Alebra albostriella and Alebra wahlbergi were found to have the same karyotype of 2n = 22 + X(0) encountered without variation in 30, 6% of studied males (N=19) and with some variation due to polymorphism in the rest of males (N=43) . The species share likewise a similar gross morphology of karyotypes. Both karyotypes are asymmetric in terms of the heterogeneity of chromosome size: one size group includes three pairs of larger chromosomes and the other group includes 8 pairs of smaller chromosomes. Within every group, chromosomes represent continuous gradation in size and therefore can not been reliably distinguished by conventional cytogenetic approaches. The X-chromosome is close by size to one of the larger chromosomes within the smaller-sized group. Chromosomes are holokinetic as in other Hemiptera; that is, the centromeric activity is dispersed along the length of each chromosome rather than concentrated at one point (Schrader 1935).

In spite of holokinetic nature of chromosomes, there are only a few Cicadellidae genera in which chromosome number has been sufficiently liable to change in the course of speciation whereas most genera have a stable number of chromosomes (Kuznetsova and Aguin-Pombo in press). As mentioned above, Alebra albostriella and Alebra wahlbergi are the first representatives of the leafhopper genus Alebra studied in respect to karyotype. The chromosome complement of 2n = 22 + X(0) found in thesespecies is fairly common in the Cicadellidae (Kuznetsova and Aguin-Pombo in press) but has never been recorded for the subfamily Typhlocybinae in which approximately 90 species in 35 genera are presently known cytologically (Kirillova 1987, Aguin-Pombo et al. 2006, Juan 2011). The only exception is probably a species referred to as Gen. nov. 6 for which Juan (2011) recorded n=12 (suggesting thus 2n = 22 + X).

Cytological analysis of male meiosis in Alebra albostriella and Alebra wahlbergi revealed that it was of the typical auchenorrhynchan type where all the chromosomes undergo segregation at anaphase I and chromatids separate at anaphase II (Halkka 1959). The small bivalents invariably had only one terminal/subterminal or, rarely, interstitial chiasma. In the larger bivalents one-two chiasmata were formed; in separate diakinesis cells up to six ring bivalents with two terminal/subterminal chiasmata were present. Halkka (1964) was the first to show that only one-two chiasmata are typically formed in male meiosis in Auchenorrhyncha species. Quite recently, Nokkala et al. (2004) have proved that the low number of chiasmata (one-two in a bivalent from a cytological standpoint) is characteristic of holokinetic chromosomes as such. The authors showed that holokinetic bivalents with multiple chiasmata entered AI but were unable to complete it as a result of wrong separation of homologues. It is noteworthy however that in our material the largest bivalents showed occasionally three and even four chiasmata. Multiple (more than two) chiasmata have also been described in other groups with holokinetic chromosomes, including in the Auchenorrhyncha (e.g. Kuznetsova et al. 2009a, b, Maryańska-Nadachowska et al. 2012), however, the work by Nokkala et al. (2004) still remains the only one in which the further fate of cells with such bivalents in meiosis was traced in detail.

In the majority of auchenorrhynchans, at least in all hitherto studied planthopper species (Fulgoroidea), the univalent X-chromosome shows a clear tendency to be arranged at the periphery of MI plate presumably forming its own meiotic spindle (Halkka 1959, Kuznetsova et al. 1998, Maryańska-Nadachowska et al. 2006). In contrast to this, in both leafhopper species studied in the present work, the univalent X-chromosome tended to locate randomly among the bivalents at MI.

Polymorphism for chromosomal rearrangements, B-chromosomes and meiotic abnormalities are not rare in nature and has been recorded for numerous species of plants and animals, including insects (White 1973). This kind of information is however very scarce in the Auchenorrhyncha. In this group, among approximately 820 species studied cytogenetically, B-chromosomes and different chromosomal rearrangements have been reported in several species only (Kuznetsova and Aguin-Pombo in press). In light of this, it is somewhat unusual to find so extensive variation which occurs in Alebra albostriella and Alebra wahlbergi from Greece. A total of 62 individuals from 13 population samples belonging to both species were examined. The studied populations inhabited different food plants (Fagus sylvatica, Castanea sativa, Quercus cerris, Acer opalus) and different altitudes ranging from 20 m to 1200 m above sea level (Table 1). In all 6 populations of Alebra albostriella, 29 males (71%; N=41) showed meiotic abnormalities and of these 4 males displayed additionally B-chromosomes. In 5 populations of Alebra wahlbergi, 11 males (52%; N=21) showed meiotic abnormalities, and of these 2 males displayed also B-chromosomes. In addition, one further male showed B-chromosomes while no meiotic abnormalities. The remaining two populations of Alebra wahlbergi, each with the only studied specimen, showed neither B-chromosomes nor meiotic abnormalities.

B-chromosomes are accessory genomic elements that are known to occur approximately in 15% of living species (Beukeboom 1994). In the Auchenorrhyncha, B-chromosomes were described in several species of planthoppers (Fulgoroidea) (Halkka 1959, Booij 1982, Kirillova and Kuznetsova 1990, den Bieman 1988) but have never been found to date in any species of leafhoppers (Cicadellidae). In leafhoppers Alebra albostriella and Alebra wahlbergi studied herein, B-chromosomes were found in low numbers (0-2) in 4 males of the first species (in 4 populations) and in 3 males of the second species (in 2 populations). In both species, B chromosomes were fairly small with the exception of male 1-AANE in which a single B-chromosome was about two times larger compared to Bs in other males (Fig. 2g). In every case however the Bs were much smaller than the univalent X-chromosome and conspicuous during meiotic prophase and metaphase I because of their negative heteropycnosis. When Bs were two in number, they did not pair and passed randomly through meiosis as univalents being still maintained in populations. Also, B-chromosome(s) did not connect to the univalent X-chromosome at MI as it was observed by Kirillova and Kuznetsova (1990) in several planthopper species from the family Delphacidae. The inter-population differences in B-chromosome distribution is believed to depend on different selective factors (Beukeboom 1994, Camacho 2004). It is interesting to note in this connection that in delphacid species Javesella pellucida (Fabricius, 1794) B-chromosomes were present only in populations inhabiting Northeast Siberia and Kamchatka whereas individuals sampled from different populations in European Russia lacked B-chromosomes (Kirillova and Kuznetsova 1990). In our study, the occurrence and frequency of B-chromosomes in males showed no relation with particular food plants on which populations inhabit. For example, Alebra albostriella males with Bs were collected both from Castanea sativa and Fagus sylvatica; Alebra wahlbergi males with Bs were sampled both from Ulmus sp. and Acer opalus. Similarly, no relationship was established between the occurrence of Bs and the habitat altitude above sea level: in Alebra albostriella Bs were present in populations inhabiting at 440 m and higher altitudes, while in Alebra wahlbergi they were found at lower altitudes (20m–520m).

In studiedpopulations, the frequency of B chromosomes was rather low. Thus, in Alebra wahlbergi they were present in 14% and in Alebra albostriella in only 10% of specimens studied. The frequency of individuals with 1B and 2Bs differed between the species. Thus, in four Alebra albostriella populations with B-chromosomes, 9, 6% of males carried 1B and 3, 2 % carried 2B, whereas in two Alebra wahlbergi populations with Bs, 11% of males had 1B and 22% had 2B. The data concerning the frequency of B chromosomes in natural populations of these species are too scarce to draw any firm conclusions. Noteworthy that the frequency of cells with B-chromosomes in 2B-males was markedly higher compared with that in 1B-carriers: 60, 7%-83, 7% against 0, 65%-1, 8% (Table 4). This observation suggests the existence of an accumulation mechanism responsible for maintaining the 2Bs in studied populations. Interesting, no males with more than two Bs were found in studied specimens. One can suppose that in Alebra populations, 1 or 2 B-chromosomes are tolerable to B-carriers and that natural selection operates by eliminating individuals with more than two Bs. In contrast, in the aforesaid planthopper species Javesella pellucida, males with up to four B-chromosomes were found (Kirillova and Kuznetsova 1990). However, in this species males with larger number of Bs were less frequent: males with 1B predominated (89%), males with 2Bs and 3Bs occurred with equal frequency (56%) whereas males with 4Bs were rare (11%).

The question of the adaptive significance of B-chromosomes in natural populations has been argued over for decades (Camacho et al. 2000), with the final position showing little if any substantial evidence to support such a role (Jones and Houben 2003). Among many others, the influence of B-chromosomes on recombination through the modulation of chiasma frequency and distribution in A-chromosomes has been recorded (e.g. in Orthoptera; Camacho et al. 2000). In Alebra albostriella and Alebra wahlbergi, intraindividual analysis demonstrated that chiasma frequency in a nucleus was independent of the occurrence and number of B-chromosomes that it contains. Similarly, there was no relationship between the occurrence of B-chromosomes and the occurrence, frequency and types of meiotic abnormalities in a male. As an example, B-chromosomes were absent in male 1-WNE which had bivalents with three chiasmata and in males 2, 3 and 4 of ASE with the highest percent of meiotic abnormalities (Tables 2 and 3). In some insects, B-carriers have shown a significant increase of the number of macrospermatids (Suja et al. 1986) however in our material such a correspondence was not observed.

As noted above, information on chromosome rearrangements and meiotic disturbances in auchenorrhynchan species is very scarce. A number of meiotic abnormalities including agmatoploidy (a result of fission of holokinetic chromosomes), aneuploidy, loose pairings of sex chromosomes and shrinkage of cytoplasm (changes in cytoplasmic volume) were described in three biotypes of the brown planthopper Nilaparvata lugens (Stål, 1854) from the family Delphacidae (Goh et al. 1992). A translocation polymorphism was encountered in the Australian leafhopper Alodeltocephalus draba Evans, 1966 (Whitten and Taylor 1969). Some other examples are presented in a review of Kuznetsova and Aguin-Pombo (in press). In Alebra albostriella and Alebra wahlbergi, one or another of meiotic abnormalities were found such as univalents, bivalent chains resulting from end-to-end non-homologous achiasmate associations, multiple translocation chains, anaphasic laggards and macrospermatids.

In males originating from different populations of Alebra albostriella and Alebra wahlbergi, end-to-end associations between bivalents were found at different stages of meiosis. The chains, involving up to four bivalents and occasionally (in Alebra albostriella) also the X-chromosome, were formed during prophase and were still intact at MI. In these cases, the persistent association was certainty non-chiasmate. Non-homologous telomeres did not touch intimately each other but unstained gaps were present between bivalents. Since in both species chromosomes display distal heterochromatic blocks (our unpublished data), one can suggest that the formation of artificial bivalent chains (pseudomultiples) is caused by heterochromatin adhesion due to which non-homologous chromosomes easily attract to one another.

Terminal associations of non-homologous bivalents without chiasma formation have been described in many plants and animals (White 1973, John and King 1982, 1985). These associations may involve from two up to all bivalents of a species. For instance, in the “holokinetic” moth species Sphinx ligustri (Linnaeus, 1758) (Lepidoptera, Sphingidae), all of the 28 bivalents were non-homologously attached to each other throughout prophase until prometaphase in females. Late in meiosis, the chains were broken down sequentially however short chains of two or three bivalents were still present at metaphase I. Like in the two Alebra species, in this moth non-homologous telomeres did not touch intimately each other but an unstained gap or some chromatin threads were seen between bivalents (Nokkala 1987).

In male 1-WANE sampled from Castanea sativa, part of nuclei had chains of several bivalents, non-homologous chromosomes showing the apparent intimate contacts by terminal chiasmata (Fig. 3g). In this male, the occurrence of heterozygotes for translocations could account for the multivalent chains formation. Since this chromosome rearrangement was observed only in some of meiotic cells, it must have happened after germinal cell development. Unfortunately, using only classical cytogenetic methods it was possible neither to affirm which chromosomes formed the chains nor to identify the orientation of separate chromosomes within a chain. Schematic representation of the possible formation of the chain-of-four caused by multiple translocations in meiotic cells is presented on Fig. 4.

In natural populations, chromosomal rearrangements arise as heterozygotes but their probability to establish is low (King 1993). Interchanges of small terminal segments of chromosomal pairs relatively frequently happen as spontaneous mutations (Hewitt 1979, Reed et al. 1992). Several studies have shown the chiasmate multivalent configurations, either rings or chains, in first meiosis as a result of heterozygosity for chromosomal fissions and fusions (e.g. in orthopterans; White 1973, John 1987, Bidau and Mirol 1988, Mirol and Bidau 1994, Colombo 2013). These configurations can lead to irregular segregation and nondisjunction in meiosis, with a consequent reduction in reproductive potential (White 1973), although sometimes they have no apparent negative influence on fertility (Mirol and Bidau 1994). Regarding insects with holokinetic chromosomes, the rearrangements can be even less deleterious since diffuse kinetochore is spreading through the length of their chromatids and products of rearrangements are able to be transmitted to daughter cells at successive cell divisions. Heterozygous translocations have occasionally been recorded in natural populations of “holokinetic” insects such as aphids (Blackman and Takada 1975, 1977, Blackman et al. 1995), heteropterans (Papeschi 1994, Bressa et al. 1998, Pérez et al. 2004) and psyllids (Grozeva and Maryańska-Nadachowska 1995, Nokkala et al. 2006). Within Auchenorrhyncha, a fascinating case of translocation polymorphism was described by Whitten and Taylor (1969) in populations of the Australian leafhopper species Alodeltocephalus draba. In all of the 8 studied populations, males showed one of the following chromosome complements: 3AA (three bivalents) + X; 4AA + X; 2AA + 1AAA (trivalent) + X; 1AA + 1AAAA (tetravalent) + X. A peculiar feature of this case is that the reduction in chromosome number has reached different stages in different localities. In one area (Lake Pedder), it was nearly or almost fixed (2 n= 7: 3AA + X), while in another area (Bruny Island) chromosome number decreased from north to south. Reduction of chromosome number following a latitudinal cline was caused by differences in the frequency of chromosome translocations. Alodeltocephalus draba was proposed to be under a process of speciation driven by the reorganization of karyotype which was initiated in some populations by the fixation of a particular chromosome fusion (Whitten and Taylor 1969).

In separate males of Alebra albostriella and Alebra wahlbergi, univalents of one-two chromosome pairs were observed at MI. The univalency involved either one of the larger or one of the smaller pairs of autosomes or occasionally both of these pairs. Although synaptic abnormalities can be responsible for the induction of abnormal chromosome segregation, no abnormal spermatids were observed in males showing univalents at metaphase I cells. This observation suggests a regular segregation of univalents in meiosis as it has been demonstrated by Nokkala (1986) for holokinetic univalents in the true bug species Rhabdomiris striatellus (Fabricius, 1794) (originally listed by Nokkala as Calocoris quadripunctatus (Vil.)) (Miridae, Hemiptera).

Macrospermatids were encountered in males of both species. Aberrant spermatids occurred in small proportion in part of spermatocysts and were twice and sometimes several times as much as normal spermatids within the same cyst. In Alebra albostriella, abnormal spermatids were more abundant being found in four populations and in about 37% of the specimens studied. Chromosomal abnormalities that affect gametogenesis are known to be one of the principle causal factors in nonbalanced gametes appearance (White 1973). If the emergence of abnormal spermatids is a reflection of meiotic disturbances, one would expect a correspondence between the amount of macrospermatids and that of meiotic abnormalities in a male. Despite of this, there was a clear discrepancy between these two parameters. Macrospermatids instead of being abundant in males with numerous abnormalities were either occasional or absent and vice versa. For instance, in Alebra albostriella, out of 15 males displaying macrospermatids, 10 had no evident meiotic abnormalities at diakinesis and MI. Notice however that the fate of abnormal meiotic configurations was not traced in any of the individuals analyzed.