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.

Only Liu et al. (2018) 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 maximumlikelihood and Bayesian inference methods, Liu et al. (2018) 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., 2018).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, 1995;Desutter-Grandcolas and Robillard, 2004;Jost and Shaw, 2006;Legendre et al., 2010;Song et al., 2015;Chintauan-Marquier et al., 2016).
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.

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 (1972).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. (1964).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 SimpleMappr, figure edition, karyotype assembly, and the chromosome ideograms were constructed using the Adobe Photoshop CC 2015 program.
The PCR assays were conducted with 50 ng of template DNA, 20 pM of each primer, 2.5 mM MgCl 2 , 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, 1996).We performed nucleotide BLAST, using a template for genes COI, 12S, and 16S in the National Center for Biotechnology Information (NCBI) (2022) 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., 2018) to align and edit the sequences.For the phylogenetic reconstructions, we used MrModeltest2 (Nylander, 2004) 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., Silva et al. 4 2011) on XSEDE in the online platform Cyberinfrastructure for Phylogenetic Research (CIPRES) (2021).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., 2010;Glick and Mayrose, 2014) 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, 1958).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.

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).
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 sexchromosome 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).

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).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, 1980;Hemp et al., 2013).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, 2005).
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, 1954(White, , 1957;  Saez, 1963;Rice, 1996;Kaiser and Bachtrog, 2010;Castillo et al., 2010;Palacios-Gimenez et al., 2015b, 2018).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, 1963;Hewitt, 1979).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., 2015b).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, 1963).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, 1951;Saez, 1963;Rice, 1996;Mesa et al., 2001).
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, 1773) (Palacios-Gimenez et al., 2015a) (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., 2015a;Buleu et al., 2019).The Y chromosome in O. pallidus is entirely heterochromatic, and the Neo-Y of R. bergii shows the same pattern (Palacios-Gimenez et al., 2015b).This pattern may be related to repeated DNA accumulation in this chromosome, changing the heterochromatin structure (Figure 3c) (Palacios-Gimenez et al., 2015b).In the bushcricket E. surinamensis, the heterochromatin showed a different pattern, occurring as dispersed blocks in the Neo-Y (Ferreira and Cella 2006;Palacios-Gimenez et al., 2015a).
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., 2005;Zefa et al., 2014a).Chromatin bridges are chromatin segments positioned parallel to the segregating chromosomes during anaphase II (Fenech et al., 2011, Bizard andHickson, 2018).Usually, they form due to dicentric chromatin manifestations, where each centromere is segregating to an opposite pole of the cell (Acilan et al., 2007;Bizard and Hickson, 2018).Chromatin bridges may cause cell instability, lead to cell death, and be related to fecundity reduction (Kirkpatrick and Barton, 2006;Bizard and Hickson, 2018).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., 2007;Fenech et al., 2011).
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., 2018).As previously found by Liu et al. (2018), 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, 1990).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. bakeri Collins et al. (2014) (Walker, 1963;Collins et al., 2014;Milach et al., 2016).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., 2021).
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, 1980;Hemp et al., 2013).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) andD. obscurus Bruner, 1900 (2n = 18) (Colombo et al., 2005).
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.

Figure 3 -
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.

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.

Table 1 -
Specimen information and number of samples.

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

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