Karyotypes of three species of Hyperophora Brunner von Wattenwyl, 1878 (Tettigoniidae, Phaneropterinae) enable morphologically similar species to be distinguished

Abstract Phaneropterinae is the largest subfamily of Tettigoniidae, distributed across the globe. There are few cytogenetic studies regarding this group, as in the case of the genus group Aniarae, which represents only two karyotyped species. The current study aims to analyze cytogenetically three species of Hyperophora Brunner von Wattenwyl, 1878 from Brazil. The male diploid number of Hyperophoraminor Brunner von Wattenwyl, 1891 and Hyperophoramajor Brunner von Wattenwyl, 1878 is 2n♂= 31, whereas Hyperophorabrasiliensis Brunner von Wattenwyl, 1878 has shown 2n♂= 29. These three species possess an X0 sex chromosome system and telo/acrocentric chromosome morphology. The only species found in the Pantanal biome, H.brasiliensis, can be chromosomally distinguished from the Cerrado biome species H.major and H.minor, due to the difference in chromosome number (2n♂= 29 and 2n♂= 31, respectively).

For some Hyperophora species there is a paucity of descriptive information regarding the intraspecific morphological variations. Rehn (1907) described a male of the species Hyperophora major Brunner von Wattenwyl, 1878, commenting that the individual is smaller than those described by Brunner von Wattenwyl (1878) and published a schematic of the male cercus along with a sketch of Hyperophora brasiliensis Brunner von Wattenwyl, 1878 cerci apparently based on individuals different from the type material. The drawings of the cerci of H. brasiliensis and H. major are slightly similar, raising doubts as to whether they are simply morphological variations, since as previously reported there is no complete description that presents other robust characteristics that allow an accurate identification.
In this work, we describe the karyotype of Hyperophora brasiliensis Brunner von Wattenwyl, 1878, Hyperophora major and Hyperophora minor Brunner von Wattenwyl, 1891, to discuss the chromosomal evolution and the cytotaxonomy of the group.

Material and methods
The specimens were collected at two localities in the state of Mato Grosso do Sul (MS), Brazil, from November 2015 to February 2017 and were deposited in the Coleção Zoológica de Referência da UFMS (ZUFMS) with the exception of one male specimen of H. major, which was used in the work of Serrão et al. (2018) (Table 1).
The individuals were anesthetized in ether, dissected and fixed in 70% ethanol, with the exception of the gonads, which were used for chromosomal preparations and Giemsa staining following the procedures of Araujo et al. (2008). Slides from all three species were submitted to Fluorescence in situ hybridization (FISH) using the telomeric probe. This process employs a peptidic nucleic acid (PNA) (AATCC) 3 probe (PNA Bio, Inc) that is complementary to the typical (TTAGG) n telomeric repeats of Orthoptera, labeled with Alexa fluor 488 (ThermoFisher Scientific). It was followed the method of Genet et al. (2013), with a hybridization time of four hours at 37 °C, without heat denaturing, and mounted with ProLong Diamond antifade containing DAPI (ThermoFisher Scientific).
All cells were photographed using a Zeiss Axioimager D2 microscope with a monochromatic AxioCam 503 camera, employing the ZEN Pro software. Chromosome morphology was determined using the free software IMAGEJ (Rasband 1997(Rasband -2018 and the LEVAN plugin (Sakamoto and Zacaro 2009), according to Levan et al. (1964) and Green and Sessions (1991), using respectively ten, nine and 18 mitotic metaphases of H. brasiliensis, H. major and H. minor.
All three species possess the SCS of the type ♂X0/♀XX and showed exclusively telo/ acrocentric chromosomes (Fig. 1), with the exception of one specimen of H. minor, which exhibited one submetacentric chromosome in all of the nine analyzed cells. (Fig. 1c).   Only the telomeric regions of all chromosomes were hybridized in the three species analyzed (Fig. 3). No interstitial telomeric sites (ITS) were observed in any of the cells submitted to telomeric FISH.

Discussion
The karyotype composed of 2n♂= 31 and ♂X0/♀XX SCS, presenting only telo/acrocentric chromosomes found in Hyperophora major and Hyperophora minor is the most common for katydids (Ferreira 1976, Warchałowska-Śliwa et al. 2011 and it was the same karyotype configuration presented in a congeneric species, H. angustipennis, the only Hyperophora species citogenetically analyzed up to now (Ferreira 1976). The heteromorphic pair found in one female of H. minor is the first recorded in Phaneropterinae, likely a result of one pericentric inversion.
Hyperophora brasiliensis showed 2n♂= 29, that is, one autosomal pair less when compared to the other Hyperophora species and the most common pattern in Tettigoniidae (2n♂= 31). In Phaneropterinae, karyotypes with 2n♂= 29 and 2n♂= 31 within the same genus were found in Holochlora Stål, 1873, Phaneroptera Serville, 1831, and Scuderia Stål, 1873(Warchałowska-Śliwa 1998. Thus, a reduction of one chromosomal pair, probably due to in tandem fusion, appears to occur independently several times within Phaneropterinae (Warchałowska-Śliwa 1998, Hemp et al. 2010). Despite the suggested chromosome fusion, no interstitial telomeric sites (ITS) were detected, which can reflect an ancient fusion event, that the telomeric region of the fused element was lost during the rearrangement, or that it is below the limit of FISH technique.
Interestingly, both species which showed 2n♂= 31 are sympatric in the Cerrado of Campo Grande, while H. brasiliensis (2n♂= 29), which was not registered in Campo Grande, was collected in the Pantanal of Corumbá (~ distance 270 Km). The cerci of H. minor differ enormously from the cerci of H. brasiliensis and H. major, thus permitting a rapid and accurate morphological identification of H. minor. In this study, it was determined that despite the morphological similarity of the cerci of H. brasiliensis and H. major (Brunner von Wattenwyl 1878), the karyotypes of H. minor and H. major are more similar to each other than those of H. brasiliensis, helping to distinguish these species.
Regarding the Aniarae group, all four Hyperophora species karyotyped (Ferreira 1976, present study) exhibited higher diploid numbers (2n♂= 31 or 2n♂= 29) than the only Aniarella species karyotyped up to now (2n♂= 21) (Ferreira and Mesa 2007). Differences of 10 or more chromosomes within karyotypes of closely related genera are uncommon among Phaneropterinae groups. However, the genus group Phyllopterae, Itarissa sp. presented 2n♂= 17, while Phylloptera fosteri Caudell, 1906(cited as Phylloptera modesta Piza, 1961 and Phylloptera sp. evidenced 2n♂= 31 (Ferreira 1977, Ferreira andMesa 2007). In both cases, the species belong to a "genus group", not a tribe. Genus groups are unreliable since there are not strict systematic studies supporting them. The clusters are allocated due to morphological similarities that could indicate the lack of a close kinship between the genera.

Conclusion
The diploid number was useful in order to distinguish on chromosome level the species of Hyperophora from the Pantanal of those from other localities. The external morphological appearance is not directly related to similarity in the chromosome number for Hyperophora. Further research of other species of the Aniarae group is fundamental for assessing karyotype patterns within the clade, however, it is possible to affirm that the reduction from 2n♂=31 to 2n♂=29 is a recurrent event in Phaneropterinae.