All specimens were identified by V.M. Gnezdilov. Several species were identified only to the genus level because of taxonomic difficulties in these genera. Only males were used for chromosome analyses. In the field, males were collected with an insect net, fixed alive in 3:1 fixative (96% ethanol: glacial acetic acid) and stored at +4 °C.

Gonads of adult males were used for chromosome analysis. Testes were dissected in a drop of 45% acetic acid and squashed. The coverslips were removed using dry ice. Prior to staining, the preparations were examined by phase contrast microscopy.

All the conventional staining techniques used herein were described in detail by Kuznetsova et al. (2009, 2010) for other issid species, i.e., Schiff-Giemsa staining, C-banding, AgNOR-banding and CMA₃-banding. All species were studied using the standard Schiff-Giemsa technique by Grozeva and Nokkala (1996), whereas the other techniques were used only for selected species (Table 1).

Chromosome banding techniques contribute to the identification of specific chromosomes within karyotypes. AgNOR-banding reveals chromosomal nucleolus organizing regions (NORs) representing the sites for the tandemly arranged 18S and 28S ribosomal RNA genes. The AgNOR-banding presumably differentiates only those NORs which were metabolically active during the preceding interphase (Howell and Black 1982). Some chromosome banding techniques, including C-banding and fluorochrome banding, are strongly dependent on the amount of heterochromatin and its distribution in chromosomes. Chromomycin A₃ (CMA₃) reveals the presence of GC-rich heterochromatin, which is usually associated with NOR regions, and thus differentiates NORs regardless of their prior metabolic activity.

This method was applied for the first time in the family Issidae. We used FISH with a (TTAGG)_n and 18S rDNA probes in 11 species from 8 genera; 9 species from Issini tribe while that one species of the Parahiraciini and Hemisphaeriini tribes (Table 1). FISH with both probes was applied as previously reported (Maryańska-Nadachowska et al. 2013, Golub et al. 2014, Kuznetsova et al. 2015b, c). In brief, chromosome preparations were treated with 100 µg/ml RNase A, and 5 mg/ml Pepsin solution was used to remove excess RNA and proteins. Chromosomes were denatured on a slide in a hybridization mixture with biotinylated 18S rDNA probe from the genomic DNA of Pyrrhocoris apterus (Linnaeus, 1758) and rhodaminated (TTAGG)_n probe with addition of salmon sperm DNA and then hybridized for 36 h. Hybridization signals were detected with NeutrAvidin-FITC.

Chromosomes were mounted in antifade medium (ProLong Gold antifade reagent with DAPI; Invitrogen) and covered with a glass coverslip. Chromosome slides were analyzed under a Leica DM 6000 B microscope. Images were taken with a Leica DFC 345 FX camera using Leica Application Suite 3.7 software with an Image Overlay module.

The testicular follicles were counted in 8 species (Table 1). The follicles were tubular and their number ranged from 6 to 18 among the species studied (here and elsewhere numbers are given per testis) and occasionally varied among males and in different testes of the same male, e.g. in Mycterodus drosopoulosi (2 males: 10/13, 17/18), Hemisphaerius sp. (2 males: 11/8, 12/12) and H. interclusus (4 males: 9/8, 11/11, 12/12, 12/12). In the only studied female of M. drosopoulosi, about 15 ovarian follicles were counted in each gonad.

Chromosome data on 10 species from 8 genera were obtained for the first time, including first observations on members of the tribes Parahiraciini and Hemisphaeriini (Table 1). Representative photographs of standard and sometimes also differentially stained meiotic karyotypes are presented in Figs 1–10. All species showed holokinetic chromosomes and the same chromosome number in males. In meiotic cells (diakinesis, metaphase I), there were 13 autosomal bivalents and a univalent X chromosome, i.e., 2n = 26 + X. Also, the karyotype structure seemed to be uniform with a pair of very large autosomes, 12 bivalents more or less gradually decreasing in size and the X chromosome as one of the smaller chromosomes of the set. The largest bivalent had a very large “secondary” constriction (a gap) in each homologue (Figs 1a, 2a, 7, 8a, 9). This constriction divided the chromosome into two unequal parts, however it was not always visible, especially when the chromosomes were more condensed (Figs 2b, 3, 4, 5a, 6a, 10). The silver staining technique used in Conosimus coelatus and Sarnus sp. produced a precipitation of silver at these regions suggesting that they harbor NORs (Figs 1b, 6b). In Thabena sp., the CMA₃/DAPI staining showed homogeneous DAPI staining (results not shown) and distinct patterns of GC-rich blocks (CMA₃-positive) in the NORs (Fig. 8b). The C-banded karyotype of Mycterodus sp. showed prominent telomeric C-bands in the largest and one of the medium-sized bivalents (Fig. 5a, b).

Figures 1–8.

Meiotic analyses of species of the tribes Issini (Figures 1–7) and Parahiraciini (Figure 8), (n = 13 bivalents + X) with different cytogenetic techniques. 1 Conosimus coelatus, a diakinesis with standard and b AgNOR-staining. Arrows point to “secondary” constrictions (gaps) (a) and empty arrows NORs (b) point to the largest autosomal pair 2 Kervillea scoleogramma, a diakinesis and b metaphase I with standard staining. Arrows point to "secondary" constrictions on the largest autosomal pair (a) 3 Latematium latifrons, metaphase I with standard staining 4Mycterodus (Mycterodus) drosopoulosi, diakinesis with standard staining 5Mycterodus (Semirodus) sp., diakinesis with C-banding. a Arrows point to C-bands on the largest and medium-sized bivalents. In the largest bivalent, C-bands are located at the terminal or b at the proximal (chiasmate) parts of chromosomes. Short arrows point to C-bands 6 Sarnus sp., a metaphase I with standard staining and b diakinesis with AgNOR-banding. Arrows point to NORs on the largest autosomal pair 7 Thionia obtusa, diakinesis with standard staining. Arrows point to “secondary” constrictions on the largest autosomal pair 8 Thabena sp. a diakinesis with standard staining, and b diplotene with CMA₃-banding. Arrows point to “secondary” constrictions (a) and asterisks mark CMA₃-positive, GC-rich regions (b) of the largest autosomal pair. Scale bar =10 µm.

Figures 9–10.

Conventionally stained meiotic karyotypes of two species of the tribe Hemisphaeriini (n = 13 bivalents + X). 9 Hemisphaerius interclusus, metaphase I with standard staining. Arrows point to “secondary” constrictions in the largest autosomal pair 10 Hemisphaerius sp., metaphase I with standard staining. Scale bar = 10 µm.

The number of testicular follicles per testis, counted here in males of eight species, ranged from 6 to 30, being the lowest in Sarnus sp. and the highest in Zopherisca tendinosa (both from Issini). In some species, the number of follicles varies among males of the same species and between testes of the same male. Specifically, variation was observed in Mycterodus drosopoulosi in which three examined males had testes with 17 and 18; 10 and 13; and 15 and 15 follicles, respectively. As in other planthopper families, in Issidae testicular follicles are of tubular shape. D’Urso et al. (2005) pointed out that Fulgoromorpha are differentiated by this pattern from Cicadomorpha in which follicles are lobular.

The evolutionary trends and the phylogenetic importance of the number of follicles in Auchenorrhyncha were repeatedly discussed in the literature (e.g., Emeljanov and Kuznetsova 1983, Kirillova 1989, D’Urso et al. 2005, Kuznetsova et al. 2009, Gnezdilov 2013b). In some groups variation in this character agrees with their taxonomy and phylogeny. For instance, the number of follicles is conserved at the level of tribes and/or subfamilies within the planthopper families Delphacidae and Dictyopharidae, with changes in this pattern correlated with their overall morphological evolution (Kirillova 1989, Kuznetsova et al. 2009). However, studies of testis structure in the Issidae documented the lability of the follicle number (Maryańska-Nadachowska et al. 2006, Kuznetsova et al. 2010). In 40 species studied so far, a wide range of follicle numbers have been reported, from four (in Palmallorcus punctulatus; but P. balearicus and P. nevadense have higher numbers, 10 or 11) and six (in Scorlupella discolor and Sarnus sp.) to 30 (in Zopherisca skaloula). Noteworthy are the unusually high numbers (24, 28 and 30) found in the three studied species of the genus Zopherisca Emeljanov, 2001. Interestingly, the number of follicles varies between closely related species (in the genera Kervillea Bergevin, 1918, Mycterodus Spinola, 1839, Palmallorcus Gnezdilov, 2003, Zopherisca) and even within the same species (e.g. Palaeolithium distinguendum, Palmallorcus balearicus and Mycterodus drosopoulosi) suggesting that evolutionary changes in the follicle number can be relatively rapid in the Issidae. In the opinion of Gnezdilov (2013b), the polymerization of seminal follicles inherent in the Issidae and also in other higher fulgoroid families, such as Nogodinidae, Ricaniidae and Flatidae (Kuznetsova et al. 1998), indicates that these families are relatively young in terms of evolution and that the testis structure has not yet been stabilized within their supraspecific taxa.

Although numbers between 9 and 18 and especially 10 (observed in one third of the species) seem to be more typical for the Issidae, there is still no conclusive evidence of the most characteristic number in this group. This problem can be resolved primarily through improved taxon sampling.

The nine species of the Issidae studied here for the first time have broadly similar karyotypes having the male diploid number (2n) of 27 chromosomes, including 13 autosomal pairs and an X(0) sex determination system. The karyotype includes a relatively small X chromosome, one pair of very long autosomes and the remaining autosomes which gradually decrease in size. Issidae, like other Auchenorrhyncha and Hemiptera, have holokinetic chromosomes. The largest bivalent is always NOR-bearing, and NORs are interstitial in the majority of species. The exceptions are Issus lauri and Zopherisca tendinosa, in which the 18S rDNA cluster is located terminally; this particular pattern probably resulted from inversions. GC-rich DNA segments labeled by CMA₃ are associated with nucleolus organizer regions.

Our study confirms that Issidae are a group characterized by the high karyotypic conservatism, with the basic karyotype of 2n = 27 (26 + X) (Maryańska-Nadachowska et al. 2006, Kuznetsova et al. 2010). At present, data on karyotypes are available for 44 species (around 4.5 % of the described species) and 27 genera (around 16 % of the recognized genera) in the three currently accepted tribes, Issini, Parahiraciini and Hemisphaeriini. With the exception of Latilica maculipes and Brahmaloka sp., both with 2n = 24 + X, and Falcidius limbatus with 2n = 24 + XY (the Issini), all species have 2n = 27 (26 + X). This makes the monophyletic origin of the latter karyotype an attractive hypothesis and, indeed, the ancestrality of this pattern has been inferred (Maryańska-Nadachowska et al. 2006, Kuznetsova et al. 2010). Every other karyotype could thus have arisen by a single tandem fusion, either between two pairs of autosomes (L. maculipes and Brahmaloka sp., 2n = 24 + X) or between an autosome and the X chromosome (F. limbatus, 2n = 24 + XY), respectively. Thus, the chromosome number decreased at least three times in the evolution of the family Issidae. Sex chromosomes of F. limbatus are most likely of the neo-XY type. Notably, neo-sex chromosome systems derived via autosome-sex chromosome fusion have been frequently reported in Auchenorrhyncha (see Kuznetsova and Aguin-Pombo 2015). This mechanism, necessarily resulting in reduced chromosome numbers, was clearly involved in sex chromosome diversification of the genus Falcidius Stål, 1866, in which the other studied species, F. doriae, has the basic chromosome complement of 2n = 27 (26 + X).

The basic karyotype appears conservative in structure within the Issidae, at least as regards the very large pair of autosomes, present in all the studied species. Based on a variety of observations (Giemsa-negative “secondary” constrictions, CMA₃, AgNOR and rDNA FISH patterns), the largest chromosomes are the NOR-bearing pair in the issid karyotypes.

C- banding has revealed unsuspected patterns of variation in the amount and distribution of constitutive heterochromatin in auchenorrhynchan karyotypes (see Kuznetsova and Aguin-Pombo 2015), and this is also true of Issidae. For example, Hysteropterum albaceticum was shown to have several bivalents easily distinguishable in meiotic cells by characteristic banding patterns (Kuznetsova et al. 2009). In the same paper, Agalmatium bilobum was shown to have C-bands on the largest and three medium-sized bivalents. Closely related species occasionally share the same or similar patterns as in Mycterodus colossicus and Mycterodus sp. (present study), both having telomeric C-bands on the largest and one of the medium-sized bivalents. On the other hand, Falcidium doriae and F. limbatus were demonstrated to differ extensively in their C-band pattern (Kuznetsova et al. 2010). Some additional examples can be found in Kuznetsova et al. (2010). Based on the data obtained, it can be stated that the gain and loss of heterochromatin is an important source of karyotype diversification in the Issidae.

Over the past decades, the FISH technique revolutionized the cytogenetic analysis providing significant advances on evolution of different insect groups with holokinetic chromosomes. At present, telomeres and the major rDNA loci are the most widely documented chromosomal regions in insects, including the order Hemiptera (e.g. Blackman et al. 2000, Manicardi et al. 2002, Monti et al. 2011a, b, Grozeva et al. 2011, 2014, Panzera et al. 2012, Chirino et al. 2013, Maryańska-Nadachowska et al. 2013, Pita et al. 2013, Bardella et al. 2013, Golub et al. 2014, 2015, Kuznetsova et al. 2012, 2015a). In addition, recent publications have shown that the number and chromosomal locations of the major rDNA multigene families are useful for the study of karyotype evolution in other insect groups (e.g. Grzywacz et al. 2011: Orthoptera; Gokhman et al. 2014: Hymenoptera; Vershinina et al. 2015: Lepidoptera; Lachowska-Cierlik et al. 2015: Mantophasmatodea; Mora et al. 2015: Coleoptera).

In Auchenorrhyncha, most cytogenetic studies were carried out by standard staining and conventional chromosome banding techniques. In this large hemipteran (= homopteran) group, FISH with rDNA and conserved insect telomeric (TTAGG)_n repeats has so far been applied to 25 species, including 8 species of the genus Philaenus Stål, 1864 from the froghopper family Aphrophoridae (Maryańska-Nadachowska et al. 2013, Kuznetsova et al. 2015c); Mapuchea chilensis (Nielson, 1996) from the leafhopper family Myerslopiidae (Golub et al. 2014); 5 species of the genus Alebra Fieber, 1872 from the leafhopper family Cicadellidae (Kuznetsova et al. 2015b); and 11 species of the planthopper family Issidae (present paper). In addition, Frydrychová et al. (2004) reported on telomeric sequences in Calligypona pellucida (Fabricius, 1794) from the planthopper family Delphacidae. In all examined species, including those studied here, the presence of the (TTAGG)_n telomeric repeat, known as the ancestral insect DNA motif of telomeres (Frydrychová et al. 2004), was detected.

The major rDNA loci were shown to vary in number (1 or 2 per haploid set) and chromosome location (autosomes, sex chromosomes or both; terminally or interstitially) in different species of Auchenorrhyncha. For example, in Mapuchea chilensis (2n = 16 +XY), the 18S rDNA clusters were present on a medium-sized pair of autosomes. In the karyotypically uniform genus Alebra (2n = 22 + X), they seem conserved and located on the largest pair of autosomes. In the genus Philaenus, which includes species with different chromosome numbers and karyotype structure, variation in number (1 or 2 per haploid set) and location (autosomes, sex chromosomes or both) of ribosomal genes was observed suggesting plasticity of the genomic organization within the genus. In the all species (11) of the Issidae from 8 genera and the three tribes, the 18S rDNA clusters were only detected in the largest autosomal pair. Basically, rDNA loci were located in an interstitial position, while in Issus lauri and Zopherisca tendinosa they were found at chromosomal ends suggesting that chromosomal rearrangements involving rDNA sequences occurred in the evolution of these unrelated species. In several karyotypes, FISH demonstrated size heteromorphism of rDNA clusters, suggesting that it can be attributed to differences in the number of ribosomal cistrons.

Among the families Caliscelidae, Acanaloniidae, Tropiduchidae and Nogodinidae, which are phylogenetically related to the Issidae, data on karyotypes and the number of follicles are still very scarce (Kuznetsova et al. 1998, 2010, Maryańska-Nadachowska et al. 2006), while molecular cytogenetic data are not yet available.

The “issidoid” families Caliscelidae, Acanaloniidae, Tropiduchidae and Nogodinidae taken together have currently only 11 species with known testis structure (Kuznetsova et al. 1998, Maryańska-Nadachowska et al. 2006). The relatively high and variable follicle numbers of the Issidae resemble the situation in the families Nogodinidae and Acanaloniidae, but not in the families Caliscelidae and Tropiduchidae, which share low and relatively stable numbers. In the four studied Nogodinidae species, numbers 5, 9 and 24 were observed, with the latter value found in two unrelated species, Biolleyana pictifrons Stål, 1864 and Pisacha sp. (Kuznetsova et al. 1998), whereas in the family Acanaloniidae, the only examined species, Acanolonia bivittata (Say, 1825), has 13 follicles per testis (Maryańska-Nadachowska et al. 2006). In the Tropiduchidae, the three studied species have either 6 or 3 follicles (Kuznetsova et al. 1998), while each of the three studied species of Caliscelidae has 6 follicles per testis (Maryańska-Nadachowska et al. 2006).

The currently available data on the families Tropiduchidae, Nogodinidae, Caliscelidae and Acanaloniidae concern just 13 species (Kuznetsova et al. 1998, 2010, Maryańska-Nadachowska et al. 2006). The 2n = 26 + X and secondarily derived 2n = 24 + XY chromosome complements are shared by Issidae and Nogodinidae. In the latter family, Bladina magnifrons Walker, 1858 and Biolleyana pictifrons have 2n = 26 + X, whereas Mindura subfasciata kotoshonis Matsumura, 1941 and Pisacha sp. share 2n = 24 + XY (Kuznetsova et al. 1998). In the Tropiduchidae, Achilorma ? bicincta Spinola, 1838 was found to have 2n = 26 + X, whereas the three other studied species have different karyotypes, i.e., 2n = 24 + X in Tambinia bizonata (Matsumura, 1914) and Barunoides albosignata Distant, 1912, while 2n = 28 + X in Varma distanti Melichar, 1914 (Kuznetsova et al. 1998). Putative ancestral issid karyotype of 2n = 26 + X (Kuznetsova et al. 2010), has not yet been found in the families Caliscelidae and Acanaloniidae (Maryańska-Nadachowska et al. 2006).

Based on the currently available data, which are still highly insufficient, we can infer that Issidae are characterized by 10 follicles per testis as the most frequent number, the presence of canonical insect telomeric repeats (TTAGG)_n, a stable karyotype constitution with the predominant karyotype of 2n = 26 + X(0), and the major rRNA gene clusters located on the largest pair of autosomes. A much broader taxonomic coverage is necessary to discuss possible implications of the above characters for the taxonomy and phylogeny of the Issidae.