The karyotypes of three species of Pyrgomorphidae grasshoppers were studied: Zonocerus elegans (Thunberg, 1815), Pyrgomorpha guentheri (Burr, 1899) and Atractomorpha lata (Mochulsky, 1866). Data on karyotypes of P. guentheri and Z. elegans are reported here for the first time. All species have karyotypes consisting of 19 acrocentric chromosomes in males and 20 acrocentric chromosomes in females (2n♂=19, NF=19; 2n♀=20, NF=20) and X0/XX sex determination system. A comparative analysis of the localization of C-heterochromatin, clusters of ribosomal DNA, and telomere repeats revealed inter-species diversity in these cytogenetic markers. These differences indicate that the karyotype divergence in the species studied is not associated with structural chromosome rearrangements, but with the evolution of repeated DNA sequences.

Pyrgomorphidae grasshoppers, karyotype, C-banding, FISH, 28S rDNA, telomeric DNA

Orthoptera is undoubtedly one of the most well cytogenetically studied groups of insects. Even at an early stage of comparative cytogenetics, they became convenient research models for analysis of mitotic and meiotic chromosomes. It was through working on Orthoptera that Robertson (1916) established the main tendencies in insect karyotype evolution through centric fusion of chromosomes, Darlington (1931, 1932) described meiosis in detail, and White (1968, 1973) proposed the chromosome speciation hypothesis.

However, the karyotypic features of various Orthoptera groups have been studied extremely unevenly. Among Acridoidea and Pyrgomorphoidea, only the family Acrididae can be considered as well studied, whereas the karyotypes of Pyrgomorphidae, Pamphagidae, Lathiceridae, Lentulidae and some other families remain poorly investigated or not studied at all. Analysis of chromosome sets within such Orthoptera groups, which have never been studied before, in conjunction with the use of new techniques for chromosome research, may therefore potentially lead to many new insights. As an example, using molecular cytogenetic methods, in-depth research of the family Pamphagidae has recently revealed new evolutionary pathways of autosomes and sex chromosomes previously unknown in this family (Bugrov et al. 2016, Jetybayev et al. 2017).

The basal chromosome set of the family Pyrgomorphidae (superfamily Pyrgomorphoidea) coincides with that of the family Pamphagidae (superfamily Acridoidea) and contains 19 acrocentric chromosomes in males, 20 in females (sex determination X0/XX) (White 1973, Hewitt 1979). In this regard, these two families with FN=19♂/20♂ differ from other Acridoidea species, the basal karyotype of which contains 23 acrocentric chromosomes in males, 24 in females (sex determination X0/XX FN=23♂/24♀). The morphological similarity of the modal chromosome set in Pamphagidae and Pyrgomorphidae was noted a long time ago (White 1973, Hewitt 1979); however, the question as to whether this similarity represents a phylogenetic signal is still unknown. This is partially related to the poor degree of karyological study of Pyrgomorphidae grasshoppers. The karyotypes of only about 30 species are known from tropical and subtropical regions of the Old World (Makino 1951, Sannomiya 1973; Nankivell 1976, John and King 1983, Fossey et al. 1989, Williams and Ogunbiyi 1995, Seino et al. 2013, Seino and Dongmo 2015). The vast majority of species have a 19-chromosome karyotype, but a few species have been shown to have a different karyotype, resulting from one, two or three Robertsonian translocations (White 1973, Fossey et al. 1989). Moreover, only in a few species the C-heterochromatin localization has been studied (Atractomorpha similis, A. hypoestes, A. austraIis; Pyrgomorpha conica) (Nankivell 1976, John and King 1983, Suja et al. 1993).

Molecular cytogenetic studies were previously performed for only one species of Pyrgomorphidae – Pyrgomorpha conica (Suja et al. 1993, López-Fernández et al. 2004, 2006).

The aim of the present study, therefore, is to reveal new features of chromosome sets in as-yet unstudied species of Pyromorphidae grasshoppers. We used standard cytogenetic techniques, as well as molecular-cytogenetic methods, to find additional markers of linear chromosome differentiation. The fluorescence in situ hybridization (FISH) method was employed to localize functionally important regions in autosomes and the sex chromosomes, containing clusters of ribosomal DNA and telomeric (TTAGG)_n repeats. The choice of these molecular markers was prompted by an awareness of their important functional role in the genome and chromosome localization of many insects including Pyrgomorphidae grasshoppers (Sahara et al. 1999, López-Fernández et al. 2004, Cabrero and Camacho 2008), and renders the data reported herein suitable for comparative analysis.

Comparative analysis of karyotypes of three species of Pyrgomorphidae grasshoppers from the Ethiopian, Mediterranean and East Asian regions confirms that 2n♂=19 (NF=19), 2n♀=20 (NF=20) (X0/XX sex determination) is the basal chromosome set in this group. However, differences from the basal chromosomal set were also observed. Some species exhibit one (Sphenarium mexicanum, 2n♂=17), three (Pyrgomorpha granulata, 2n♂=13) or four (Pyrgomorpha rugosa, 2n♂=11) Robertsonian translocations (White 1973, Fossey et al. 1989). Another variant of non-basal karyotype was described in Pyrgomorpha sp. White (1973), referring to his unpublished data, mentions that the 2n=18, XX♀/XY♂ karyotype in this species resulted from centric fusion of acrocentric X-chromosome and acrocentric autosome. In all the cases mentioned above, the fundamental karyotype number remains constant: NF♂=19, NF♀=20.

Searching for new karyotype variants in this poorly studied group holds the potential to turn up interesting findings. For instance, recently, a new model of the Y-chromosome evolution was proposed based on studies in Pamphagidae grasshoppers. It was shown that in Pamphagidae grasshoppers centric fusion of the X chromosome and autosome occurred independently in two phylogenetic branches, and due to further evolution the neo-Y chromosome exhibited different stages of degradation process (Bugrov et al. 2016, Jetybayev et al. 2017).

The Pyrgomorphidae and Pamphagidae both have NF♂=19, NF♀=20, while Acridoidea has NF♂=23, NF♀=24. This gives rise to a question about the monophyly or homoplasy of Pyrgomorphidae and Pamphagidae. However, further detailed analysis of linear chromosome differentiation in these families is needed to shed light on this issue.

The present study revealed the difference in size and localization of C-positive blocks of chromosomes between the species studied. Furthermore, in A. lata and P. guentheri the difference observed on homologous chromosomes suggests the presence of the polymorphism in population of these species. A high level of interpopulation polymorphism of C-positive regions was previously reported for three Pyrgomorphidae species from Australia, Papua-New Guinea and Indonesia (Nankivell 1976, John and King 1983). Different populations of Atractomorpha crenaticeps, A. similis and A. australis were found to show polymorphism in terms of the size and localization of C-blocks in pericentromeric, interstitial, and telomeric regions in large and medium chromosomes. Furthermore, in some populations of A. australis additional arms were found, consisting of very large C-heterochromatin. Later some supernumerary heterochromatic segments in two chromosome pairs were revealed in Pyrgomorpha conica (Suja et al. 1993).

Such diversity in terms of the size and localization of C-positive blocks within different species of Pyrgomorphidae grasshoppers indicates that the evolution of repeated DNA sequences plays an important role in the divergence of karyotypes in this group.

However, molecular cytogenetic studies of repetitive sequences in chromosomes of Pyrgomorphidae grasshoppers were carried out only in Pyrgomorpha conica (Suja et al. 1993, López-Fernández et al. 2004, 2006). These methods showed that supernumerary heterochromatic segments derived from amplification of rRNA genes (Suja et al. 1993) and telomeric repeats enrich pericentric C-positive blocks (López-Fernández et al. 2006).

The current study represents comparative analysis of localization of 28S rDNA and telomeric (TTAGG)_n sequences in this group. Telomeric repeats exhibited very conservative localization, only in terminal areas of all chromosomes, and no interstitial telomeric sites. This may indicate that the karyotype evolution of these species did not include chromosome structural reorganizations involving terminal regions of chromosomes (for example pericentric inversions). However, interstitial telomeric sequences have previously been reported for Acrididae grasshoppers; such localization of clusters of telomeric DNA may be the result of such chromosomal reorganizations (Jetybayev et al. 2012). The observed polymorphism in the size of the telomeric cluster in A. lata correlates with C-block polymorphism in S₉ chromosome. Previously the same kind of polymorphism in terms of size was reported for Pyrgomorpha conica (López-Fernández et al. 2006). The C-blocks consist of amplified repetitive sequences, and sometimes amplification could involve telomeric or rDNA repeats.

Fluorescence hybridization in situ (FISH) of the rDNA fragment revealed a consistent pattern of rDNA distribution in chromosomes of the Pyrgomorphidae family. Ribosomal DNA clusters may be found in one pair (S₉ in Z. elegans), two pairs (M₇, S₉, in A. lata) or in all chromosomes (the pericentric regions of chromosomes in P. guentheri). However, in P. guentheri most of the rDNA clusters were very small and only clusters on the chromosomes M₇, M₈ and S₉ were significantly larger. This might be the result of a recent expansion of rDNA repeats in pericentric heterochromatin and the newly arisen rDNA clusters may be silent (Suja et al. 1993, Cabrero and Camacho 2008, Jetybayev et al. 2012).

The diversity in the rDNA distribution itself apparently reflects the degree of divergence in the species studied, which belong to different tribes of Pyrgomorphidae. Comparing the patterns of rDNA distribution in the karyotypes of the species studied here with known data on rDNA distribution in karyotypes of other Orthoptera, we may suggest that Pyrgomorphidae are close to the Acrididae family of Orthoptera. In this family, distribution of rDNA is basically limited to one or two pairs of chromosomes in the karyotype (Cabrero and Camacho 2008). In single cases, clusters of rDNA were revealed in the pericentric heterochromatin of all chromosomes in the set (Jetybayev et al. 2012). In contrast to Pyrgomorphidae and Acrididae grasshoppers, multiple localization of rDNA clusters on one chromosome in the Pamphagidae family has been shown (Bugrov et al. 2016, Jetybayev et al. 2017). Perhaps, the revealed differences in the localization of rDNA in Pyrgomorphidae and Acrididae on the one hand, and Pamphagidae on the other hand, may contain a certain phylogenetic signal. However, we still lack enough data, especially for the Pyrgomorphidae family, to approach the problem of the origin of the modal 19-chromosome karyotype of Pyrgomorphidae and Pamphagidae from a molecular-cytogenetic position. Nevertheless, intensive development of molecular-cytogenetic methods gives us hope that more species examined will allow further reconsideration of the pathways of Orthoptera chromosome evolution, which led to the formation of similar karyotype structure of Pyrgomorphidae and Pamphagidae grasshoppers.

The authors are grateful to the Center for Microscopy of Biological Objects (Institute of Cytology and Genetics, Novosibirsk, Russia). The authors are also grateful to Master of Biology Aleksejs Cernihs (Riga, Latvia) for the translation of the Russian version of this manuscript into English. The authors would like to thank Dr. František Marec and the anonymous reviewer for their valuable comments and helpful suggestions for the manuscript.

Funding

This work was funded by the research grants from the Russian Foundation for Basic Research #15-04-04816-aproject and the project #0324-2016-0003 of the Federal Research Center Institute of Cytology and Genetics SB RAS.