Four antlion species (only males), namely Palpares libelluloides, Distoleon tetragrammicus, Macronemurus bilineatus, Myrmecaelurus trigrammus, and male owlfly Bubopsis hamatus, were used in the present study. The specimens were collected from May to October 2013 in the Republic of Dagestan (North-East Caucasus, Russia). The material was collected by G. Khabiev. Collection sites, sampling dates, and the numbers of studied males are given in Table 1. In the field, adult individuals were fixed in a solution of 96% alcohol and glacial acetic acid (3:1) and then stored at 4 °C until required.

Air-dried preparations were made by macerating testicular follicles in a drop of 45% acetic acid on a glass microscope slide and squashing under a cover slip. The preparations were frozen using dry ice, the cover slips were removed with a razor blade, and the preparations were dehydrated in fresh fixative (3:1) for 20 min and air dried. Slides were first examined under a phase-contrast microscope to check for the availability of meiotic divisions and quality of chromosome spreads. Counts were based on samples of one to 23 individuals. The preparations and remains of the specimens are stored at the Department of Karyosystematics, Zoological Institute, RAS.

Meiotic chromosomes were stained using the Feulgen-Giemsa method developed by Grozeva and Nokkala (1996).

Chromosome preparations were analyzed under a Leica DM 4000B microscope with a 100x objective. Images were taken with a Leica DFC 345 FX camera using Leica Application Suite 3.7 software with an Image Overlay module.

In the Myrmeleontidae, with the original data presented here, karyotype data have been made available for 37 species and 21 genera in 9 out of 12 subfamilies accepted by Krivokhatsky (2011). Having regard to 1500 valid species and 191 valid genera in this family (Stange 2004), the proportion of the studied species and genera is approximately 2.5% and 11% respectively. The karyotypes (chromosome numbers and sex chromosome systems) are presently known for the subfamilies Palparinae (3 species/2 genera), Pseudimarinae (1/1), Dendroleontinae (2/2), Nemoleontinae (6/4), Glenurinae (2/2), Myrmeleontinae (11/3), Brachynemurinae (7/3), Myrmecaelurinae (2/1), and Acanthaclisinae (3/3). The family demonstrates a relatively high diversity of karyotypes, with diploid chromosome numbers (2n) of 37 studied species ranging from 14 to 26 including four intermediate counts, i.e. 16, 18, 22 and 24. The highest numbers, 26, 24 and 22, occur only in the subfamily Palparinae, in three species of the genera Palpares Rambur, 1842 and Indopalpares Insom & Carfi, 1988. Other numbers, 2n = 14, 16 and 18, are encountered in the remaining subfamilies. In the Pseudimarinae, the only studied species, Palparidius concinnus, exhibits the next highest number found in antlions, i.e. 2n = 18. In the Nemoleontinae, with the exception of Distoleon tetragrammicus and Creoleon lugdunensis displaying 2n = 18, all studied species, including three Macronemurus Costa, 1855 species, have karyotypes with 2n = 16. The latter value is also found in all studied Dendroleontinae and Glenurinae. The subfamilies Brachynemurinae, Myrmecaelurinae and Acanthaclisinae include species both with 2n = 16 and 2n = 14, whereas Myrmeleontinae show 2n = 14 in all the studied species. It is noteworthy that, with the exception of Palpares (but see below), all these genera do not show interspecific variation in the chromosome number. This is especially remarkable for those genera where several species have been studied, e.g. Macronemurus (Nemoleontinae) and Scotoleon Banks, 1913 (Brachynemurinae). In each of these genera, three studied species share 2n = 16. Moreover, in Myrmeleon Linnaeus, 1767 (Myrmeleontinae) all eight studied species have 2n = 14. It is noteworthy that closely related genera, Baliga Navás, 1912 and Euroleon Esben-Petersen, 1918 in the Myrmeleontini, show the same karyotype with 2n = 14. It is unclear at present whether the chromosome number varies within the genus Palpares. The highest chromosome number, 2n = 26, is found in P. libelluloides, the type species of the genus. Palpares pardus asanai Kuwayama, 1933 with 2n = 24 (Oguma and Asana 1932) is treated here as a member of Indopalpares. Additionally, there is a possibility that Palpares sobrinus with 2n = 22 (Klok and Chown 1993) represents in fact Pseudopalpares sparsus (McLachlan, 1867). Although few members of the Palparinae are studied at present, karyotypic differences between the genera of this subfamily probably occur.

Compared to the Myrmeleontidae, karyotypes of the Ascalaphidae are less studied. The chromosome numbers are currently known in only seven owlfly species from the genera Ascalohybris Sziraki, 1998, Ogcogaster Westwood, 1847, Libelloides Schaeffer, 1766, Bubopsis McLachlan, 1898, and Glyptobasis McLachlan, 1871, all presently classified within the subfamily Ascalaphinae. The species studied show relatively high chromosome numbers, i.e. 2n = 18 in Bubopsis hamatus, 20 in Libelloides corsicus, and 22 in all the remaining species, including two other studied members of the genus Libelloides.

Although Myrmeleontidae and Ascalaphidae show a similar range of chromosome numbers (2n = 14 - 26 in the former and 18 - 22 in the latter), these families differ in the modal numbers. Of 37 species studied in the Myrmeleontidae, 19 species display 2n = 14, and 12 species have 2n = 16. On the other hand, five of seven species studied in the Ascalaphidae display 2n = 22. Other chromosome numbers occur only occasionally within the families except for high numbers characteristic of the antlion subfamily Palparinae.

In different eukaryotic organisms, evolutionary changes in the chromosome number happen via polyploidy, aneuploidy or fusion/fission events. In animals polyploidy is known to be rare, whereas chromosomal fusions and fissions are common. As stated above, most Myrmeleontidae possess lower chromosome numbers, 2n = 14 and 2n = 16, which are encountered in all subfamilies, with the only exception of the Palparinae. The latter is the only subfamily characterized by higher numbers, 2n = 26, 24, and 22, and the higher number, 2n = 18, is also found in the only studied species of the related subfamily Pseudimarinae. The higher numbers, 2n = 22, 20 and 18, are also characteristic of the sister family Ascalaphidae. Since Palparinae represent a basal phylogenetic lineage of the Myrmeleontidae (Krivokhatsky 2011), it is hypothesized that higher chromosome numbers are ancestral for antlions. Most likely, the higher chromosome numbers were inherited from the common ancestor of Myrmeleontidae + Ascalaphidae. It was preserved in the subfamily Palparinae (Myrmeleontidae) but changed via chromosomal fusions toward lower numbers, 2n = 18, 16 and 14, in other subfamilies.

Knowledge of the chromosome morphology in the low-numbered and high-numbered chromosome complements would help in understanding the karyotype evolution in the Myrmeleontidae and Ascalaphidae and testing the above hypothesis. Unfortunately, despite several efforts to identify chromosomal morphology within particular karyotypes (e.g. Asana and Kichijo 1936, Hughes-Schrader 1983, present study), this important question remains unresolved. Special staining methods, e.g. C-banding, are therefore needed to identify the centromeric position in the chromosomes and thus their morphology. However, these techniques have never been used in neuropteran cytogenetics, and therefore this is the most serious objective in the chromosome research of antlions and owlflies.

All Myrmeleontidae and Ascalaphidae species, including those studied here, exhibit a simple sex chromosome system XY/XX, which is characteristic of the whole order Neuroptera (White 1973, Blackman 1995). Both antlions and owlflies demonstrate a very peculiar behavior of sex chromosomes in males (Naville and de Beaumont 1933, 1936, Asana and Kichijo 1936, Hughes-Schrader 1983, Klok and Chown 1993, present paper). In spermatocyte meiosis of those insects, sex chromosomes take up positions at opposite halves of the meiotic spindle at metaphase I before segregating into the daughter spermatocytes. It means that the X and Y chromosomes get segregated to opposite poles of the spindle long before the autosomal half-bivalents disjoin at anaphase I and move to the poles. The same pattern, the so-called “distance pairing” of sex chromosomes first discovered by Naville and de Beaumont (1933) in antlions, is known to be characteristic of the related neuropteran families Chrysopidae, Mantispidae, Sisyridae, Osmylidae, and Hemerobiidae (Naville and de Beaumont 1936, Hughes-Schrader 1969, 1975b, 1980, Nokkala 1986) and probably of the order Neuroptera in general. The biological role of this unusual behavior of sex chromosomes is unclear. In any case, this mechanism observed in brown lacewings (Hemerobiidae) showed no significance for the regular segregation of the sex chromosomes in meiosis (Nokkala 1986).

The order Neuroptera belongs to the superorder Neuropterida, which comprises another two orders, namely, Raphidioptera with two extant families, Raphidiidae and Inocelliidae, and Megaloptera with two extant families, Corydalidae and Sialidae (Aspöck and Aspöck 2007). Interestingly, the Neuroptera share the “distance pairing” of sex chromosomes with Raphidioptera (Naville and de Beaumont 1936, Hughes-Schrader 1975a) but not with Megaloptera. In the latter group, all hitherto studied species, which belong to the single family Corydalidae, show another very specific “parachute-like” sex bivalent in spermatocyte meiosis (Hughes-Schrader 1980, Takeuchi et al. 2002). In this case, the X and Y chromosomes form a pseudo-bivalent that is situated together with the autosomes on the equator of the spindle and segregates synchronously with them at the first meiotic anaphase. This unique meiotic sex chromosome configuration called Xy_p (Smith 1950) is the well-known characteristic feature of the related order Coleoptera, and is encountered in almost all coleopteran families. Therefore Xy_p is considered ancestral for beetles (Smith 1950), at least for the suborder Polyphaga (Petitpierre 1987).

The variety and distribution of sex chromosome systems in different orders of the class Insecta have been comprehensively reviewed by Blackman (1995). The X(0) system was shown to predominate in the lower orders and is considered as ancestral condition for several major groups and for Insecta as a whole. The XY systems when occur are all derived from an X(0) one. The sex chromosome systems seem to provide useful phylogenetic evidence. Within Holometabola orders, besides simple X(0) and XY, there are some peculiar systems, e.g., those involving female heterogamety (XY/XX or ZW/ZZ) shared by Lepidoptera and Trichoptera, haplodiploid sex determination characteristic of Hymenoptera, and some others. Of these, distance pairing of the X and Y chromosomes in spermatocyte meiosis and the parachute Xy_p system are hypothesized to be synapomorphies respectively of the clade Neuroptera + Raphidioptera and of the clade Megaloptera + Coleoptera (Blackman 1995, Takeuchi et al. 2002).