species. Trans Amer Entomol Soc 121:87–152.). Native grasses, for instance Trachypogon sp. (Poaceae), constitute the preferred food of grasshoppers (Silva et al., 2006Silva RA, Jesus CR, Silva WR and Costa NL (2006) Ocorrência de gafanhotos em áreas de cerrados de Mazagão, Amapá. Embrapa Amapá, Macapá, 5 pp.). Therefore, the large offer of grasses in the localities sampled in this study and the absence of effective geographic barriers between the populations are fundamental factors for the wide dispersion of the analyzed specimens. The feeding preference of R. brasiliensis is, which is strongly related to its mandibular structure of graminivorous type, is also present in other Gomphocerinae such as Achurum carinatum (Walker, 1870), Dichromorpha viridis (Scudder, 1862) and Orphulella pelidna (Burmaister, 1838) (Smith and Capinera, 2005Smith TR and Capinera JL (2005) Mandibular morphology of some floridian grasshoppers (Orthoptera: Acrididae). Florida Entomol 88:204–207.). For other grasshopper species, for instance Mioscirtus wagneri (Eversmann, 1859) and Ramburiella hispanica (Rambur, 1838), a positive relationship was also observed between the distribution of plants used as food and the genetic connectivity between populations (Ortego et al., 2010Ortego J, Aguirre MP and Cordero PJ (2010) Population genetics of Mioscirtus wagneri, a grasshopper showing a highly fragmented distribution. Mol Ecol 19:472–483., 2015Ortego J, Aguirre MP, Noguerales V and Cordero PJ (2015) Consequences of extensive habitat fragmentation in landscape-level patterns of genetic diversity and structure in the Mediterranean esparto grasshopper. Evol Appl 8:621–632.).

Although the populations of Pernambuco and other states occupy different altitudes (range 0 to 999 m), as well as potential geographic barriers (Borborema Plateau, Chapada do Araripe and São Francisco river), R. brasiliensis individuals seem to be capable to overcome these obstacles and maintain a positive gene flow among the analyzed populations. This way, no correlation was observed between the occurrence of B chromosomes and the distribution of R. brasiliensis in different landscapes. For this species, the altitude of the analyzed populations did not seem to influence the frequency of B chromosomes either. This observation differs from that of Manrique-Poyato et al. (2015)Manrique-Poyato MI, López-León MD, Cabrero J, Gómez R, Perfectti F and Camacho JPM (2015) Geographical barriers impeded the spread of a parasitic chromosome. PLoS One 10:e0131277., who stated that individuals with B chromosomes have low tolerance to high-altitude environments as a result of delay in meiosis due to the presence of B chromosomes that may negatively select the host (Hewitt and East, 1978Hewitt GM and East TM (1978) Effects of B chromosomes on development in grasshopper embryos. Heredity 41:347–356.; Harvey and Hewitt, 1979Harvey AW and Hewitt GM (1979) B chromosomes slow development in a grasshopper. Heredity 42:397–401.).

Based on literature evidence that B chromosomes are only transmitted vertically, it is suggested that, for the species R. brasiliensis, the broad distribution of B chromosomes indeed arises from the extant gene flow among the analyzed populations. The migratory habit coupled to the high flying capacity of R. brasiliensis, besides the wide offer of food resources in the sampled localities, are factors that certainly contributed to the genetic connectivity of its populations, and consequently for the broad dispersion of the B chromosome. A variant B chromosome, with CH blocks in the pericentromeric and terminal regions, was detected and is probably currently in the regeneration stage of the cycle. The presence of one such variant B chromosome reinforces the hypothesis of the non-recent origin of the B chromosome, considering that successive generations are necessary for such a chromosome to be nearly neutralized, accumulate modifications and be dispersed among distinct populations.

Acknowledgments

We would like to thank all those who have collaborated to the collections, in particular Mônica Pereira Santos (in memoriam), who also contributed to the cytogenetic analysis. We further thank Dr. Ulisses Montarroyos for the colaboration with the statistical analysis. Finally, we thank the funding agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brasil, Finance code 001) for the PNPD scholarship of GASC (20130558); Fundação de Amparo a Ciência e Tecnologia de Pernambuco (FACEPE) for the doctorate degree scholarship of ASM (IBPG-0769-2.02/14) and the BCT scholarship of GASC (00632.02/14); and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq–Brazil) for the PQ scholarship of RCM (process no. 305298/2014-3).

References

Publication Dates

History