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Study of four Neotropical species of tree crickets Oecanthus Serville, 1831 (Orthoptera, Gryllidae) using cytogenetic and molecular markers

Abstract

Karyotypes in the worldwide subfamily Oecanthinae show variations in diploid number, chromosome morphology, and sex-chromosome system. This study described the chromosome set and phylogenetic relationships of four Neotropical species, Oecanthus lineolatus, O. valensis, O. pallidus, and O. pictus. We used classical cytogenetics and Bayesian Inference for phylogenetic reconstruction, using the mitochondrial genes COI, 12S rRNA, and 16S rRNA; and analyzed the phylogenetic patterns of changes in chromosome numbers, using ChromEvol. We observed differences in chromosome number among species and two different sex-chromosome systems. Oecanthus pictus showed 2n = 21, X0♂/22, XX♀; O. lineolatus, 2n = 20, XY♂/XX♀; and O. valensis and O. pallidus, 2n = 18, XY♂/XX♀. The karyotype of Oecanthus was asymmetric, one group with large chromosomes and variation in heterochromatin distribution, and another with small acrocentric chromosomes. The phylogenetic tree recovered two main groups: one with the Palearctic species and another with species from different bioregions, but with low posterior probability. The Neotropical species grouped separately, O. valensis and O. pictus with Nearctic and Ethiopian species, and O. pallidus and O. lineolatus in another, well-supported clade. Together, the phylogenic and chromosome data suggest descending dysploidy events during the evolution of the group.

Keywords:
Insect; karyotype; Chromosome; Bayesian Inference; Oecanthinae

Introduction

The order OrthopteraOrthoptera Species File (2022) Orthoptera Species File Online, Orthoptera Species File (2022) Orthoptera Species File Online, http://Orthoptera.SpeciesFile.org .(accessed 29 October 2020)
http://Orthoptera.SpeciesFile.org...
contains more than 28,000 described species, with a worldwide distribution. Species have been used as model organisms in several studies of cytogenetics, bioacoustics, and evolution (Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Bidau and Martí, 2010Bidau CJ and Martí DA (2010) 110 years of orthopteran cytogenetics, the chromosomal evolutionary viewpoint, and Michael White’s signal contributions to the field. J Orthoptera Res 19:165-182.; Blackmon et al., 2017Blackmon H, Ross L and Bachtrog D (2017) Sex determination, sex chromosomes, and karyotype evolution in insects. J Hered 108:78-93.; Cigliano et al., 2021Cigliano MM, Braun H, Eades DC and Otte D (2021) Orthoptera Species File Online. Version 5.0/5.0.). Members of the suborder Ensifera show wide variation in chromosome number, ranging from 2n = 15 to 37 in Tettigoniidea (Warchałowska-Śliwa, 1998Warchałowska-Śliwa E (1998) Karyotype characteristics of katydid orthopterans (Ensifera, Tettigoniidae) and remarks on their evolution at different taxonomic levels. Folia Biol - Prague 46:143-176.) and from 2n = 7 to 29 in Gryllidea (White, 1973White MD (1973) Animal cytology and evolution. 3rd edition. Cambridge University Press, London , 468 p.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Mesa et al., 1982Mesa A, Ferreira A, Carbonell CS (1982) Cariologia de los Acridiodeos neotropicales: estado actual de su conocimiento y nuevas contribuciones. Ann Soc Entomol Fr 18:507-526.). Most of the species have the sex-chromosome system X0♂-XX♀, with the X chromosome usually showing positive heteropycnosis compared to the autosomes during prophase I of meiosis (White, 1978White MJD (1978) Chain processes in chromosomal speciation. Syst Zool 27:285-298.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Palacios-Gimenez et al., 2018Palacios-Gimenez OM, Milani D, Lemos B, Castillo ER, Martí DA, Ramos E, Martins C and Cabral-de-Mello DC (2018) Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis. BMC Evol Biol 18:2.). This differential heteropycnosis occurs through the earlier condensation of chromosome X in the initial phases of cell division (Bidau and Martí, 2010Bidau CJ and Martí DA (2010) 110 years of orthopteran cytogenetics, the chromosomal evolutionary viewpoint, and Michael White’s signal contributions to the field. J Orthoptera Res 19:165-182.). X/autosome rearrangements may give rise to derived sex systems, from the original X0♂/XX♀ to Neo-XY♂/XX♀, X1X2Y♂-X1X1X2X2♀, and X1X20♂-X1X1X2X2♀ (White, 1957White MJD (1957) Cytogenetics and systematic entomology. Annu Rev Entomol 2:71-90.; Saez, 1963Saez FA (1963) Gradient of heterochromatinization in the evolution of the sexual system “neo-X neo-Y”. Port Acta Biol Ser A 7:111-138.; Mesa et al., 2002Mesa A, García-Novo P and Dos Santos D (2002) X1X2O (male)-X1X1X2X2 (female) chromosomal sex-determining mechanism in the cricket Cicloptyloides americanus (Orthoptera, Grylloidea, Mogoplistidae). J Orthoptera Res 11:87-90.; Zefa et al., 2014bZefa E, Redu DR, Costa MKM, Fontanetti CS, Gottschalk MS, Padilha GB, Silva AF and Martins LP (2014b) A new species of Endecous Saussure, 1878 (Orthoptera, Gryllidae) from northeast Brazil with the first X1X20 chromosomal sex system in Gryllidae. Zootaxa 3847:125-132.; Palacios-Gimenez and Cabral-de-Mello, 2015Palacios-Gimenez OM and Cabral-de-Mello DC (2015) Repetitive DNA chromosomal organization in the cricket Cycloptiloides americanus: A case of the unusual X1X20 sex chromosome system in Orthoptera. Mol Genet Genomics 290:623-631.).

Members of Oecanthinae are commonly known as “tree crickets”. Oecanthus Serville, 1831 is the largest genus, with 74 described species and a worldwide distribution (Walker, 1962Walker TJ (1962) The taxonomy and calling songs of United States tree crickets (Orthoptera: Gryllidae: Oecanthinae). I. The genus Neoxabea and the niveus and varicornis groups of the genus Oecanthus. Ann Entomol Soc Am 55:303-322.; Cigliano et al., 2021Cigliano MM, Braun H, Eades DC and Otte D (2021) Orthoptera Species File Online. Version 5.0/5.0.). Described karyotypes of oecanthine crickets show variations in the chromosome number, morphology, and sex-chromosome system, although the chromosome sets of only eight species have been studied so far (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1935Ohmachi F (1935) A comparative study of chromosome complements in the Grylloidea in relation to taxonomy. Bull Mie Imp Coll Agric For 5:1-48.; Kitada, 1949Kitada S (1949) Preliminary notes on the chromosomes of Oecanthus indicus. Kromosomo 5:227-228.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.; Zefa et al., 2018Zefa E, Acosta RC, Timm VF, Szinwelski N, Marinho MAT and Da Costa MKM (2018) The Tree Cricket Neoxabea brevipes Rehn, 1913 (Orthoptera: Gryllidae: Oecanthinae) from the Brazilian southern Atlantic Forest: Morphology, bioacoustics, and cytogenetics. Zootaxa 4531:554-566.). One of these is Neoxabea brevipes Rehn, 1913, with 2n = 24 autosomes + two sex chromosomes (XY or X1X20) (Zefa et al., 2018Zefa E, Acosta RC, Timm VF, Szinwelski N, Marinho MAT and Da Costa MKM (2018) The Tree Cricket Neoxabea brevipes Rehn, 1913 (Orthoptera: Gryllidae: Oecanthinae) from the Brazilian southern Atlantic Forest: Morphology, bioacoustics, and cytogenetics. Zootaxa 4531:554-566.); the other seven species belong to the genus Oecanthus (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1935; Kitada, 1949; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.). Neoxabea brevipes and O. valensis Milach and Zefa, 2016 inhabit the Neotropical bioregion; O. nigricornis Walker, 1869 and O. quadripunctatus Beutenmüller, 1894 the Nearctic; O. longicauda Matsumura, 1904 and O. pellucens (Scopoli, 1763) the Paleoarctic; and O. indicus Saussure, 1878 and Oecanthus sp. the Oriental (Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.; Cigliano et al., 2021Cigliano MM, Braun H, Eades DC and Otte D (2021) Orthoptera Species File Online. Version 5.0/5.0.).

The karyotypes for O. indicus, O. nigricornis, and O. quadripunctatus are 2n = 19, X0♂; for O. longicauda, and O. pellucens, 2n = 20, XY♂; and Oecanthus sp. and O. valensis, 2n = 18, XY♂. The species share an asymmetric karyotype that forms two groups of chromosomes according to size. The first group comprises large chromosomes, consisting of two or three autosome pairs and the X chromosome. The second comprises small (dot-like) chromosomes, including five, six, or seven autosome pairs and the Y chromosome, when the sex-chromosome system is XY (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1927Ohmachi F (1927) Preliminary note on cytological studies on Grylloidea. Proc Imp Acad 3:451-456., 1935Ohmachi F (1935) A comparative study of chromosome complements in the Grylloidea in relation to taxonomy. Bull Mie Imp Coll Agric For 5:1-48.; Kitada, 1949Kitada S (1949) Preliminary notes on the chromosomes of Oecanthus indicus. Kromosomo 5:227-228.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.).

Only Liu et al. (2018Liu XT, Jing J, Xu Y, Liu YF and He ZQ (2018) Revision of the tree crickets of China (Orthoptera: Gryllidae: Oecanthinae). Zootaxa 4497:535-546.) previously dealt with the molecular evolution of the genus, reconstructing the phylogenetic relationships of the Cytochrome c Oxidase subunit I (COI) gene among species of Oecanthus from China. Using maximum-likelihood and Bayesian inference methods, Liu et al. (2018Liu XT, Jing J, Xu Y, Liu YF and He ZQ (2018) Revision of the tree crickets of China (Orthoptera: Gryllidae: Oecanthinae). Zootaxa 4497:535-546.) found that the first separation occurred between Oecanthus ssp. and Xabea levissima Gorochov, 1992, both from the same subfamily. Within the genus, O. antennalis Liu, Yin and Xia, 1994 was the first to diverge and showed a close relationship to O. longicauda and O. similator Ichikawa, 2001; probably O. similator originated from the O. longicauda group (Liu et al., 2018Liu XT, Jing J, Xu Y, Liu YF and He ZQ (2018) Revision of the tree crickets of China (Orthoptera: Gryllidae: Oecanthinae). Zootaxa 4497:535-546.). In other phylogenetic studies, species of Oecanthus have been included in analyses to elucidate phylogenetic relationships in Ensifera, aiming to clarify the evolution of acoustic communication (Gwynne, 1995Gwynne DT (1995) Phylogeny of the Ensifera (Orthoptera): A hypothesis supporting multiple origins of acoustical signalling, complex spermatophores and maternal care in crickets, katydids, and weta. J Orthoptera Res 4:203-218.; Desutter-Grandcolas and Robillard, 2004Desutter-Grandcolas L and Robillard T (2004) Acoustic evolution in crickets: need for phylogenetic study and a reappraisal of signal effectiveness. An Acad Bras Cienc 76:301-315.; Jost and Shaw, 2006Jost MC and Shaw KL (2006) Phylogeny of Ensifera (Hexapoda: Orthoptera) using three ribosomal loci, with implications for the evolution of acoustic communication. Mol Phylogenet Evol 38:510-530.; Legendre et al., 2010Legendre F, Robillard T, Song H, Whiting MF and Desutter‐Grandcolas L (2010) One hundred years of instability in ensiferan relationships. Syst Entomol 35:475-488.; Song et al., 2015Song H, Amédégnato C, Cigliano MM, Desutter‐Grandcolas L, Heads SW, Huang Y, Otte D and Whiting MF (2015) 300 million years of diversification: elucidating the patterns of orthopteran evolution based on comprehensive taxon and gene sampling. Cladistics 31:621-651.; Chintauan-Marquier et al., 2016Chintauan‐Marquier IC, Legendre F, Hugel S, Robillard T, Grandcolas P, Nel A, Zuccon D and Desutter‐Grandcolas L (2016) Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): A multilocus phylogenetic analysis. Cladistics 32:54-81.).

The cytogenetics and phylogenetics of Oecanthus are little investigated, even though they show interesting chromosome variations and wide distributions, with species occurring in all bioregions. This study aimed to gain a more comprehensive insight into the evolutionary history of Oecanthus, describing the chromosome sets and phylogenetic relationships of O. valensis, O. pallidus Zefa, 2012, O. lineolatus Saussure, 1897, and O. pictus Milach and Zefa, 2015. We identified the chromosome number, sex-chromosome system, and heterochromatic regions using classical cytogenetic methods. Regarding molecular analysis, we used Bayesian Inference for phylogenetic reconstruction, using the mitochondrial genes. We then inferred phylogenetic relationships for the group and the pattern of changes in chromosome number during the course of evolution.

Material and Methods

Samples

Individuals of O. valensis were collected with a sweep net in shrubs and grasses, bordering highway BR101 alongside the conservation area “Reserva Natural Vale”, municipality of Linhares, state of Espírito Santo, Brazil on July 28, 2012 (Table 1). The specimens of O. pictus, O. pallidus, and O. lineolatus were collected in tobacco fields and on shrubs in the São João da Reserva district, municipality of São Lourenço do Sul, state of Rio Grande do Sul, Brazil in March 2012 (Table 1).

Table 1-
Specimen information and number of samples.

Cytogenetic analyses

We obtained the chromosomes from testis follicles of males and from midguts of females and males, previously injected with 0.05% colchicine solution for 5 h, next in 0.075 KCl hypotonic solution for 5-10 min, and then fixed in Carnoy I (3 ethyl alcohol: 1 glacial acetic acid). We squashed the fixed material on the slide in 45% acetic acid and stained the chromosomes with 0.5% lacto-acetic orcein.

We used the C-banding technique of Sumner (1972Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75:304-306.). The slides were dipped into hydrochloric acid solution (0.1 N HCl) for 30 min at room temperature and rinsed with distilled water. Slides were then treated with 5% barium hydroxide at 60 °C for 3 min, washed in 0.2 N HCl for 2 min, and rinsed with distilled water. Next, slides were dipped in 2 x SSC solution at 60 °C for 45 min, washed with distilled water, and stained with 2% Giemsa in phosphate buffer (pH 6.8) for 10 min.

Meiosis and mitosis phases were selected and photographed with a Nikon S3200 digital camera mounted on an Olympus CX21 optical microscope. We calculated the centromere index according to Levan et al. (1964Levan A, Fredga K and Sandberg AA (1964) Nomenclature for centromeric position on chromosomes. Hereditas 52:201-220.). For C-banding, slides were analyzed and photographed under a Zeiss Axiophot microscope using ZEN blue edition software. The generated map was constructed in the online platform SimpleMapprSimpleMappr, SimpleMappr, https://www.simplemappr.net/ (accessed 8 February 2021).
https://www.simplemappr.net/...
, figure edition, karyotype assembly, and the chromosome ideograms were constructed using the Adobe Photoshop CC 2015 program.

Molecular and phylogenetic analyses

DNA was extracted from the cricket hind femur and treated with a phenol/chloroform protocol, according to Jowett (1986Jowett T (1986) Preparation of nucleic acids. In Roberts DB (ed) Drosophila: A practical approach. Oxford University Press, Oxford, pp 347-372.). We amplified the genetic material using specific primers for Cytochrome c oxidase I (COI), 12S ribosomal RNA (rRNA), and 16S ribosomal RNA (rRNA), through the polymerase chain reaction (PCR). The primers used were HCO2198 and LCO1490 (Folmer et al., 1994Folmer O, Black M, Hoeh W, Lutz R and Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294-299.), 12SF and 12SR (Kambhampati, 1995Kambhampati S (1995) A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes. Proc Natl Acad Sci U S A 92:2017-2020.), and 16SAG and 16SBG (Robillard and Desutter-Grandcolas, 2006Robillard T and Desutter-Grandcolas L (2006) Phylogeny of the cricket subfamily Eneopterinae (Orthoptera, Grylloidea, Eneopteridae) based on four molecular loci and morphology. Mol Phylogenet Evol 40:643-661.) for COI, 12S rRNA, and 16S rRNA, respectively (Table 2).

Table 2 -
Primers used for PCR amplification and sequencing, indicating the gene, described name, sequence, and source of each sequence primer.

The PCR assays were conducted with 50 ng of template DNA, 20 pM of each primer, 2.5 mM MgCl2, and 1 μL Taq DNA polymerase in a total volume of 50 μL. The reactions were amplified under the following conditions: first denaturation at 95 °C for 1 min, then 35 denaturation cycles at 95 °C for 1 min, 45 s for primer annealing at temperatures of 47-48 °C for COI, 44-45 °C for 12S, and 48-49 °C for 16S, then extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min.

PCR products were visualized in 1% agarose gel and then purified with the EXO-SAP (UAB) enzymatic method for sequencing. The sequencing was performed both ways by the Sanger sequencing method at Macrogen Inc. (Seoul, South Korea). The chromatograms obtained were assembled and inspected using the Staden Package (Staden, 1996Staden R (1996) The Staden sequence analysis package. Mol Biotechnol 5:233-241.). We performed nucleotide BLAST, using a template for genes COI, 12S, and 16S in the National Center for Biotechnology Information (NCBI) (2022National Center of Biotechnology Information (NCBI) (2022) Welcome to NCBI, National Center of Biotechnology Information (NCBI) (2022) Welcome to NCBI, https://www.ncbi.nlm.nih.gov/ (accessed 19 January 2021).
https://www.ncbi.nlm.nih.gov/...
) to select Oecanthus sequences. We included in the phylogenetic analysis all the sequences available in GenBank for Oecanthus and for the outgroups, Ceuthophilus sp. Scudder, 1862 (Ensifera) and Locusta migratoria (Linnaeus, 1758) (Caelifera) (Table 3). We concatenated the sequences in head-to-tail sequence alignment, and for the species with unavailable genes, these were considered missing data. We used the software MEGA X 10.1 (Kumar et al., 2018Kumar S, Stecher G, Li M, Knyaz C and Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547-1549.) to align and edit the sequences.

Table 3 -
Species and accesses numbers of each sequence used in molecular analysis.

For the phylogenetic reconstructions, we used MrModeltest2 (Nylander, 2004Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.) to determine the best-fit evolutionary model of substitution for each gene - the three partitions, according to the values of the Akaike information criterion (AIC). The best model for COI was GTR+I+G, for 12S rRNA was GTR+G, and for 16S rRNA was GTR+I+G. The analysis was run from 30 million generations, sampling every 30,000 generations, discarding the first 25% of the samples as burn-in. We performed the Bayesian Inference (BI) analysis in the program MrBayes 3.2.6 (Ronquist et al., 2011Ronquist F, Huelsenbeck J and Teslenko M (2011) Draft MrBayes version 3.2 manual: Tutorials and model summaries. 105 p.) on XSEDE in the online platform Cyberinfrastructure for Phylogenetic Research (CIPRES) (2021Cyberinfrastructure for Phylogenetic Research (CIPRES) (2021) The CIPRES Science Gateway V. 3.3, 3, http://www.phylo.org/index.php/ (accessed 20 January 2021).
http://www.phylo.org/index.php/...
). In addition, to corroborate the findings in the BI, we performed a Neighbor-Joining analysis and phylogenetic reconstruction, employing each gene separately (data not shown).

Chromosome number evolution

We used the software ChromEvol (Mayrose et al., 2010Mayrose I, Barker MS and Otto SP (2010) Probabilistic models of chromosome number evolution and the inference of polyploidy. Syst Biol 59:132-144.; Glick and Mayrose, 2014Glick L and Mayrose I (2014) ChromEvol: Assessing the pattern of chromosome number evolution and the inference of polyploidy along a phylogeny. Mol Biol Evol 31:1914-1922.) to infer the chromosome evolution of Oecanthus along the phylogenetic tree recovered from the BI analysis. This software compares the fit of different models to biological data and may make it possible to gain insight into the pathways of chromosome-number evolution. For our data, the best evolution model determined by the program was DYS (dysploidy) according to the AIC values. The input files for analysis were the Bayesian phylogenetic tree, and the chromosome counts, with the name of each species and the haploid chromosome number (n). We included the L. migratoria outgroup chromosome information, with 2n = 23, X0 (Wei, 1958Wei H (1958) Cytological studies on migratory locust hybrid, Locusta migratoria migratoria L. Locusta migratoria manilensis Meyen. Acta Zool Sinica 10:53-59.). We accepted two possible numbers for species with different haploid numbers for males and females, assuming a frequency of 0.5 for each one and that the proportion between males and females is the same. For taxa with an unknown chromosome number, we used the symbol “X” and considered this as missing data. Table 3 lists all species used in the molecular analysis and Table 4 lists the chromosome numbers. In Table 4, O. indicus and Oecanthus sp. were excluded from the ChromEvol analysis due to missing molecular data and no species specification, respectively.

Table 4 -
Available literature information: new results of karyotypes in Oecanthus, describing the diploid number, sex system, and morphologies for large chromosomes, the sexual X and Y.

Results

Karyotyping and C-banding

Oecanthus lineolatus showed a diploid number of 2n = 20, XY♂/XX♀, with two pairs of large metacentric autosomes (Table 4), pair 2 with a secondary constriction in the interstitial region, and seven pairs of small chromosomes (Figure 1a). The X chromosome was large and submetacentric (Table 4), and the Y chromosome was one of the smallest (Figure 1a). During meiosis I, the sex chromosomes behaved as bivalents, forming chiasma in prophase I (Figure 2a, b), positioning together in the equatorial plate in metaphase I, and each migrating to opposite poles of the cell in anaphase I (Figure 2c). In pachytene, the sex chromosomes were heterochromatic at the ends and with a euchromatic region between them. In diplotene, the chromosome of pair 2 showed elastic constrictions, which may correspond to secondary constrictions (Figure 2b).

Figure 1 -
Mitotic karyotype of (a) Oecanthus lineolatus with 2n = 20, XY♂/ XX♀; (b) O. valensis with 2n = 18, XY♂/ XX♀ and one chromosome B in the male karyotype; (c) O. pallidus with 2n = 18, XY♂/ XX♀; and (d) O. pictus with 2n = 21, X0♂/ 22, XX♀. Scale bar = 10 µm.

Figure 2 -
Meiotic phases of male individuals indicating the behavior of the sex chromosomes: a-c) Oecanthus lineolatus during (a) Pachytene, (b) Diplotene, and (c) Metaphase I; d-f) O. valensis during (d) Diplotene, (e) Metaphase I, and (f) Anaphase II (arrow indicates the chromatin bridge observed); g-i) O. pallidus during (g) Pachytene, (h) Diplotene, and (i) Metaphase I; and j-l) O. pictus during (j) Diplotene, (k) Metaphase I, and (l) Anaphase I. Scale bar = 10 µm.

Oecanthus valensis had 2n = 18, XY♂/XX♀, with two pairs of large meta/submetacentric autosomes and six pairs of small chromosomes (Figure 1b and Table 4). The sex-chromosome system had a large submetacentric X (Table 4) and a small Y chromosome (Figure 1b), both attached by a terminal chiasma during prophase I. The X was more heteropycnotic than the Y, and both showed a gradual increase in heterochromatinization during prophase I (Figure 2d, e). Some cells of one individual exhibited a B chromosome (Figure 1b), and in another individual the cells formed a chromatin bridge during anaphase/telophase II (Figure 2f).

Oecanthus pallidus had 2n = 18, XY♂/XX♀, with two pairs of large metacentric chromosomes (Table 4) and six small autosomal pairs (Figure 1c). The X chromosome was large and metacentric, and the Y was small and subtelocentric (Figure 1c and Table 4). We observed the morphology of chromosome Y only in O. pallidus because it was larger and it was possible to locate the centromere position. In contrast, the Y was not well defined in the other species, showing a dot-like morphology. In pachytene, chromosome X was heterochromatic and Y was euchromatic, and they appeared not to be paired. In diplotene I, the sex chromosomes behaved as heteromorphic bivalents and were heteropycnotic positive, with a euchromatic segment between them (Figure 2g). There was a gradual increase in heterochromatinization of X and Y segments in prophase I (Figure 2g, h), and in metaphase I they moved together on the equatorial plate (Figure 2i).

In specimens of O. pictus, the diploid number was 2n = 21, X0♂, and 2n = 22, XX♀, with three pairs of large autosomes, one metacentric, one subtelocentric, and one acrocentric; and seven pairs of small autosomes (Figure 1d and Table 4). The X chromosome was large and metacentric (Table 4), behaving as univalent during cell division (Figure 2j, k, l) and migrating to one of the cell poles in anaphase I (Figure 2l). In diplotene I, the sex chromosome showed positive heteropycnosis in comparison with the autosomes (Figure 2j).

The C-banding pattern showed that in all species, the small chromosomes were acrocentric with a small pericentromeric C-band at one end, except for pair 3 in O. lineolatus, O. valensis, and O. pallidus that showed a heterochromatic block (Figure 3a, b, c). The chromosomes X had a high degree of heterochromatinization differing between the species (Figure 3). Variations were observed in the large autosomes, such as in O. lineolatus, where pair 1 had an interstitial band and pair 2 exhibited a heterochromatic block in a secondary constriction (Figure 3a). Oecanthus valensis had an interstitial band in the bivalents of pair 1 and a heterochromatic block in pair 2 (Figure 3b). Oecanthus pallidus had a C-band in the telomeric region of pair 1 and a pericentric heterochromatin block in pair 2, and the Y chromosome was heterochromatic (Figure 3c). C-banding in O. pictus showed high heterochromatinization of the three large chromosomes (Figure 3d).

Figure 3-
Identification of C-banding markers (gray) in mitotic metaphase of females of (a) Oecanthus lineolatus and (b) O. valensis, and males of (c) O. pallidus and (d) O. pictus. Indication of chromosome pairs with C-banding markers. Scale bar = 10 µm.

Phylogenetic reconstructions

The consensus tree (obtained from COI, 12S rRNA, and 16S rRNA concatenated mitochondrial fragments) enabled us to infer the phylogenetic relationships of O. lineolatus, O. pallidus, O. valensis, and O. pictus with the other species. The phylogenetic tree showed two main groups: one composed only of species from the Palearctic bioregion (except O. antennalis), and the other group composed of the remaining species from different bioregions, although this separation was not strongly supported (p.p. value = 0.55). In the Palearctic group, high posterior probabilities and close relationships were recovered mainly between O. similator and O. longicauda, and among O. euryelytra Ichikawa, 2001, O. sinensis Walker, 1869, and O. rufescens Serville, 1838.

Regarding the Neotropical species, O. pallidus and O. lineolatus showed a close, strongly supported relationship (p.p. value = 1.00), and O. valensis and O. pictus grouped with species from the Nearctic and Ethiopian regions. In this group, only O. quadripunctatus, O. nigricornis, and O. celerinictusWalker, 1963Walker TJ (1963) The taxonomy and calling songs of United States tree crickets (Orthoptera: Gryllidae: Oecanthinae). II. The nigricornis group of the genus Oecanthus. Ann Entomol Soc Am 56:772-789. from the Nearctic region were closely related and showed high posterior probability (p.p. value = 1.00) (Figure 4).

Figure 4-
Bayesian Inference using mitochondrial concatenated data (COI, 12S rDNA, and 16S rDNA) in Oecanthus species. Colors indicate each bioregion: green, species from the Palearctic bioregion; purple, Nearctic; red, Neotropical; and blue, Ethiopian. The outgroups were Ceuthophilus sp. and Locusta migratoria. Above each branch are indicated the posterior probabilities; (N) represents the node names and the ancestral haploid chromosome number inferred by ChromEvol software. Chromosome haploid number of living species, and (-) represents missing data for karyotype.

Chromosome evolution along the phylogenetic tree

Concerning karyotype evolution, we used ChromEvol and based the analysis on chromosome number and molecular markers. Chromosome data (Table 4) indicated that the transition occurring in the genus is descending dysploidy, indicating a process of chromosome loss along the tree. There were four main loss events with significances greater than 0.5, in the ancestral nodes N2 (0.51) and N3 (0.58) and in the species O. valensis (0.65) and O. pallidus (1.00) (Figure 4). The program inferred that the ancestral node N2 may have an n = 11 and for the ancestral N3 was then reduced, to n = 10. The ancestral nodes along the branches maintained the haploid number of n = 10, until a significant loss in O. valensis and O. pallidus, both with n = 9 (Figure 4).

Discussion

Cytogenetic studies with Oecanthus comprise species from four bioregions, Palearctic, Nearctic, Neotropical, and Oriental (Figure 5) (Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.; Cigliano et al., 2021Cigliano MM, Braun H, Eades DC and Otte D (2021) Orthoptera Species File Online. Version 5.0/5.0.). We described for the first time the karyotypes of O. pallidus, O. lineolatus, and O. pictus, all from the Neotropical region (Figure 1). We found that the sex-chromosome system and diploid number were the same for O. valensis and O. pallidus, with 2n = 18, XY♂; O. lineolatus had the same system, with a different diploid number, 2n = 20, XY♂; and O. pictus had the most distinct karyotype, with 2n = 21, X0♂ (Figure 1).

Figure 5 -
Map showing the distribution of Oecanthus species with karyotypes analyzed. In green, O. longicauda and O. pellucens from the Palearctic bioregion, both with 2n = 20, XY♂ (Hewitt 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Ohmachi 1927Ohmachi F (1927) Preliminary note on cytological studies on Grylloidea. Proc Imp Acad 3:451-456.; Makino 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.). In purple, O. nigricornis and O. quadripunctatus from the Nearctic bioregion, both with 2n = 19, X0♂ (Johnson 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Beaudry 1973Beaudry JR (1973) Une analyse des complements chromosomiques de certains orthopteres du Quebec et sa signification taxonomique et evolutionnaire. Can J Genet Cytol 15:155-170.). In orange, species from the Oriental bioregion, O. indicus with 2n = 19, X0♂, and Oecanthus sp. with 2n = 18, XY♂ (Kitada 1949Kitada S (1949) Preliminary notes on the chromosomes of Oecanthus indicus. Kromosomo 5:227-228.; Nakamura and Kitada 1955Nakamura K and Kitada JI (1955) Chromosomes of some orthopteroid insects, with special reference to sex-chromosomes. Cytologia 20:119-132.; Aswanianarayana and Ashwath 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.). In red, species from the Neotropical bioregion, O. lineolatus with 2n = 20, XY♂; O. valensis and O. pallidus with 2n = 18, XY♂; and O. pictus with 2n = 21, X0♂.

We found that O. pallidus had the smallest diploid number, 2n = 18, as previously reported for O. valensis and Oecanthus sp., and O. pictus had the highest chromosome number in the genus, with 2n = 21. The other species did not show wide chromosome variability, with diploid numbers ranging from 2n = 19 to 20 (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1927Ohmachi F (1927) Preliminary note on cytological studies on Grylloidea. Proc Imp Acad 3:451-456., 1935Ohmachi F (1935) A comparative study of chromosome complements in the Grylloidea in relation to taxonomy. Bull Mie Imp Coll Agric For 5:1-48.; Kitada, 1949Kitada S (1949) Preliminary notes on the chromosomes of Oecanthus indicus. Kromosomo 5:227-228.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.). The sex-chromosome system was as previously described for the other species of the genus (X0 and XY) (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1927Ohmachi F (1927) Preliminary note on cytological studies on Grylloidea. Proc Imp Acad 3:451-456., 1935; Kitada, 1949; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.).

The Neotropical species showed two pairs of large metacentric chromosomes, similar to the Nearctic species and different from the Oriental and Palearctic species with large acrocentric chromosomes (Figure 5 and Table 4) (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1927Ohmachi F (1927) Preliminary note on cytological studies on Grylloidea. Proc Imp Acad 3:451-456., 1935Ohmachi F (1935) A comparative study of chromosome complements in the Grylloidea in relation to taxonomy. Bull Mie Imp Coll Agric For 5:1-48.; Kitada, 1949Kitada S (1949) Preliminary notes on the chromosomes of Oecanthus indicus. Kromosomo 5:227-228.; Nakamura and Kitada, 1955Nakamura K and Kitada JI (1955) Chromosomes of some orthopteroid insects, with special reference to sex-chromosomes. Cytologia 20:119-132.; Montalenti et al., 1965Montalenti G, Rocchi A and Fontana PG (1965) IL Corredo cromosomico di Oecanthus pellucens (ORTHOPTERA GRYLLOIDEA). Rend Lincei-Mat Appl 39:237.; Beaudry, 1973Beaudry JR (1973) Une analyse des complements chromosomiques de certains orthopteres du Quebec et sa signification taxonomique et evolutionnaire. Can J Genet Cytol 15:155-170.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.). The large chromosomes in O. pictus differed from other karyotypes, with one metacentric, one submetacentric, and one acrocentric pair (Figure 5 and Table 4). Our results for O. valensis were congruent with those of Milach et al. (2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.) (Table 4). B chromosomes were observed in only two species of the genus; in O. valensis they were small and larger than chromosome Y (Figure 1b), and in O. pellucens were small and similar in size to chromosome Y (Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.).

Both kinds of sex-chromosome systems, X0 and XY, occurred in the Neotropical species. This variation was also seen in the species from the Oriental Region, while the Nearctic species possess only the X0 mechanism, and in the Palearctic only the XY (Figure 5) (Johnson, 1931Johnson HH (1931) Centrioles and other cytoplasmic components of the male germ cells of the Gryllidae. Z Wiss Zool 140:115-165.; Makino, 1932Makino S (1932) An unequal pair of idiochromosomes in the tree-cricket, Oecanthus longicauda Mats. J Fac Sci Hokkaido Univ Ser VI Zool 2:1-35.; Ohmachi, 1927Ohmachi F (1927) Preliminary note on cytological studies on Grylloidea. Proc Imp Acad 3:451-456., 1935Ohmachi F (1935) A comparative study of chromosome complements in the Grylloidea in relation to taxonomy. Bull Mie Imp Coll Agric For 5:1-48.; Kitada, 1949Kitada S (1949) Preliminary notes on the chromosomes of Oecanthus indicus. Kromosomo 5:227-228.; Nakamura and Kitada, 1955Nakamura K and Kitada JI (1955) Chromosomes of some orthopteroid insects, with special reference to sex-chromosomes. Cytologia 20:119-132.; Beaudry, 1973Beaudry JR (1973) Une analyse des complements chromosomiques de certains orthopteres du Quebec et sa signification taxonomique et evolutionnaire. Can J Genet Cytol 15:155-170.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.; Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164., Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.).

Evolutionarily, it is expected that fusions will occur between chromosomes, reducing the diploid number and forming bi-armed chromosomes (metacentric or submetacentric). In chromosome changes, fusion processes are expected to be more common than fissions (Baker and Bickham, 1980Baker RJ and Bickham JW (1980) Karyotypic evolution in bats: evidence of extensive and conservative chromosomal evolution in closely related taxa. Syst Biol 29:239-253.; Hemp et al., 2013Hemp C, Heller KG, Warchałowska-Śliwa E and Hemp A (2013) The genus Aerotegmina (Orthoptera, Tettigoniidae, Hexacentrinae): Chromosomes, morphological relations, phylogeographical patterns and description of a new species. Org Divers Evol 13:521-530.). Considering this and the analysis of chromosome evolution along the phylogenetic tree, the chromosome set of O. valensis and O. pallidus appears to be the most derived, with the smallest diploid number in the group and an XY sex-chromosome system. Although Oecanthus sp. (Oriental) shows the same diploid number and sex-chromosome system as both Neotropical species, the chromosome morphology set (acrocentric) indicated a less-derived condition (Aswanianarayana and Ashwath, 2005Aswanianarayana NV and Ashwath S (2005) Karyotype characteristics of forty-one species of Orthoptera and their evolutionary trends at the family level. Rec Zool Surv India 104:157-164.).

The XY sex-chromosome system of O. valensis, O. pallidus, and O. lineolatus probably derived from a centric fusion rearrangement between a large X-acrocentric chromosome with a small bivalent pair (White, 1954White MJD (1954) Animal cytology and evolution. 2nd edition. Cambridge University Press, London., 1957White MJD (1957) Cytogenetics and systematic entomology. Annu Rev Entomol 2:71-90.; Saez, 1963Saez FA (1963) Gradient of heterochromatinization in the evolution of the sexual system “neo-X neo-Y”. Port Acta Biol Ser A 7:111-138.; Rice, 1996Rice WR (1996) Evolution of the Y sex chromosome in animals. Bioscience 46:331-343.; Kaiser and Bachtrog, 2010Kaiser VB and Bachtrog D (2010) Evolution of sex chromosomes in insects. Annu Rev Genet 44:91-112.; Castillo et al., 2010Castillo ER, Marti DA and Bidau CJ (2010) Sex and neo-sex chromosomes in Orthoptera: A review. J Orthoptera Res 19:213-231.; Palacios-Gimenez et al., 2015bPalacios-Gimenez OM, Marti DA and Cabral-de-Mello DC (2015b) Neo-sex chromosomes of Ronderosia bergi: Insight into the evolution of sex chromosomes in grasshoppers. Chromosoma 124:353-365., 2018Palacios-Gimenez OM, Milani D, Lemos B, Castillo ER, Martí DA, Ramos E, Martins C and Cabral-de-Mello DC (2018) Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis. BMC Evol Biol 18:2.). The X chromosome and the autosomes undergo breaks and fusion, forming a metacentric and a small chromosome; the latter is composed of centromere regions and is usually lost during cell divisions. Chromosome X becomes a bi-armed chromosome, formed by fusion of the acrocentric X and the autosome, and the free autosome starts to behave similarly to the Y chromosome (Saez, 1963Saez FA (1963) Gradient of heterochromatinization in the evolution of the sexual system “neo-X neo-Y”. Port Acta Biol Ser A 7:111-138.; Hewitt, 1979Hewitt GM (1979) Orthoptera: Grasshoppers and crickets. In: Jolui B (ed) Animal Cytogenetics 3. Insecta I. Gebrüder-Borntraeger, Berlin-Stuttgart, pp 170.). During the meiotic prophase, the Y chromosome will pair with its homologue, which fused with the X chromosome, as occurs during pachytene and diplotene of the grasshopper Ronderosia bergii (Stål, 1878) (Palacios-Gimenez et al., 2015bPalacios-Gimenez OM, Marti DA and Cabral-de-Mello DC (2015b) Neo-sex chromosomes of Ronderosia bergi: Insight into the evolution of sex chromosomes in grasshoppers. Chromosoma 124:353-365.). In contrast, O. pictus has the X0 mechanism, and the X is metacentric and smaller than in the other three species.

In the XY sex-chromosome system of the Neotropical species of Oecanthus, a euchromatic segment occurs between two heterochromatic segments in the initial phases of meiosis. The euchromatic part is referent to the chiasma between the Y chromosome and its homologue fused with the X chromosome. The X/autosome rearrangement accompanied a gradual loss of crossing over between autosomal homologues and gradual heterochromatinization of the autosomal arm on the X chromosome (Saez, 1963Saez FA (1963) Gradient of heterochromatinization in the evolution of the sexual system “neo-X neo-Y”. Port Acta Biol Ser A 7:111-138.). This process of heterochromatinization is typical in the evolution of sex chromosomes and indicates that the greater the degree of heterochromatinization in the segments of the XY mechanism, the older the origin of the rearrangement (White, 1951White MJD (1951) Cytogenetics of orthopteroid insects. Adv Genet 4:267-330.; Saez, 1963Saez FA (1963) Gradient of heterochromatinization in the evolution of the sexual system “neo-X neo-Y”. Port Acta Biol Ser A 7:111-138.; Rice, 1996Rice WR (1996) Evolution of the Y sex chromosome in animals. Bioscience 46:331-343.; Mesa et al., 2001Mesa A, Fontanetti CS and García-Novo P (2001) Does an x-autosome centric fusion in Acridoidea condemn the species to extinction? J Orthoptera Res 10:141-146.).

Using the C-banding technique for the first time in chromosomes of Oecanthus, we found different patterns in the large chromosomes among species (Figure 3). For O. lineolatus, we observed a large heterochromatic block in the secondary constriction of pair 2, as also seen for the karyotypes of Gryllus assimilis (Fabricius, 1775) and Eneoptera surinamensis (De Geer, 1773Zefa E, Cordeiro J, Blauth M, Piumbini M, Silva AF, Costa MKM and Martins LDP (2014a) Expanding the geographic cytogenetic studies in the bush crickets Eneoptera surinamensis (De Geer, 1773) (Orthoptera, Gryllidae, Eneopterinae) from Brazilian Atlantic and Amazon Forest. Zootaxa 3860:396-400.) (Palacios-Gimenez et al., 2015aPalacios-Gimenez OM, Carvalho CR, Soares FAF and Cabral-de-Mello DC (2015a) Contrasting the chromosomal organization of repetitive DNAs in two Gryllidae crickets with highly divergent karyotypes. PLoS One 10:e0143540.) (Figure 3a). Oecanthus lineolatus, O. valensis, and O. pallidus showed heterochromatic bands for pairs 1 and 2. Pair 1 in O. lineolatus and O. valensis was in the interstitial region, and in O. pallidus was in the telomere (Figure 3b, c). The telomere bands also differed from the findings for the cricket G. assimilis and the grasshopper Paracinipe sp. Descamps and Maunassif, 1972, where they occurred only in medium and small chromosomes (Palacios-Gimenez et al., 2015aPalacios-Gimenez OM, Carvalho CR, Soares FAF and Cabral-de-Mello DC (2015a) Contrasting the chromosomal organization of repetitive DNAs in two Gryllidae crickets with highly divergent karyotypes. PLoS One 10:e0143540.; Buleu et al., 2019Buleu OG, Jetybayev IY, Chobanov DP and Bugrov AG (2019) Comparative analysis of C-heterochromatin, ribosomal and telomeric DNA markers in chromosomes of Pamphagidae grasshoppers from Morocco. Comp Cytogenet 13:61-74.). The Y chromosome in O. pallidus is entirely heterochromatic, and the Neo-Y of R. bergii shows the same pattern (Palacios-Gimenez et al., 2015bPalacios-Gimenez OM, Marti DA and Cabral-de-Mello DC (2015b) Neo-sex chromosomes of Ronderosia bergi: Insight into the evolution of sex chromosomes in grasshoppers. Chromosoma 124:353-365.). This pattern may be related to repeated DNA accumulation in this chromosome, changing the heterochromatin structure (Figure 3c) (Palacios-Gimenez et al., 2015bPalacios-Gimenez OM, Marti DA and Cabral-de-Mello DC (2015b) Neo-sex chromosomes of Ronderosia bergi: Insight into the evolution of sex chromosomes in grasshoppers. Chromosoma 124:353-365.). In the bushcricket E. surinamensis, the heterochromatin showed a different pattern, occurring as dispersed blocks in the Neo-Y (Ferreira and Cella 2006Ferreira A and Cella DM (2006) Chromosome structure of Eneoptera surinamensis (Orthoptera, Grylloidea, Eneopterinae) as revealed by C, NOR and N banding techniques. Chromosome Sci 9:47-51.; Palacios-Gimenez et al., 2015aPalacios-Gimenez OM, Carvalho CR, Soares FAF and Cabral-de-Mello DC (2015a) Contrasting the chromosomal organization of repetitive DNAs in two Gryllidae crickets with highly divergent karyotypes. PLoS One 10:e0143540.).

The chromatin bridge in anaphase II of O. valensis occurs in other species of Orthoptera, usually related to chromosome breaks and rearrangements (Figure 2f) (Warchałowska-Śliwa et al., 2005Warchałowska-Śliwa E, Niklińska M, Görlich A, Michailova P and Pyza E (2005) Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas. Environ Pollut 133:373-381.; Zefa et al., 2014aZefa E, Redu DR, Costa MKM, Fontanetti CS, Gottschalk MS, Padilha GB, Silva AF and Martins LP (2014b) A new species of Endecous Saussure, 1878 (Orthoptera, Gryllidae) from northeast Brazil with the first X1X20 chromosomal sex system in Gryllidae. Zootaxa 3847:125-132.). Chromatin bridges are chromatin segments positioned parallel to the segregating chromosomes during anaphase II (Fenech et al., 2011Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, Norppa H, Eastmond DA, Tucker JD and Thomas P (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26:125-132., Bizard and Hickson, 2018Bizard AH and Hickson ID (2018) Anaphase: A fortune-teller of genomic instability. Curr Opin Cell Biol 52:112-119.). Usually, they form due to dicentric chromatin manifestations, where each centromere is segregating to an opposite pole of the cell (Acilan et al., 2007Acilan C, Potter DM and Saunders WS (2007) DNA repair pathways involved in anaphase bridge formation. Genes Chromosomes Cancer 46:522-531.; Bizard and Hickson, 2018Bizard AH and Hickson ID (2018) Anaphase: A fortune-teller of genomic instability. Curr Opin Cell Biol 52:112-119.). Chromatin bridges may cause cell instability, lead to cell death, and be related to fecundity reduction (Kirkpatrick and Barton, 2006Kirkpatrick M and Barton N (2006) Chromosome inversions, local adaptation and speciation. Genetics 173:419-434.; Bizard and Hickson, 2018Bizard AH and Hickson ID (2018) Anaphase: A fortune-teller of genomic instability. Curr Opin Cell Biol 52:112-119.). Also, when the bridge breaks, it usually generates daughter cells with unbalanced copies of genes due to uneven breaking of the chromatin segment and rearrangements between chromosomes such as translocations and deletions (Acilan et al., 2007Acilan C, Potter DM and Saunders WS (2007) DNA repair pathways involved in anaphase bridge formation. Genes Chromosomes Cancer 46:522-531.; Fenech et al., 2011Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, Norppa H, Eastmond DA, Tucker JD and Thomas P (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26:125-132.).

Using molecular analysis, this study is the first to recover the phylogenetic relationships of Oecanthus from different bioregions. According to the BI, O. longicauda and O. similator are phylogenetically close, and probably O. similator originated from a group of O. longicauda (Liu et al., 2018Liu XT, Jing J, Xu Y, Liu YF and He ZQ (2018) Revision of the tree crickets of China (Orthoptera: Gryllidae: Oecanthinae). Zootaxa 4497:535-546.). As previously found by Liu et al. (2018Liu XT, Jing J, Xu Y, Liu YF and He ZQ (2018) Revision of the tree crickets of China (Orthoptera: Gryllidae: Oecanthinae). Zootaxa 4497:535-546.), O. antennalis was positioned separately from other species from the Palearctic region. The Neotropical species O. pallidus and O. lineolatus shared the same distribution and showed a close phylogenetic relationship. Oecanthus pictus, also from southern Brazil, appeared to be little related to these species. Oecanthus valensis, from southeastern Brazil, was more closely associated with the Nearctic than the Neotropical species.

Species of Oecanthus have an uncertain phylogenetic position within Oecanthinae; they appear close to the Neoxabea-Xabea group due to their general form, which may be related to adaptive issues. Therefore, the morphologic pattern found in this genus could be highly conserved, independently of their distribution (Desutter-Grandcolas, 1990Desutter-Grandcolas L (1990) Etude phylogénétique biogéographique et écologique des Grylloidea néotropicaux (Insectes Orthoptères). M. Sc. Thesis, Université Paris-Saclay, Paris, 347 p.). Such as the pigmented spots on the legs of O. valensis that are observed in only a few species, among them O. niveus, O. celerinictus, and O. bakeriCollins et al. (2014Collins N, van den Berghe E and Carson L (2014) Two new species of Neoxabea, three new species of Oecanthus, and documentation of two other species in Nicaragua (Orthoptera: Gryllidae: Oecanthinae). T Am Entomol Soc 140:163-184.) (Walker, 1963Walker TJ (1963) The taxonomy and calling songs of United States tree crickets (Orthoptera: Gryllidae: Oecanthinae). II. The nigricornis group of the genus Oecanthus. Ann Entomol Soc Am 56:772-789.; Collins et al., 2014Collins N, van den Berghe E and Carson L (2014) Two new species of Neoxabea, three new species of Oecanthus, and documentation of two other species in Nicaragua (Orthoptera: Gryllidae: Oecanthinae). T Am Entomol Soc 140:163-184.; Milach et al., 2016Milach EM, Costa MKM, Martins LP, Nunes LA, Silva DSM, Garcia FRM, De Oliveira EC and Zefa E (2016) New species of tree cricket Oecanthus Serville, 1831 (Orthoptera: Gryllidae: Oecanthinae) from Reserva Natural Vale, Espírito Santo, Brazil, with chromosome complement. Zootaxa 4173:137-146.). All these species occur in the Nearctic and Neotropical bioregions, and O. valensis grouped in the same clade as O. niveus and O. celerinictus (Cigliano et al., 2021Cigliano MM, Braun H, Eades DC and Otte D (2021) Orthoptera Species File Online. Version 5.0/5.0.).

The analysis to identify patterns of change in chromosome number in the course of evolution showed four loss events, with high significance, indicating a reduction in the chromosome number. These events were highly important for the reduced diploid number found in O. valensis and O. pallidus. The decrease may be due to rearrangements and fusion processes between chromosomes (Baker and Bickham, 1980Baker RJ and Bickham JW (1980) Karyotypic evolution in bats: evidence of extensive and conservative chromosomal evolution in closely related taxa. Syst Biol 29:239-253.; Hemp et al., 2013Hemp C, Heller KG, Warchałowska-Śliwa E and Hemp A (2013) The genus Aerotegmina (Orthoptera, Tettigoniidae, Hexacentrinae): Chromosomes, morphological relations, phylogeographical patterns and description of a new species. Org Divers Evol 13:521-530.). Similar processes occurred in other orthopteran species, as in the genus Dichroplus Stål, 1873, where the accumulation of fusions between autosome-autosome and X-autosome led to modifications of the ancestral chromosome set of 2n = 22 + X0♂/XX♀ to the reduced karyotypes of D. pratensis Bruner, 1900 (2n = 20) and D. obscurus Bruner, 1900 (2n = 18) (Colombo et al., 2005Colombo P, Cigliano MM, Sequeira AS, Lange CE, Vilardi JC and Confalonieri VA (2005) Phylogenetic relationships in Dichroplus Stål (Orthoptera: Acrididae: Melanoplinae) inferred from molecular and morphological data: testing karyotype diversification. Cladistics 21:375-389.).

The present study is the first to describe the karyotypes of O. pallidus, O. lineolatus, and O. pictus, and also to use banding techniques in karyotypes of Oecanthus and analyze the relationship of this group using individuals from different bioregions. We found variations in the diploid number and two sex-chromosome systems in the genus. Among the species, O. pictus shows distinct chromosome characteristics in the diploid number and morphology. Two of the species that have been studied are Palearctic, two Nearctic, one Neotropical, and two Oriental. The molecular and cytogenetic data indicated that the process of descending dysploidy is the most probable event for chromosome evolution along the phylogenetic tree. Future cytogenetic and molecular studies involving more species of Oecanthus are needed to comprehend the chromosome and group evolution.

Acknowledgments

We thank CAPES and CNPQ for their financial support. The Programa de Pós-Graduação em Biologia Animal for the grants, and the Programa de Pós-Graduação em Genética e Biologia Molecular for the opportunity to conduct this study. We also thank the Universidade Federal do Rio Grande do Sul and Universidade Federal de Pelotas for the infrastructure. Our gratitude to the Cytogenetics and Evolution Laboratory of the Universidade Federal do Rio Grande do Sul, coordinated by professor Thales Renato Ochotorena de Freitas, for material support. We thank Henrique Moreira for help with the phylogenetic analysis.

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Edited by

Associate editor:

Louis Bernard Klaczko

Data availability

Data citations

Cigliano MM, Braun H, Eades DC and Otte D (2021) Orthoptera Species File Online. Version 5.0/5.0.

Orthoptera Species File (2022) Orthoptera Species File Online, Orthoptera Species File (2022) Orthoptera Species File Online, http://Orthoptera.SpeciesFile.org (accessed 29 October 2020)

Publication Dates

  • Publication in this collection
    29 Apr 2022
  • Date of issue
    2022

History

  • Received
    22 July 2021
  • Accepted
    31 Jan 2022
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