ORIGINAL ARTICLE

U1 snDNA clusters in grasshoppers: chromosomal dynamicsand genomic organization

A Anjos1, FJ Ruiz-Ruano2, JPM Camacho2, V Loreto3, J Cabrero2, MJ de Souza3 and DC Cabral-de-Mello1

The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturationthrough intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusterswere observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed inthe first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome.By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosomepairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis inEyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogeniclineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor toboth species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersedelements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements.Heredity (2015) 114, 207–219; doi:10.1038/hdy.2014.87; published online 24 September 2014

INTRODUCTION

In higher eukaryotes, most protein-coding transcripts contain multipleintrons that need to be removed from the nascent RNA throughsplicing, an essential mechanism for mRNA maturation (West, 2012).The spliceosome consists of a set of proteins associated with U smallnuclear RNAs (snRNAs), which are crucial components of thismacromolecular complex. The major spliceosome complex is encodedby a multigene family including the U1, U2, U4, U5 and U6 snRNAgenes (Gilbert, 1978; Busch et al., 1982; Bringmann and Lührmann,1986; Nilsen, 2003; Valadkhan, 2005). Among U snRNA genes, the U1snDNA is a multigene family showing variable number of repeats pergenome and is conserved in sequence; however, distinct U1 snDNAintragenomic variants have been found in several organisms, such astoad (Forbes et al., 1984), fruit flies (Lo and Mount, 1990), pea(Hanley and Schuler, 1991), silk moths (Sierra-Montes et al., 2003),Oreochromis niloticus (Cabral-de-Mello et al., 2012) and humans(O’Reilly et al., 2012).The U1 snRNA gene is tandemly arranged in some species, such as

the toad Xenopus laevis (Zeller et al., 1984), the sea urchin Strongy-locentrotus purpuratus (Yu et al., 1991) and the tapeworm Echinococcusmultilocularis (Bretagne et al., 1991). In addition, the U1 snRNA islinked to other gene repeats (for example, 5S rDNA) in the crustaceanAsellus aquaticus (Pelliccia et al., 2001), the fish Solea senegalensis

(Manchado et al., 2006) and 10 razor shell species (Vierna et al., 2011).At the chromosomal level, the mapping of U1 snRNA genes has beenperformed only in a few species, for instance, humans (Lund et al.,1983), mice (Lund and Nesbitt, 1988), crustaceans (Barzotti et al.,2003) and fishes (Cabral-de-Mello et al., 2012). Such endeavors haverevealed that these genes are usually conserved with regard to locationamong related species, harbored on one or a few chromosome pairs.Chromosomal studies in grasshopper species have revealed high

karyotype conservatism, with most species showing 2n= 23♂/24♀acrocentric chromosomes and an X0♂/XX♀ sex chromosome system.However, derived karyotypes showing reductions in diploid number,derived sex chromosomes and supernumerary elements have also beenreported (White, 1978; Hewitt, 1979; Mesa et al., 1982; Castillo et al.,2010). Current knowledge about repetitive DNA organization ingrasshopper chromosomes is still scarce; concerning multigenefamilies, only the 45S and 5S rDNAs and H3 and H4 histone geneshave been mapped in multiple species, mainly in Acrididae represen-tatives, which represent the most diverse family worldwide distributed,and together with Ommexechidae and Romaleidae are consideredmodern grasshoppers. In a lesser extent, chromosomal mapping forrDNAs and/or distinct histone genes was also performed in closelysister families of Acrididae, that is, Romaleidae and Ommexechidaeand in the earliest diverging basal lineage of Acridomorpha

1Departamento de Biologia, Instituto de Biociências/IB, UNESP—Universidade Estadual Paulista, Rio Claro, Brazil; 2Departamento de Genética, Facultad de Ciencias,UGR—Universidad de Granada, Granada, Spain and 3Departamento de Genética, Centro de Ciências Biológicas/CCB, UFPE—Universidade Federal de Pernambuco, Recife,BrazilCorrespondence: Dr DC Cabral-de-Mello, Departamento de Biologia, Instituto de Biociências/IB, UNESP—Universidade Estadual Paulista, CEP 13506-900 Rio Claro, São Paulo,Brazil.E-mail: mellodc@rc.unesp.brReceived 24 March 2014; revised 14 August 2014; accepted 18 August 2014; published online 24 September 2014

Heredity (2015) 114, 207–219& 2015 Macmillan Publishers Limited All rights reserved 0018-067X/15www.nature.com/hdy

(Eumastacoidea) represented by Proscopiidae (Flook et al., 1999;Cabrero and Camacho, 2008; Cabrero et al., 2009; Cabral-de-Melloet al., 2011a, b; Carvalho et al., 2011; Jetybayev et al., 2012; Leavittet al., 2013; Neto et al., 2013; Palacios-Gimenez et al., 2013).Grasshoppers show gigantic genomes, reaching 16.93 pg in Podisma

pedestris (Westerman et al., 1987). The recent full genome sequencingin Locusta migratoria (genome size ca. 6 pg) has revealed that 60% ofDNA in this species is repetitive (Wang et al. 2014), a figure whichcould be even higher in species with larger genomes. However, thechromosomal organization of such a major genomic component ismostly unknown despite being important information complementingfull-genome sequencing projects. We show here that the integration ofchromosomal and genomic analyses unveils new details on theorganization and evolution of repetitive DNA families. We have thusperformed fluorescence in situ hybridization (FISH) mapping of theU1 snRNA gene in 70 species of grasshoppers belonging to fivedistantly related families, including the most representative groups,inferred from molecular phylogenetic studies (Leavitt et al., 2013):Proscopiidae, Ommexechidae, Pyrgomorphidae, Romaleidae andAcrididae (the latter including 10 subfamilies). We also analyzed U1snDNA genomic organization in E. plorans, which is one of the speciesshowing U1 clusters in seven chromosome pairs, using data obtainedthrough 454 pyrosequencing, aiming to unveil possible causes for U1snDNA intragenomic spread. In addition, we analyzed the U1 snDNAsequences in the recently published full genome of L. migratoria(Wang et al., 2014), a species showing only one U1 locus. This hasrevealed the colocalization of U1 snDNA with other repetitive DNAfamilies being suggestive of possible spreading mechanism that couldbe generally operating in grasshopper genomes.

MATERIALS AND METHODS

Chromosomes, probes and FISHAdult males and females of 63 species of grasshoppers belonging to familiesProscopiidae (4 species), Ommexechidae (2 species), Pyrgomorphidae(2 species), Romaleidae (11 species) and Acrididae (44 species belonging to10 subfamilies) were sampled from distinct localities in Spain, Paraguay,Argentina and Brazil (see Supplementary Material S1). Testes were fixed in 3:1absolute ethanol:acetic acid and stored at − 20 °C until use. Embryos ofE. plorans were obtained as described by Camacho et al. (1991), and adultfemale gastric ceca were removed and fixed following Castillo et al. (2011). Thedifferent tissues were macerated in a drop of 50% acetic acid, and the slideswere dried using a hot plate at 40–45 °C. In addition, previous results forAbracris flavolineata (Bueno et al., 2013) and six other species (Palacios-Gimenez et al., 2013) were also included for analysis.The DNA probe for the U1 snDNA sequence was obtained by PCR using

DNA of Rhammatocerus brasiliensis with the primers described by Cabral-de-Mello et al. (2012); 5S rDNA was obtained from a previously cloned fragmentisolated from the E. plorans genome. The PCR product for U1 snDNA waspreviously sequenced, and the sequence was deposited in GenBank under theaccession number KC896793 (Bueno et al., 2013). U1 snDNA and the 5S rDNAfragment were labeled with digoxigenin-11-dUTP (Roche, Mannheim,Germany) or biotin-14-dUTP (Invitrogen, San Diego, CA, USA) using PCRor nick translation. The FISH procedures were performed according to Pinkelet al. (1986), with some modifications (Cabral-de-Mello et al., 2010). FiberFISH was performed as described in Muñoz-Pajares et al. (2011). In brief,cerebral ganglia were homogenized in 60% acetic acid and centrifuged for10min at 1000 r.p.m. After removing the supernatant, we added 500 μl of 1:3acetic acid: ethanol and resuspended the cells. Fixed cells were then spread onclean moist slides and, before the complete evaporation of the fixative, the slideswere placed in phosphate-buffered saline solution (pH= 7) for 1 min. To relaxchromatin, the slides were drained on a paper towel and immediately treatedwith 0.05 M NaOH in 30% ethanol as follows: 200 μl solution was placed onone end of a long coverslip that was then moved along the horizontal slide at an

angle of about 30°. Then, we added a few drops of absolute ethanol (500ml)and left it dry. Finally, to dehydrate the material, the slides were placed in a 70,90 and 100% ethanol series and left it dry. Double FISH was performed asdescribed in Cabrero et al. (2003). The probes were detected using anti-digoxigenin-rhodamine (Roche) or Streptavidin Alexa Fluor 488 conjugate(Invitrogen). The chromosomes were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted using Vectashield (Vector, Burlingame,CA, USA).The chromosomes and signals were observed using an Olympus microscope

(Tokyo, Japan) BX61 equipped with a fluorescence lamp and appropriatefilters. The photographs were recorded using a DP70 cooled digital camera, andimages were merged and optimized for brightness and contrast using AdobePhotoshop CS2. Finally, the signals were analyzed and organized into fivecategories: centromeric (c), proximal (pr), interstitial (i), subdistal (sd) anddistal (d), as shown in the diagram in Supplementary Material S2. To facilitatethe comparative analysis, we numbered the autosome pairs in each species inorder of decreasing size, but this does not necessarily imply chromosomehomology among the species.

Genomic organization of U1 snDNA in E. plorans and L. migratoriaAs a first approach to unveiling the genomic organization of the U1 snDNA ingrasshopper genomes, we analyzed two species showing very different patternsat the chromosomal level: E. plorans, showing U1 clusters in seven chromosomepairs, and L. migratoria, showing it in a single chromosome pair. For thispurpose, we performed an analysis of the intragenomic variation of U1 snDNAin E. plorans by constructing a whole-genome sequencing library, which wassequenced using 3/8 of a 454 GS FLX Plus plate (accession numbersSRR1200829 and SRR1200835) generated by Macrogen Inc. (Seoul, Korea).We checked the quality of the reads with FASTQC (Andrews, 2012) andperformed a quality trimming with the Roche’s 454 GS Assembler. Afterexcluding reads shorter than 200 nt, we splitted the remaining reads in as manyfragments of 200 nt as possible, for example, from a 537 nt long read weincluded in the analysis, a sequence corresponded to the 1–200 nt and anotherone to the 201–400. Then, we performed a graph-based clustering and assemblyof these sequences to search for genes associated with U1 snDNA in thegenome using the RepeatExplorer software (Novák et al., 2013). The contigsincluding the U1 snDNA were submitted to GenBank (accession numbersKJ606066–70). We then extracted the reads used to assemble each U1 carriercontig separately with SeqGrapheR (Novák et al., 2010) to map them againsttheir contig and manually discard the U1 snDNA spacers to select the completecopies of U1 using Geneious v4.8 (Drummond et al., 2009).In addition, we got copies of the U1 snDNA present in the assembled

genome of L. migratoria recently published by Wang et al. (2014), which isavailable at GenBank with accession number AVCP000000000. We searched allthe contigs matching one of the U1 snDNA copies previously found inE. plorans by a local BLASTN v2.2.28 search (Altschul et al., 1997) with defaultoptions. To extract only the U1 snDNA region in all the matched contigs, wesearched the U1 snDNA copies of E. plorans using RepeatMasker (AFA Smit,R Hubley, P Green, unpublished data, current version: open-4.0.3 (RMLib:20130422 & Dfam: 1.2)) and, with a custom Python script, we extracted thematching regions. We aligned the resulting sequences with MAFFT v7.157b(Katoh and Standley, 2013) with LINSI options organizing by identity. We thenprepared a consensus sequence from this alignment of L. migratoria andrepeated the previous procedure using this sequence as a reference. Weconsidered all complete U1 copies and those truncated copies being foundthree or more times. To get representative lineages of complete copies, weperformed a clustering analysis with UCLUST v6.0.307_linux32 (Edgar, 2010)using the ‘–cluster-fast’ option and 0.96 identity threshold, as this was theidentity between the two functional lineages in E. plorans. We only consideredas lineages those groups of sequences with two or more sequences, and werepresented them by their consensus sequence. Truncated copies sharing thesame deletion were included into the same lineage.A minimum spanning tree was built with Arlequin v3.5 (Excoffier and

Lischer, 2010) for the U1 snDNA sequences found in the contigs of bothspecies. As an outgroup, we used the U1 sequence of the mountain pine beetleDendroctonus ponderosae obtained in a local BLASTN search (Altschul et al.,

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1997) against NCBI NR database. The analysis for sequence diversity wasconducted using DNASP v5.10 (Librado and Rozas, 2009) excluding positionswith gaps in the alignments. The G+C content was calculated with the MEGAv5.0 (Tamura et al., 2011) software, and the prediction of secondary structureand Gibbs free energy (ΔG) for the different sequence lineages was performedwith RNAFold (Hofacker, 2003), applying a constrained folding to avoid thepairing of the four conserved motifs, according to Vierna et al. (2013).To find other repetitive elements associated with U1, in the contigs generated

by RepeatExplorer in E. plorans and L. migratoria, we annotated them by meansof a search in RepBase using the CENSOR software (Kohany et al., 2006) andalso by means of a BLASTN search (Altschul et al., 1997) in the NCBI NRdatabase. We considered only annotations obtained by both methods. More-over, to visualize the relationship between the different contigs obtained fromthe E. plorans genome, including U1 snDNA and other repetitive sequences, werepresented them graphically with the SeqGrapheR software (Novák et al.,2010). We also searched for repeated regions inside the selected contigs andcompared each contig with itself applying a thereshold dotplot analysisintegrated in Geneious v4.8 (Drummond et al., 2009).

RESULTS

Chromosomal mapping of U1 snDNAThe diploid chromosome number and sex chromosome system for the70 species (some reported here for the first time) are shown inSupplementary Material S1. A total of 168 U1 snDNA sites weredetected by FISH in the 70 species included, with an average of2.4 sites per haploid genome. Most of these sites (162) were located onautosomes, whereas only 5 were on the X chromosome and 1 ona neo-Y chromosome. Most sites were located proximal tothe centromere (60 sites, 35.7%), and fewer were interstitial(39, 23.2%), centromeric (35, 20.9%), subdistal (20, 11.9%) or distal(14, 8.3%; Table 1). Although most species carried U1 snDNA clusterson only one autosomal pair, variable patterns were also observed, evenincluding two species with clusters on all chromosome pairs.Furthermore, in several species, one (for example, Ommexecha virens,Agriacris auripennis, Chorthippus apicalis, R. brasiliensis and Eumastusiakoebelei koebelei) or two (for example, Omocestus bolivari, Omocestusburri, Omocestus panteli, and Stenobothrus festivus) chromosome pairscarried two or more clusters of U1 snDNA on the same chromosome(Table 1).The five grasshopper families analyzed showed distinct patterns of

distribution for the U1 snDNA clusters. In Proscopiidae, all fourspecies analyzed showed a single interstitial cluster on the fourthlargest autosome pair (Figures 1a and b). Similarly, the two Pyrgo-morphidae species analyzed showed a single cluster proximally locatedon the third largest autosome pair (Figures 1c and d). The twoOmmexechidae representatives also showed a coincident pattern but,in this case, showed three sites per species, with one of themproximally located on the largest autosome pair and the two otherat centromeric and interstitial regions on the third largest autosomepair (Figure 1e).Among the Romaleidae, 7 out of the 11 species analyzed showed a

single cluster of U1 snDNA on the third largest autosome pair, whichwas proximal in 6 species and subdistal in 1 species (Figure 2).However, A. auripennis showed two clusters (centromeric andproximal) on the eighth autosome pair (Figure 2a), and the twoChromacris species showed clusters on a single pair, namely, proximalon the fifth autosome of C. nuptialis and subdistal on the fourthautosome of C. speciosa (Figures 2c and d). Finally, Brasilacris gigasshowed a single interstitial cluster on the seventh largest autosome pair(Figure 2b).In the Acrididae family, the most representative sample analyzed

with 51 species from 10 subfamilies, we found 144 U1 snDNA clusters,

that is, 2.82 per haploid genome (Figures 3 and 4; Table 1). Thisanalysis showed that 46 of these sites were proximal (32.0%), 34 wereinterstitial (23.6%), 32 were centromeric (22.2%), 18 were subdistal(12.5%) and 14 were distal (9.7%). About 40% of the species (21)showed U1 snDNA on a single chromosome pair, but some speciesshowed it on two or more pairs, with two species (Oedipodafuscocincta and Sphingonotus caerulans) carrying it on all chromosomepairs (see Figure 3l). In several species, two or more clusters werelocated on the same chromosome (Figure 4; Table 1).The three best-represented acridid subfamilies were Leptysminae

(7 species), Melanoplinae (13 species) and Gomphocerinae (16species). The Leptysminae species showed 1–3 clusters that werepredominantly interstitial (six clusters) or proximal (four) but rarelycentromeric (one) and never subdistal or distal, with two species(Cylindrotettix obscurus and Stenopola sp) showing U1 snDNA clusterson the X chromosome (Figure 3e; Table 1). Almost all Melanoplinaespecies showed a single autosome pair carrying the genes for U1snRNA, but they never showed a centromeric location (Table 1).Propedies auriculares was the only Melanoplinae species carrying twoclusters on the same chromosome (Figure 3i; Table 1). Finally,Gomphocerinae species showed 1–6 U1 snDNA clusters, always onautosomes, with all types of location but higher frequency in proximal(21 clusters), interstitial (18) and subdistal (14) locations and lowerfrequency in centromeric (7) and distal (6) locations (Figure 4;Table 1). Remarkably, all Chorthippus and Omocestus species carried2, 3 or more U1 snDNA clusters (see Figure 4) on the longestautosome, which could have been present in their common ancestor.The average number of U1 snDNA clusters per haploid genome was1.6, 1.3 and 4.12 in the subfamilies Leptysminae, Melanoplinae andGomphocerinae, respectively.Finally, some acridid species carried B chromosomes, including

E. plorans, C. attenuatus, Eu. k. koebelei, S. dorsalis, R. brasiliensis,A. flavolineata (Bueno et al., 2013), Orthoscapheus rufipes and Vilernarugulosa, but no clusters of U1 snDNA were observed on any of theseelements (see, for example, Figures 3f, j and k).

U1 snDNA and other repetitive elements in the E. plorans andL. migratoria genomesUsing graph-based clustering, we obtained a complex cluster inE. plorans with U1 snDNA in the central position and a multitudeof ‘loops’ and ‘branches’ connected to it (Figure 5), as expected fromits multiple locations in this genome. This cluster constituted 0.222%of the genomic sequences analyzed. The assembly of all DNAsequences included in the cluster resulted in a total of 63 contigs,5 of them carrying U1 snDNA. On the basis of its coding region, wefound complete U1 snRNA genes in two contigs (lineages 1 and 2),but there were several others showing different mutations, such as adeletion in the middle (lineage 3), a 5′ truncation of 34 nt (lineage 4)and a deletion and an insertion close to the 5′ end (lineage 5;Figure 6a). Nucleotide diversity was zero for lineages 1 and 3, but veryhigh for lineages 2 (π= 0.01536), 4 (π= 0.03946) and 5 (π= 0.01258;Table 2). We predicted the secondary structure of the five U1 snDNAconsensus sequences, as this can be an indicator of its functionality orpseudogenicity (Figure 6b). Lineages 1 and 2 contained the fourconserved sites described in Vierna et al. (2013) (Figure 6b), and theyshowed the same pattern, with four helices, as the consensussecondary structure of U1 snRNA reported in Vierna et al. (2013).The secondary structure of lineage 1 showed higher stability(ΔG=− 56.0 kcal mol− 1) than lineage 2 (ΔG=− 54.6 kcal mol− 1).In contrast, lineages 3, 4 and 5 showed secondary structures lackingthe conserved motifs present in the two other lineages (Figure 6b).

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Table 1 Chromosomal location of U1 snDNA in 70 grasshopper species belonging to five families

Family

Subfamily

SpeciesChromosome no (in order of decreasing size)

1 2 3 4 5 6 7 8 9 10 11 Sex chromosomes

ProscopiidaeProscopiinae

Scleratoscopia protopeirae — — — 1i — — — — — — — —

Scleratoscopia spinosa — — — 1i — — — — — — — —

Scleratoscopia silvae — — — 1i — — — — — — — —

Stiphra robusta — — — 1i — — — — — — — —

PyrgomorphidaePyrgomorphinae

Algete brunneri — — 1pr — — — — — — — — —

Omura congrua — — 1pr — — — — — — — — —

OmmexechidaeOmmexechinae — — — — — — — — — — — —

Ommexecha gracilis 1pr — 1c/1pr — — — — — — — — —

Ommexecha virens 1pr — 1c/1pr — — — — — — — — —

RomaleidaeRomaleinae

Agriacris auripennis — — — — — — — 1c/1pr — — — —

Brasilacris gigas — — — — — — 1i — — — — —

Chromacris nuptialis — — — — 1pr — — — — — — —

Chromacris speciosa — — — 1sd — — — — — — — —

Helionotus mirabilis — — 1pr — — — — — — — — —

Phaeopharia megacephala — — 1pr — — — — — — — — —

Radacridium nordestinum — — 1pr — — — — — — — — —

Tropidacris collaris — — 1pr — — — — — — — — —

Xestotrachelus robustus — — 1sd — — — — — — — — —

Xyleus discoideus angulatus — — 1pr — — — — — — — — —

Xyleus discoideus discoideus — — 1pr — — — — — — — — —

— — — — — — — — — — — —

AcrididaeAcridinae

Metaleptea adspersa — — — — 1pr 1pr — — — — — —

Orphula pagana — — 1d 1d — — — — — — — —

Catantopinae

Pezotettix giornae — — — — — 1pr — — — — — —

Copiocerinae

Adimantus ornatissimus — — — — — — — 1pr — — — —

Cyrtacanthacridinae

Anacridium aegyptium 1i — — — — — — — — — — —

Schistocerca flavofasciata 1i 1i — — — — — — — — — —

Schistocerca pallens 1i 1i — — — — — — — — — —

Eyprepocnemidinae

Eyprepocnemis plorans# 1pr — — 1pr 1pr 1pr — 1pr 1pr — 1pr —

Gomphocerinae

Amblytropidia australis — — 1i — — — — 1pr/1i — — — —

Amblytropidia sp. 1pr 1sd — — 1sd — 1i 1i — — — —

Chorthippus apicalis 1pr/1d — — — — — — — — — — —

Chorthippus binotatus 1pr/1i/1sd — — — — — — — — — — —

Chorthippus jacobsi 1pr/1i/1sd — 1c — 1pr — — — — — — —

Chorthippus jucundus 1pr/1i/1sd — 1sd — — — — — — — — —

Chorthippus nevadensis s 1d 1pr 1pr — — — — — — — —

Chorthippus parallelus 2pr/2i 1sd — — 1i — — — — — — —

Dociostaurus maroccanus — — 1c — 1c — 1c — 1c — — —

Euchorthippus pulvinatus — 1pr/2i/1sd — — — — — 1pr — — — —

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These results suggest the possibility that lineages 3–5 are pseudogenic.The number of sequences obtained for each lineage (shown inTable 2) indicates that 45% were functional and 55% werepseudogenic.In the case of L. migratoria, we found 130 U1 copies in 109 different

contigs from the genome assembled by Wang et al. (2014), but only 55(42.3%) were complete, with 47 of them grouping into four differentlineages (Figure 6a) and the remaining 8 being singletons, which werediscarded for subsequent analysis. All these 47 copies of U1 werefound in differents contigs. In addition, we found 15 sequencesshowing the same truncation of 34 nt in the 5′ end, and theywere considered a fifth lineage (Supplementary Material S3). Thesecondary structure of the lineages 1–4 (Figure 6b) suggested their

functionality, with stability values (ΔG) ranging between − 57.9 and− 61.3 kcal mol− 1. The truncation of lineage 5 makes it clearlypseudogenic (ΔG=− 41.3 kcal mol− 1; Figure 6b). The lowest nucleto-tide diversity was shown by lineage 2 (π= 0.00781) and the highestone was observed in lineage 5 (π= 0.15366; Table 2).We built a minimum spanning tree with the five U1 snDNA

lineages observed in E. plorans and the five ones observed inL. migratoria (Figure 6b). Because NCBI’s nucleotide database lacksfull-length copies of U1 snDNA from Orthoptera, we searched thesequences of lineages 1 and 2 of E. plorans in the NR database of NCBIand chose the U1 snDNA sequence showing the lowest e-value forboth lineages, which corresponded to that in the mountain pine beetleDendroctonus ponderosae (accession number APGK01030738.1,

Table 1 (Continued )

Family

Subfamily

SpeciesChromosome no (in order of decreasing size)

1 2 3 4 5 6 7 8 9 10 11 Sex chromosomes

Omocestus bolivari 2pr/1i/1sd 1pr/1d — — — — — — — — — —

Omocestus burri 1pr/1i/1sd 1pr/1d — — — — — — — — — —

Omocestus panteli 1pr/1sd 1pr/1d — — — — — — — — — —

Orphulella punctata — — — — — 1d — — — — — —

Rhammatocerus brasiliensis# — — — 1i — 1c/1sd 1i — — — — —

Stenobothrus festivus 2i/1sd 1c/1sd — — 1pr — — — — — — —

Leptysminae

Cornops frenatum frenatum — — — — — — — — — 1i — —

Cylindrotettix attenuatus# — — — — — — — — — 1i — —

Cylindrotettix obscurus — — 1i — — — — — — — — 1pr(X)

Eumastusia koebelei koebelei — — 1c/1pr — — — — — — — — —

Stenopola sp — — — 1i — — — — — 1pr — 1pr(X)

Stenopola dorsalis# — — 1i — — — — — — — — —

Tucayaca parvula — — — — — — — — — 1i — —

Melanoplinae — — — — — — — — — — — —

Baeacris pseudopunctulatus — 1i — — — — — — — — — —

Baeacris punctulatus — — — 1sd — — — — — — — —

Chlorus chiquitensis* — — 1d — — — — — — — — —

Chlorus vittatus* — — — 1d — — — — — — — —

Dichromatos lilloanus* — 1i — — — — — — — — — —

Dichromatos schrottkyi* — — 1d — — — — — — — — —

Dichroplus fuscus — — 1sd 1pr — — — — — — — —

Eurotettix brevicerci* — — — 1d — — — — — — — —

Eurotettix minor* — — — 1d — — — — — — — 1i(XR); 1i(Y)

Parascopas obesus — — — — — — 1sd — — — — —

Parascopas sanguineus — — — — — — 1sd — — — — —

Propedies auricularis — — 1pr/1sd — — — — — — — — —

Propedies viriosus — — 1pr — — — — — — — — —

Ommatolampidinae

Abracris flavolineata*,# — — — — — — 1pr — — — — —

Orthoscapheus rufipes# — — — — 1pr 1pr — — — — — —

Vilerna rugulosa# 1i — 1pr — — — — — — — — —

Oedipodinae

Aiolopus strepens — — — — — — — 1pr — — — —

Locusta migratoria# — — 1pr — — — — — — — — —

Oedipoda fuscocincta 1c 1c 1c 1c 1c 1c/1pr 1c 1c 1c 1c 1c 1c(X)

Sphingonotus caerulans 1c 1c 1c 1c 1c 1c 1c 1c 1c 1c 1c 1c(X)

Abbreviations: c, centromeric; d, distal; i, interstitial; pr, proximal; s, spread; sd, subdistal.Bold `—' correspond to absent chromosomes in species with less than 23 chromosomes (males). * indicates results for species previously published and # species in which the individuals analyzedcarried B chromosomes.

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positions 13746–13908). The minimum spanning tree shows lineage 1in E. plorans as the most connected and directly joined to theoutgroup, suggesting that it is ancestral in the genome of this species.In addition to U1 snDNA, we found other identifiable repeated

elements in both species. In E. plorans, the contig with the highestcoverage included lineage 4 for U1 snDNA and also 5S rDNA. Bothwere present in the same orientation in the 3191-bp-long contig, andthe dotplot analysis showed both ends as direct repetitions, suggestinga tandem distribution of these elements in the genome. Aftertrimming one of the ends, the resulting sequence was 2879-bp long,with a spacer 1 of 1209 bp and a spacer 2 of 1421 bp. Spacer 1 alsoshowed a tandemly duplicated region of ~ 350 bp. In addition, wefound two more contigs, including a region annotated as 5S rDNA(Figure 5). This association of U1 snDNA with 5S rDNA repeats wascorroborated by two-color FISH in mitotic metaphase and indistended fibers (fiber FISH; Figure 7). In addition to 5S rDNA, wefound some regions that were annotated as SINE-like elementsassociated with the U1 snDNA cluster. For instance, one contig wasannotated as tRNA (transfer RNA)-thr/pseudogenic and anothershowed homology with ALPINE/SINE (Figure 5).In the L. migratoria genome, the contig with accession number

AVCP010084704 showed a copy of lineage 2 U1 snDNA and also acopy of 5S rDNA in the same direction, and a direct repetition in bothends suggesting a tandem structure in the genome, likewise that forlineage 4 in E. plorans (see above). No other repetitive elements werefound associated with the U1 snDNA in the L. migratoria genome, atleast in the available contigs.

DISCUSSION

U1 snDNA organization in grasshopper chromosomesThe occurrence of evident signals in all species studied indicates that,at least, part of U1 snDNA clusters among grasshoppers are tandemlyarrayed, as observed in some other animals (Zeller et al., 1984;Bretagne et al., 1991; Yu et al., 1991; Cabral-de-Mello et al., 2012), thispattern being a common placement in the group. Although, theoccurrence of dispersed (non-tandemly arrayed) sites, not detectedthrough FISH could not be completely ruled out, as revealed in othereukaryotes through molecular analysis (see, for example, Mount et al.,2007; Marz et al., 2008; Vierna et al., 2011). A comparative analysis ofthe results for the five families indicated that the presence of a singlecluster for U1 snDNA on one autosomal pair is the modal and mediannumber, as it was found in approximately half of the species analyzed.Therefore, we hypothesize that this is the ancestral pattern in grass-hoppers and that the divergent situations observed in specific groupscould be the result of molecular mechanisms moving some U1 snRNAgenes between non-homologous chromosomes, as observed for othermultigene families (see discussion below). The ancestrality of a singleU1 snDNA cluster per haploid genome is reinforced by the fact thatthis pattern was invariant in Proscopiidae, which is included in thesuperfamily Eumastacoidea and is considered the earliest diverginglineage of Acridomorpha, with a basal placement in molecularphylogenies (Leavitt et al., 2013). Similar to grasshoppers, theoccurrence of a single site of U1 snDNA per haploid genome hasalso been reported in mice (Lund and Nesbitt, 1988), crustaceans(Barzotti et al., 2003) and fish (Cabral-de-Mello et al., 2012).

Figure 1 FISH using the U1 snDNA as a probe in species of Proscopiidae (a and b), Pyrgomorphidae (c and d) and Ommexechidae (e), shown in metaphaseI (a, c and e), metaphase II (b) and diplotene (d) cells. The chromosomes carrying U1 snDNA signals and the X chromosome are indicated. c centromerelocation. The inset in e corresponds to pair 3 from a metaphase II cell, showing the location of the two U1 snDNA clusters (centromeric and proximal).Bar=5 μm.

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Grasshopper genomes typically consist of a gradual series ofmorphologically similar chromosomes slightly differing in size, suchthat it is difficult to identify each chromosome pair (except for the Xand megameric bivalent chromosomes because of their heteropycnoticbehavior during meiosis). This situation makes it impossible todetermine whether chromosomes occupying the same size–orderposition (for example, the third) in two different species are actuallyhomeologous. This implies that the chromosome numbers in Table 1are only strictly valid per species and that extrapolation among speciesneeds to be performed with extreme care. With this problem in mind,we can reach two main conclusions: (i) the single U1 snDNA clusterin the ancestor of the grasshopper species analyzed here was notlocated on the X chromosome because this pattern was extremely rare.(ii) It is likely that the ancestral location of the U1 snDNA cluster wason an autosome of a size being about the third in the genome becausethis was the size–order position found most frequently and in distinctdistantly related families. In addition, adjacent positions (2nd and 4th)were also very frequently found (14 and 17 species); bearing in mindthe uncertainty of determining homeology, this suggests that autosome2 or 4 could actually be derived from ancestor chromosome 3 and thatits size changed due to some genomic events, such as a higherproliferation for a given mobile element or other repetitive DNA. Onthis basis, chromosome 4 in Proscopiidae could be homologous tochromosome 3 in Romaleidae. Nonetheless, ascertaining preciselywhich of the chromosome pairs originally harbored the U1 snDNA

cluster in ancestral grasshoppers requires a comparative analyses ofshared synteny for multiple genetic markers among species. A firsttopic to investigate would be whether chromosome size–order isassociated with genetic content.For the family Proscopiidae, the conservative organization

evidenced for U1 snRNA, that is, an interstitial cluster located onpair no. 4 and the occurrence of H3 histone and 5S rRNA genes onthe same chromosome (Cabral-de-Mello et al., 2011b), indicate thatthis chromosome could have been maintained without gross rearran-gements after the speciation process. In a similar way, at the familylevel, this conservative pattern for the distribution of U1 snDNAclusters was also found in Pyrgomorphidae (pair 3), Ommexechidae(pairs 1 and 3) and Romaleidae (pair 3). Furthermore, similarity wasalso observed at the genus level, such as in Ommexechidae (O. gracilisand O. virens), Romaleidae (Xyleus discoideus angulatus and Xy.d. discoideus) and Acrididae representatives, that is, Parascopas(P. obesus and P. sanguineus) and Schistocerca (S. flavofasciata andS. pallens), which may be indicative of a possible commondescendent and conservation of U1 snRNA gene distribution in thesegenera.In Acrididae, several species from some subfamilies showed clusters

on one or two autosomal bivalents (for example, Acridinae, Catanto-pinae, Copiocerinae, Cyrtacanthacridinae and Ommatolampidinae),although no specific pattern of distribution on a given chromosomeemerged in these groups. In general, the variation observed for the U1

Figure 2 Chromosomal location of U1 snDNA in nine Romaleidae species, shown in metaphase I (a, b, d, f, h and i), metaphase II (c) and diplotene(e and g) cells. The chromosomes carrying U1 snDNA signals and the X chromosome are indicated. c centromere location. The inset in a corresponds to theeighth chromosome from an anaphase I cell, showing U1 clusters at centromeric and proximal/proximal locations. Bar=5 μm.

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snDNA number and location does not show a phylogenetic relation-ship beyond the genus level, except in the case of Gomphocerinae inwhich U1 snDNA spread throughout the genome was observed inmost representatives (see below). Examples of this independentvariation were most evident in the Oedipodinae subfamily, with

Aiolopus strepens and Locusta migratoria showing U1 snDNA clustersrestricted to one chromosome pair, whereas O. fuscocincta andS. caerulans carry clusters on all chromosomes. Similarly, eachOmmatolampidinae species showed a specific pattern of U1 snDNAlocation.

Figure 3 Chromosome location of U1 snDNA in 12 species of Acrididae grasshoppers, belonging to seven subfamilies, shown in diplotene (a, c, d, f, i and l),female mitotic metaphase (b) and metaphase I (e, g, h, j and k) cells. The chromosomes carrying U1 snDNA signals and sex and B chromosomes areindicated. c centromere location. Chromosomes carrying small U1 snDNA clusters are shown enlarged within the insets. Note the extensive variation for thenumber and location of U1 snDNA clusters. Bar=5 μm.

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Gomphocerinae constitutes one of the most diverse subfamilieswithin Acrididae (Contreras and Chapco, 2006) and includes a groupof species with a reduction in diploid number to 2n= 17 due to threecentric fusion events (Hewitt, 1979). We also observed the highestnumber of U1 snDNA clusters per haploid genome (4.12) withinAcrididae, which is comparatively much higher than those found inLeptysminae and Melanoplinae. Apparently, this capacity of move-ment and multiplication for U1 snDNA in Gomphocerinae is basal inthe group and preceded the occurrence of diploid number reductionbecause multiple U1 clusters are also present in some species with2n= 23, such as R. brasiliensis. In addition, in the species with 2n= 17,autosome pairs 1 and 2, which resulted from centric fusions, showmultiple U1 snDNA clusters, suggesting either a higher spread rate inthese metacentric chromosomes or that some of the acrocentricchromosomes involved in the fusions already carried multiple U1snRNA clusters in the ancestor species.

Possible causes of U1 snDNA variation and its organization in thegenome of E. plorans and L. migratoriaThe high variation in the number of chromosome pairs carrying U1snDNA clusters (1–12) clearly indicates that this DNA has spreadbetween non-homologous chromosomes in many species, especially inAcrididae. This situation resembles the extensive variation in thenumber of 45S and 5S rDNA loci previously reported in Acrididaegrasshoppers (Cabrero and Camacho, 2008; Cabral-de-Mello et al.,2011a). In all three cases, the spread was paralleled by an increase inthe number of genomic clusters, suggesting that the mechanisms

involved should entail duplication events. Therefore, ectopicrecombination, the involvement of extrachromosomal circular DNAand the action of transposable elements have frequently been cited(Charlesworth et al., 1994; Coyne and Orr, 1998; Raskina et al., 2008;Nguyen et al., 2010; Cabral-de-Mello et al., 2011c; Panzera et al.,2012).DNA sequencing in E. plorans and L. migratoria allowed a more

precise analysis of the U1 snDNA genomic structure and evolution.Both species show similar association between U1 snDNA and 5SrDNA, even though one of them (L. migratoria) shows U1 genes in asingle chromosome pair, whereas the other (E. plorans) carries them inseven chromosome pairs. The similar structure for U1 and 5S in bothspecies suggests the possibility that this pattern is quite old ingrasshoppers. In addition, the presence of SINE-like elements associ-ated with U1 and 5S in the species where the U1 cluster has spread upto seven chromosomes (E. plorans) but their absence in the specieswhere the U1 is restricted to one chromosomal pair (L. migratoria)suggests possible transposition events as responsible for such intrage-nomic spread, as previously suggested for the 5S rDNA (Drouin andMoniz de Sá, 1995; Cabral-de-Mello et al., 2011a). Supporting thispossibility, there is evidence that pseudogenic copies of 5S rDNA couldacquire SINE behavior (Kapitonov and Jurka, 2003; Gogolevsky et al.,2009). Genomic linkage for these multigene families has also beenreported, for example, in crustaceans (Pelliccia et al., 2001), molluscs(Vierna et al., 2011) and fish (Manchado et al., 2006).The remarkable similarity shown by the pseudogenic lineage 4 in

E. plorans and lineage 5 in L. migratoria suggests their common

Figure 4 Chromosome location of U1 snDNA clusters in eight Gomphocerinae (Acrididae) grasshoppers, shown in metaphase I (a, b, g and h) and diplotene(c, d and f) cells. (e and e’) Selected chromosome no. 1 from metaphase I of C. nevadensis showing DAPI pattern (e) and U1 snDNA location (e’). Thechromosomes carrying U1 snDNA signals and the X and B chromosomes are indicated. c centromere location. Note the presence of multiple sites on thelong (1 and 2) chromosomes of the 2n=17 species (a–e) and the presence of a 2n=23 species with a single U1 snDNA cluster (g) or four clusters on threechromosomes (h). Bar=5 μm.

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ancestry, as the independent origin of so many sequences (21 inE. plorans and 15 in L. migratoria) with the same truncation is highlyunlikely. Given that these two species belong to different Acrididsubfamilies, we can conclude that this truncated (pseudogenic) lineageis quite old in grasshopper genomes and is probably present in otherspecies too.The extensive genomic variation observed for U1 snDNA in the two

grasshopper genomes analyzed, even for putatively functional units,and the presence of pseudogenic copies in both species (55–58% ofthem), is consistent with the birth-and-death model for the evolutionof gene families (Nei and Rooney, 2005), as previously observed inother insect genomes, for example, Aedes aegypti (Mount et al., 2007).Alternatively, the highly homogenized copies found for lineages 1 and3 in E. plorans could suggest the occurrence of concerted evolution,but we believe that this could also be explained by recent intragenomicspread of these lineages. In addition, concerted evolution would hardlyexplain the long persistence of the truncated pseudogenic copies inboth species. A mixed process of concerted evolution and birth-and-death evolution (Nei and Rooney, 2005) has been proposed for othermultigene families, such as 5S rDNAs in distinct groups (Freire et al.,2010; Pinhal et al., 2011; Merlo et al., 2012; Vierna et al., 2013), butthe U1 family in grasshoppers fits better to the birth-and-death model.

CONCLUSIONS

The analysis of five distinct families and some subfamilies from themost specious grasshopper family, that is, Acrididae, allowed a more

precise depiction of the organization of U1 snDNA clusters ingrasshoppers, providing a detailed knowledge of the karyotypeorganization of this sequence in the group as a whole. The highgenomic dynamism of U1 snDNA clusters contrasts with the extensiveconservation of macro-chromosomal structure in grasshoppers,indicating that the extensive variation observed in the number of U1snDNA clusters per genome is not associated with major chromo-somal rearrangements. As demonstrated by the analysis of the 454sequences in the E. plorans genome, and that of the L. migratoriaassembled genome, the high dynamism of this gene in the Acrididaecould be the consequence of its association with transposableelements. The integration of these two kinds of data, that is,cytogenetic mapping and genomic information, permitted a moredetailed analysis for understanding patterns of U1 snDNA evolution.The existence of several U1 snDNA lineages in the same genome,including several pseudogenic lineages, opens new interestingquestions. For instance, does the U1 snDNA cluster on a givenchromosome show a single lineage or a mixture of several lineages,some functional and some pseudogenic? If the different clusters arehomogeneous (that is, they contain either functional or pseudogeniccopies), the search for the original ancestral location of the U1 clusterwould be delimited to only those clusters showing functional U1copies. Another interesting question is whether the pseudogeniclineages are more spread throughout the genome than the functionallineages. The fact that the analyzed cluster in E. plorans containspseudogenic lineage 4 suggests this possibility, but the coverage of our

Figure 5 Schematic representation of the cluster containing five U1 snDNA lineages in the genome of E. plorans (shown in different colors). In addition toU1 snDNA, we found homology with several elements, such as incomplete 5S rDNA and SINEs (indicated as boxes). A full color version of this figure isavailable at the Heredity journal online.

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Figure 6 (a) Alignment of the consensus sequence for the U1 snDNA lineages in E. plorans and in L. migratoria. (b) Minimum spanning tree (MST) builtwith the consensus sequence of the nine U1 lineages, also depicting their predicted secondary structure. Three types of changes are represented withdifferent filled forms: circle, triangle and square, corresponding to substitutions, deletions and insertions, respectively (the number of involved nucleotides isalso indicated for the latter two). Deletions and insertions are considered as single mutational events, and the number of involved positions in the alignmentis also indicated beside the corresponding symbol. The mean Gibbs free energy (ΔG) for the U1 secondary structure, expressed in kcal mol−1, is depicted inbrackets. In b, the conserved functional domains for lineages 1 and 2 are highlighted in light green. A full color version of this figure is available at theHeredity journal online.

Table 2 Analysis of diversity and functionality within each lineage of U1 snDNA.

n s H Hd π (s.d.) Θw G+C (s.d.)

E. plorans L1 11 168 1 0.000 0.00000 (0.00000) 0.00000 54.8 (0.4)

E. plorans L2 16 166 10 0.933 0.01536 (0.00223) 0.02072 53.9 (0.5)

E. plorans L3 5 134 1 0.000 0.00000 (0.00000) 0.00000 55.2 (0.0)

E. plorans L4 21 133 13 0.938 0.03946 (0.01018) 0.07721 55.9 (0.5)

E. plorans L5 7 162 5 0.857 0.01258 (0.00387) 0.01797 51.9 (0.6)

L. migratoria L1 30 163 16 0.899 0.01943 (0.00259) 0.02942 55.7 (0.6)

L. migratoria L2 11 163 4 0.491 0.00781 (0.00377) 0.01466 52.1 (0.3)

L. migratoria L3 3 163 2 0.667 0.02045 (0.00964) 0.02045 56.4 (0.0)

L. migratoria L4 3 163 3 1.000 0.02045 (0.00578) 0.02045 55.4 (0.9)

L. migratoria L5 15 132 15 1.000 0.15366 (0.01373) 0.18349 54.0 (2.2)

Analysis of diversity and functionality within each lineage of U1 snDNA found in the genome of E. plorans and L. migratoria, showing the number of sequences (n), number of sites (s), number ofhaplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), number of segregating sites per site by means of Watterson’s estimator (Θw) and mean percentage of G+C.Note that all five sequences for lineage 3 showed exactly the same sequence; thus, π and Θw were equal to zero. Lineage 1, however, showed five haplotypes differing for some insertions from theconsensus sequence but not for substitutions, such that the former parameters were also equal to zero because they were calculated including only those nucleotide positions present in all DNAsequences.

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analysis was actually very low. These questions can be technicallyapproached, and similar genomic analysis in other species showingdifferent degrees of U1 spread would help very much to unveil thegenomic evolution of this gene.

DATA ARCHIVING

Contigs including the U1 snDNA were obtained from sequencedgenome of Eyprepocnemis plorans available from GenBank, accessionnumbers: KJ606066–KJ606070.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

We are grateful to Dr Carlos Salvador Carbonell (Universidad de Montevideo,

Uruguay), Dr Felipe Pascual (Universidad de Granada, Spain) and Dr Ricardo

Gómez (Universidad de Castilla La Mancha, Spain) for the taxonomic

identification of some specimens used here. This study was supported by the

Programa Primeiros Projetos (PROPe, UNESP), Fundação de Amparo a

Pesquisa do Estado de São Paulo-FAPESP (2011/19481-3), Conselho Nacional

de Desenvolvimento Científico e Tecnológico-CNPq (475308/2011-5), the

Spanish Ministerio de Ciencia e Innovación (CGL2009-11917) and Plan

Andaluz de Investigación (CVI-6649), and was partially performed by FEDER

funds. AA and FJR-R were supported by scholarships from the Brazilian CNPq

and the Spanish Junta de Andalucía, respectively.

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Supplementary Information accompanies this paper on Heredity website (http://www.nature.com/hdy)

U1 snDNA clusters in grasshoppersA Anjos et al

219

Heredity

62

Supplementary material S1. Grasshopper species studied in this work with their respective sampling site and karyotype.Family

SubfamilySpecies

Locality Karyotype (male) Reference

Proscopiidae

Proscopiinae

Scleratoscopia protopeirae Gravatá, Pernambuco, BR 19, X0 Moura et al. (1996)

Scleratoscopia silvae Surubim, Pernambuco, BR 19, X0 Moura et al. (1996)

Scleratoscopia spinosa Araripina, Pernambuco, BR 19, X0 Moura et al. (1996)

Stiphra robusta Araripina, Pernambuco, BR 19, X0 Souza and Moura (2000)

Pyrgomorphidae

Pyrgomorphinae

Algete brunneri Rio Formoso, Pernambuco, BR 20, X0 Mesa and Fontanetti (1983)

Omura congrua Goiana, Pernambuco, BR 20, X0 Mesa et al. (1982)

Ommexechidae

Ommexechinae

Ommexecha gracilis Barra do Garça, Mato Grosso, BR 23, X0 This work

Ommexecha virens Rio de Contas, Bahia, BR 23, X0 Mesa et al. (1982)

63

Romaleidae

Romaleinae

Agriacris auripennis Cabo de Santo Agostinho,Pernambuco, BR

23, X0 This work

Brasilacris gigas Arco Verde, Pernambuco, BR 23, X0 Souza and Kido (1995)

Chromacris speciosa Bonito, Pernambuco, BR 23, X0 Mesa et al. (1982)

Chromacris nuptialis Vitória de Santo Antão,Pernambuco, BR

23, X0 Loreto et al. (2005)

Helionotus mirabilis Bezerros, Pernambuco, BR 23, X0 This work

Phaeopharia megacephala Igarassu, Pernambuco, BR 23, X0 Pereira and Souza (2000)

Radacridium nordestinum Surubim, Pernambuco, BR 23, X0 Rocha et al. (1997)

Tropidacris collaris Goiana, Pernambuco, BR 23, X0 This work

Xestotrachelus robustus Buíque, Pernambuco, BR 23, X0 Mesa et al. (1982)

Xyleus discoideus angulatus Surubim, Pernambuco, BR 23, X0 Mesa et al. (1982)

Xyleus discoideus discoideus Rio Claro, São Paulo, BR 23, X0 Mesa et al. (1982)

Acrididae

Acridinae

Metaleptea adspersa Bezerros, Pernambuco, BR 23, X0 Mesa et al. (1982)

64

Orphula pagana Cabo de Santo Agostinho,Pernambuco, BR

23, X0 This work

Cantatopinae

Pezotettix giornae Granada, ES 23, X0 Hewitt (1979)

Copiocerinae

Adimantus ornatissimus Rio Claro, São Paulo, BR 23, X0 Mesa et al. (1982)

Cyrtacanthacridinae

Anacridium aegyptium Granada, ES 23, X0 Tamayo and Grande (1959)

Schistocerca flavofasciata Rio Claro, São Paulo, BR 23, X0 Mesa et al. (1982)

Schistocerca pallens Goiana, Pernambuco, BR 23, X0 Mesa et al. (1982)

Eyprepocnemidinae

Eyprepocnemis plorans Granada, ES 23, X0 John and Lewis (1965)

Gomphocerinae

Amblytropidia australis Rio Claro, São Paulo, BR 23, X0 Mesa et al. (1982)

Amblytropidia sp São Lourenço da Mata,Pernambuco, BR

23, X0 This work

Chorthippus apicalis Granada, ES 17, X0 Cabrero and Camacho (1986)

Chorthippus binotatus Granada, ES 17, X0 Cabrero and Camacho (1986)

65

Chorthippus jacobsi Granada, ES 17, X0 López-León et al (1992)

Chorthippus jucundus Granada, ES 17, X0 John (1973)

Chorthippus nevadensis Granada, ES 17, X0 Pascual et al. (1980)

Chorthippus parallelus Granada, ES 17, X0 Hewitt (1963)

Dociostaurus maroccanus Granada, ES 23, X0 Camacho (1980)

Euchorthippus pulvinatus Granada, ES 17, X0 Arana et al. (1980)

Omocestus bolivari Granada, ES 17, X0 Camacho et al. (1981)

Omocestus burri Granada, ES 17, X0 Santos et al. (1993)

Omocestus panteli Granada, ES 17, X0 Cabrero and Camacho (1986)

Orphulella punctata Araripina, Pernambuco, BR 23, X0 Mesa et al. (1982)

Rhammatocerus brasiliensis Bezerros, Pernambuco, BR 23, X0 Loreto et al. (2008)

Stenobothrus festivus Granada, ES 17, X0 Cabrero and Camacho (1986)

Leptysminae

Cornops frenatum frenatum Cabo de Santo Agostinho,Pernambuco, BR

23, X0 Mesa et al. (1982)

Cylindrotettix attenuatus Rio Claro, São Paulo, BR 23, X0 This work

Cylindrotettix obscurus Goiana, Pernambuco, BR 23, X0 Confalonieri and Bidau (1986)

66

Eumastusia koebelei koebelei Serrolândia, Pernambuco, BR 23, X0 Mesa and Fontanetti (1983)

Stenopola sp Rio Claro, São Paulo, BR 23, X0 This work

Stenopola dorsalis Cabo de Santo Agostinho,Pernambuco, BR

23, X0 Mesa et al. (1982)

Tucayaca parvula Caruaru, Pernambuco, BR 23, X0 Rocha et al. (2004)

Melanoplinae

Baecris pseudopunctulatus Rio Claro, São Paulo, BR 23, X0 This work

Baecris punctulatus Goiana, Pernambuco, BR 23, X0 This work

Chlorus chiquitensis Corumba, Mato Grosso, BR 19, X0 Palacios-Gimenez et al. (2013)

Chlorus vittatus Ybycuí, PY 23, X0 Mesa et al. (1982)

Dichromatos lillioanus Eldorado, AR 21, X1X2Y Mesa (1962)

Dichromatos schrottkyi Eldorado, AR 21, X1X2Y Mesa et al. (1982)

Dichroplus fuscus Ouricuri, Pernambuco, BR 23, X0 Mesa et al. (1982)

Eurotettix brevicerci Botucatu, São Paulo, BR 23, X0 Palacios-Gimenez et al. (2013)

Eurotettix minor Paraguarí, PY 22, XY Mesa et al. (1982)

Parascopas obesus Rio Claro, São Paulo, BR 23, X0 Mesa et al. (1982)

Parascopas sanguineus Rio Claro, São Paulo, BR 23, X0 Mesa et al. (1982)

67

Propedies auriculares Corumbá, Mato Grosso, BR 23, X0 This work

Propedies viriosus Colina, São Paulo, BR 23, X0 This work

Ommatolampidinae

Abracris flavolineata Rio Claro, São Paulo, BR 23, X0 Ferreira et al. (1980)

Orthoscapheus rufipes Toritama, Pernambuco, BR 23, X0 Rocha et al. (2011)

Vilerna rugulosa Rio Claro, São Paulo, BR 23, X0 This work

Oedipodinae

Aiolopus strepens Granada, ES 23, X0 Cabrero and Camacho (1982)

Locusta migratoria Granada, ES 23, X0 White (1935)

Oedipoda fuscocincta Granada, ES 23, X0 Cabrero and Camacho (1982)

Sphingonotus caerulans Granada, ES 23, X0 Gosalves et al. (1985)

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[TAB]

Supplementary material S3. U1 sequences obtained after a RepeatMasker alignment with the L. migratoria reference full DNA sequence available in the GenBank with accession number AVCP000000000. It shows the name of the contig, the starting and ending positions matching in the query, the orientation (“+” if it matchs in the same orientation or “C” if it match with the complement of the reference), the starting and ending positions matching in the reference, whether the U1 copy was found complete and the number of lineage assigned for the diversity analysis. Sing.= Singleton.

query repeatquery sequence begin end orientation begin endgb|AVCP010014975.1| 3882 4044 C 1 163gb|AVCP010014983.1| 2463 2625 C 1 163gb|AVCP010031325.1| 5133 5295 + 1 163gb|AVCP010037409.1| 11975 12137 + 1 163gb|AVCP010037763.1| 658 820 + 1 163gb|AVCP010073722.1| 1412 1574 C 1 163gb|AVCP010154568.1| 16660 16822 C 1 163gb|AVCP010204212.1| 2545 2707 + 1 163gb|AVCP010244617.1| 43592 43754 C 1 163gb|AVCP010254383.1| 6867 7029 + 1 163gb|AVCP010380362.1| 3115 3277 C 1 163gb|AVCP010433965.1| 7249 7411 + 1 163gb|AVCP010435505.1| 84 246 + 1 163gb|AVCP010436757.1| 5019 5181 C 1 163gb|AVCP010450792.1| 1897 2059 + 1 163gb|AVCP010451404.1| 696 858 C 1 163gb|AVCP010493624.1| 8764 8926 + 1 163gb|AVCP010548845.1| 13352 13514 C 1 163gb|AVCP010585250.1| 1543 1705 C 1 163gb|AVCP010596121.1| 3870 4032 + 1 163gb|AVCP010615875.1| 3141 3303 C 1 163gb|AVCP010699836.1| 6695 6857 + 1 163gb|AVCP010707697.1| 9035 9197 + 1 163gb|AVCP010724817.1| 877 1039 C 1 163gb|AVCP010787618.1| 5293 5455 + 1 163gb|AVCP010829925.1| 17757 17919 C 1 163gb|AVCP010899246.1| 12177 12339 + 1 163gb|AVCP010955751.1| 5737 5899 C 1 163gb|AVCP010962097.1| 362 524 C 1 163gb|AVCP011046390.1| 9285 9447 C 1 163gb|AVCP010084704.1| 2248 2410 + 1 163gb|AVCP010084706.1| 1402 1564 + 1 163gb|AVCP010084709.1| 2719 2881 + 1 163gb|AVCP010110797.1| 9925 10087 + 1 163gb|AVCP010235426.1| 10924 11086 C 1 163gb|AVCP010588408.1| 5538 5700 + 1 163gb|AVCP010742909.1| 2093 2255 C 1 163gb|AVCP010806087.1| 275 437 + 1 163gb|AVCP010819873.1| 1480 1642 C 1 163gb|AVCP010899379.1| 2579 2741 + 1 163gb|AVCP010948047.1| 1430 1592 + 1 163

Page [PAGE]

[TAB]

gb|AVCP010230861.1| 144 306 + 1 163gb|AVCP010342364.1| 2551 2713 C 1 163gb|AVCP010965528.1| 2395 2557 C 1 163gb|AVCP010046729.1| 6008 6170 + 1 163gb|AVCP010388281.1| 1346 1508 + 1 163gb|AVCP010590413.1| 630 792 C 1 163gb|AVCP010151244.1| 11339 11466 + 35 163gb|AVCP010436757.1| 4145 4270 C 35 163gb|AVCP010450792.1| 2373 2502 + 34 163gb|AVCP010451403.1| 11166 11292 C 36 163gb|AVCP010455750.1| 4626 4755 C 35 163gb|AVCP010786782.1| 9007 9135 C 35 163gb|AVCP010899246.1| 14557 14683 + 36 163gb|AVCP010901144.1| 369 497 C 35 163gb|AVCP010946228.1| 17556 17684 C 35 163gb|AVCP010946228.1| 18319 18447 C 35 163gb|AVCP010949074.1| 2655 2783 + 35 163gb|AVCP010975620.1| 8807 8935 + 35 163gb|AVCP010992131.1| 718 844 C 36 163gb|AVCP011011555.1| 12078 12205 + 35 163gb|AVCP011148319.1| 6541 6668 + 35 163gb|AVCP010249683.1| 989 1151 + 1 163gb|AVCP010339046.1| 4655 4817 C 1 163gb|AVCP010595729.1| 2598 2760 C 1 163gb|AVCP010615875.1| 4922 5083 C 1 163gb|AVCP010897776.1| 4214 4376 + 1 163gb|AVCP010936987.1| 28738 28900 + 1 163gb|AVCP010994015.1| 18181 18343 C 1 163gb|AVCP011023427.1| 4 165 + 1 163gb|AVCP010007270.1| 1018 1141 + 40 163gb|AVCP010055223.1| 14726 14863 + 1 163gb|AVCP010066843.1| 1015 1172 + 6 163gb|AVCP010073722.1| 1047 1106 C 103 162gb|AVCP010084704.1| 139 267 + 35 163gb|AVCP010112984.1| 7473 7592 + 43 162gb|AVCP010149806.1| 6132 6254 C 41 163gb|AVCP010151244.1| 6016 6046 + 1 31gb|AVCP010179932.1| 14163 14265 + 61 163gb|AVCP010202223.1| 2666 2727 C 35 97gb|AVCP010244617.1| 37854 37964 C 37 163gb|AVCP010247761.1| 106 223 + 42 159gb|AVCP010271156.1| 20767 20859 C 35 129gb|AVCP010277455.1| 110 181 + 42 113gb|AVCP010304374.1| 2519 2599 + 36 116gb|AVCP010316666.1| 1049 1149 + 36 137gb|AVCP010316666.1| 1345 1382 + 125 163gb|AVCP010354458.1| 8296 8395 C 49 148gb|AVCP010388281.1| 1644 1781 + 26 163gb|AVCP010433214.1| 1367 1466 + 34 133gb|AVCP010433965.1| 7497 7577 + 35 122

Page [PAGE]

[TAB]

gb|AVCP010447664.1| 3086 3182 + 59 156gb|AVCP010457491.1| 7705 7767 + 100 162gb|AVCP010460783.1| 6505 6587 C 35 115gb|AVCP010471353.1| 695 843 + 1 163gb|AVCP010520228.1| 7809 7958 C 16 162gb|AVCP010522967.1| 9132 9250 C 46 163gb|AVCP010531584.1| 14041 14080 C 55 94gb|AVCP010531584.1| 14152 14191 C 55 94gb|AVCP010549296.1| 602 760 + 5 162gb|AVCP010575064.1| 2240 2366 C 35 160gb|AVCP010600720.1| 577 734 C 6 163gb|AVCP010607081.1| 512 678 C 4 163gb|AVCP010615875.1| 486 517 + 1 32gb|AVCP010615875.1| 670 766 + 40 160gb|AVCP010632977.1| 2898 3043 + 2 156gb|AVCP010663158.1| 6946 7094 C 12 161gb|AVCP010663158.1| 7112 7263 C 12 163gb|AVCP010699836.1| 7129 7199 + 35 105gb|AVCP010708480.1| 30225 30336 + 40 158gb|AVCP010725378.1| 3484 3555 C 42 113gb|AVCP010742909.1| 1763 1888 C 38 163gb|AVCP010831839.1| 1649 1798 + 12 163gb|AVCP010851813.1| 8267 8326 + 1 53gb|AVCP010851813.1| 9589 9708 + 45 163gb|AVCP010895105.1| 980 1094 + 40 154gb|AVCP010899246.1| 12430 12488 + 35 105gb|AVCP010899376.1| 11167 11269 + 61 163gb|AVCP010901630.1| 5459 5568 + 35 145gb|AVCP010904642.1| 20 134 C 49 163gb|AVCP010921710.1| 6243 6353 C 35 163gb|AVCP010972085.1| 7400 7547 + 3 154gb|AVCP011021620.1| 2025 2078 C 110 163gb|AVCP011049012.1| 988 1131 + 20 163gb|AVCP011079010.1| 6369 6440 + 32 107gb|AVCP011079010.1| 6617 6701 + 80 163gb|AVCP011142977.1| 2862 2993 + 32 163gb|AVCP011149022.1| 239 394 C 8 163gb|AVCP011263311.1| 1 36 C 1 36gb|AVCP011289050.1| 87 207 + 43 163

Page [PAGE]

[TAB]

Supplementary material S3. U1 sequences obtained after a RepeatMasker alignment with the L. migratoria reference full DNA sequence available in the GenBank with accession number AVCP000000000. It shows the name of the contig, the starting and ending positions matching in the query, the orientation (“+” if it matchs in the same orientation or “C” if it match with the complement of the reference), the starting and ending positions matching in the reference, whether the U1 copy was found complete and the number of lineage assigned for the diversity analysis. Sing.= Singleton.

Complete? LineageY 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 1Y 2Y 2Y 2Y 2Y 2Y 2Y 2Y 2Y 2Y 2Y 2

Page [PAGE]

[TAB]

Y 3Y 3Y 3Y 4Y 4Y 4N 5N 5N 5N 5N 5N 5N 5N 5N 5N 5N 5N 5N 5N 5N 5Y Sing.Y Sing.Y Sing.Y Sing.Y Sing.Y Sing.Y Sing.Y Sing.N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --

Page [PAGE]

[TAB]

N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --N --

Page [PAGE]

62

Supplementary material S1. Grasshopper species studied in this work with their respective sampling site and karyotype.

Supplementary material S2. Classification adopted for the distinct location patterns of U1 snDNA clusters. (a) Schematic representation of a

mitotic chromosome and (b) diplotene acrocentric chromosome showing the possible distinct positions for U1 snDNA (distinct colors). (c)

Schematic representation of the diplotene acrocentric chromosome presented in (b) showing the points of contact for this bivalent (arrowheads).

c centromere, t telomere. In the case of a biarmed chromosome, the same classification for the cluster positions was followed.

Supplementary material S3. U1 sequences obtained after a RepeatMasker alignment with the L. migratoria reference full DNA sequence

available in the GenBank with accession number AVCP000000000. It shows the name of the contig, the starting and ending positions matching

in the query, the orientation (“+” if it matchs in the same orientation or “C” if it match with the complement of the reference), the starting and

ending positions matching in the reference, whether the U1 copy was found complete and the number of lineage assigned for the diversity

analysis. Sing.= Singleton.