Mediterranean Species of the Spittlebug Genus Philaenus: Modes of Chromosome Evolution Author(s): Anna Maryańska-Nadachowska, Valentina G. Kuznetsova, Dorota Lachowska and Sakis Drosopoulos Source: Journal of Science, 12(54):1-17. 2012. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1673/031.012.5401 URL: http://www.bioone.org/doi/full/10.1673/031.012.5401

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Mediterranean species of the spittlebug genus Philaenus: Modes of chromosome evolution Anna Maryańska-Nadachowska1a*, Valentina G. Kuznetsova2b, Dorota Lachowska3c, Sakis Drosopoulos4d 1Institute of Systematics and Evolution of , Polish Academy of Sciences, Poland 2Zoological Institute, Russian Academy of Sciences, Russia 3Department of Entomology, Institute of Zoology, Jagiellonian University, Poland 4Agricultural University, Athens, Greece Abstract The evolution of karyotypes and sex determination system of Philaenus Stål (: ) species is studied here in detail. The most plausible scenario of chromosomal rearrangements accompanying phylogenetic differentiation in Philaenus is advanced. It is postulated that the ancestral karyotype of Philaenus was 2n = 24 + X0. Karyotype changes occurred several times independently in the genus. The karyotype of 2n = 22 + X0 (P. spumarius and P. tesselatus) originated from 2n = 24 + X0 by fusion between two autosomal pairs. The neo–XY system (P. arslani, P. loukasi, P. signatus, P. maghresignus, and P. tarifa) also originated from the 24 + X0 karyotype by means of independent fusions between autosomes and the original X chromosome. The neo–X1X2Y system (P. italosignus) evolved from the 2n = 22 + neo–XY karyotype by an additional fusion between the Y chromosome and one more autosomal pair. The neo–XnY system of P. italosignus is the first reported case of an evolutionarily fixed multiple sex chromosome system in Auchenorrhyncha.

Keywords: cytogenetics, karyotype evolution, sex chromosome system Correspondence: a [email protected], b [email protected], c dorota.lachowska- [email protected], d [email protected], *Corresponding author Editor: Igor Sharakhov was Editor of this paper. Received: 16 June 2011, Accepted: 26 August 2011 Copyright : This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 12, Number 54 Cite this paper as: Maryańska-Nadachowska A, Kuznetsova VG, Lachowska D, Drosopoulos S. 2012. Mediterranean species of the spittlebug genus Philaenus: Modes of chromosome evolution. Journal of Insect Science 12:54 available online: insectscience.org/12.54

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Introduction The Mediterranean species of Philaenus were shown to be sympatric with P. spumarius, The spittlebug genus Philaenus Stål while partly allopatric with each other. At (Auchenorrhyncha: Aphrophoridae) has long present, eight species are recognized in the attracted the particular interest of biologists genus Philaenus. The current taxonomy of due to its high color polymorphism. The this genus accepts a division of these species nature and origin of this polymorphism and its into two groups based on morphological possible contribution to the evolution of similarities in the male anal tube: the reproductive isolation and sympatric “spumarius” group (P. spumarius, P. speciation have been extensively documented tesselatus, P. loukasi,andP. arslani), and the for P. spumarius. This species is widely “signatus” group (P. signatus, P. italosignus, distributed, covering most of the Palaearctic P. maghresignus, and P. tarifa) (Drosopoulos region and extending into the Nearctic, as well and Remane 2000). According to larval food as most other temperate regions of the earth plant preferences, the genus is subclassified and many oceanic islands (Halkka and Halkka into the three groups developing: (1) on the 1990; Stewart and Lees 1996; Drosopoulos lily, Asphodelus aestivus (= A. microcarpus) 2003; Drosopoulos et al. 2010). Philaenus (P. signatus, P. italosignus, P. maghresignus, spumarius is a highly polyphagous insect, and and P. tarifa), (2) on xerophilic plants (P. has become a pest of fodder plants and loukasi and P. arslani), and (3) on various strawberries in areas where it is not a native dicotyledonous and monocotyledonous plants species (Halkka et al. 1967; Zając and Wilson (P. spumarius and P. tesselatus) (Drosopoulos 1984). Due to outstanding polymorphism in 2003). The results of a recent phylogenetic adult dorsal color/pattern, more than 50 study of Philaenus using nucleotide sequences synonyms have been given to P. spumarius from two mitochondrial (COI and CytB) (Nast 1972). Until the late 1980s, only three genes and one nuclear (ITS2) region is in species were recognized in the genus general agreement both with morphological Philaenus: the Holarctic P. spumarius, the and food plant preference classifications, with Mediterranean species P. signatus (which the exception of P. maghresignus, placed as a inhabits the Balkans and Middle East), and P. sister taxon to all remaining Philaenus species tesselatus (southern Iberia and Maghreb). (Maryańska-Nadachowska et al. 2010). Philaenus tesselatus was often treated as a subspecies (Wagner 1959) or a synonym of P. Over 90% of speciation events are suggested spumarius (Nast 1972). However, more recent to be accompanied with chromosomal studies suggested that it is a valid species rearrangements (White 1978). distinct from P. spumarius (Drosopoulos and Auchenorrhyncha possess holokinetic Quartau 2002). Since the 1990s, as a result of chromosomes; that is, their chromosomes do purposeful morphological studies on not have a primary constriction (the Philaenus in the Mediterranean region, five centromere) (Halkka 1959). Because of the further species have been described: P. absence of the centromere as a morphological loukasi (southern Balkans), P. arslani (Middle marker, and also because of the paucity of East), P. maghresignus (Maghreb and convenient differential techniques, the southern Spain), P. italosignus (southern Italy interchromosomal (particularly and Sicily), and P. tarifa (southern Iberia). intrachromosomal) rearrangements cannot be

Journal of Insect Science | www.insectscience.org 2 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. detected in holokinetic chromosomes. These FISH mapping of genes, which are currently chromosomes have thus no distinctive features used in some economically important for individual identification in a karyotype, holokinetic organisms (Mandrioli et al. 2003; besides size differences, if present. In Mandrioli and Borsatti 2007; Marec et al. holokinetic chromosomes, a kinetochor plate 2010), are not yet developed or available for (to which the spindle microtubules attach) Auchenorrhyncha (Kuznetsova et al. 2010). covers all or the majority of the chromosome Over the past several decades, a number of surface (Wolf 1996). Theoretically, the large studies have used conventional banding kinetochor plate facilitates karyotype techniques (C-, AgNOR-, DAPI/CMA3- evolution by means of fusion and fission of banding) for the study of auchenorrhynchan holokinetic chromosomes, since on the one karyotypes (Noda and Tatewaki 1990; hand there is no risk of the formation of Perepelov et al. 2002; Kuznetsova et al. 2003, dicentric chromosomes and, on the other 2009a, 2009b, 2010; Maryańska- hand, even relatively small chromosome Nadachowska et al. 2006); however, these fragments can have a part of the kinetochore studies developed only a few chromosomal plate and thus be attached to the spindle. For markers. Additionally, their limited taxonomic the reasons mentioned above, these representation failed to provide rearrangements are conventionally accepted as comprehensive insight into the comparative the most common mechanisms of cytogenetics of the group (Kuznetsova et al. chromosome evolution in holokinetic groups. 2010). However, contrary to what may be expected, Auchenorrhyncha seem to be characterized by The family Aphrophoridae, to which the stable or only slightly variable karyotypes at genus Philaenus belongs, is a group with the levels of genera, tribes, and families fairly diversified karyotypes. In 29 studied (Halkka 1959; Kirillova 1986, 1988; species assigned to nine genera within Emeljanov and Kirillova 1989, 1991) Aphrophoridae, the number of autosomes suggesting that chromosomal fusions/fissions ranges from 11 to 30 including all possible have not played a key role in karyotype values of the diploid number (Kirillova 1986; evolution and speciation within this group. Kuznetsova et al. 2003; Maryańska- For example, almost all species of the tribes Nadachowska et al. 2008, 2010). Almost Issini () and Almanini () without exception, the evidence today were found to have 2n = 26 + XX/X0 concerns just the number of chromosomes and (Maryańska-Nadachowska et al. 2006; the type of sex determination, the data being Kuznetsova et al. 2010) and 2n = 24 + neo– too few in number in each genus studied for XY (Kuznetsova et al. 1986, 2009a), any conclusions to be reached. In the respectively. comparatively better–studied genus Aphrophora, all species (probably with the Theoretically, in holokinetic chromosomes, only exception of A. quadrinotata) have 28 rearrangements can be detected if advanced autosomes in the diploid complement. In techniques of molecular cytogenetics are used contrast, the genus Philaenus demonstrates a to establish chromosomal markers. However, wide variety of different karyotypes that have the most informative techniques, such as been briefly described by Maryańska- immunofluorescence, chromosome painting, Nadachowska et al. (2010). At the present genomic in situ hybridization (GISH), and time, P. spumarius and P. arslani are the only

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two aphrophorid species comprehensively fluorochromes CMA3 and DAPI. For studied using chromosome banding methodological details see Kuznetsova et al. techniques (Kuznetsova et al. 2003; (2003). Analysis of slides was performed Maryańska-Nadachowska et al. 2008). using a Nikon Eclipse E400 light microscope (www.nikon.com) at 1000× magnification. The aims of this study were to apply these Photomicrographs were taken using a Nikon techniques to the six other Philaenus species DS-U1 camera. All voucher specimens are and used cytogenetic markers for preserved in the Institute of Systematics and understanding the taxonomy and intrageneric Evolution of Animals, Polish Academy of relationships of the Philaenus and elucidating Sciences in Kraków, Poland. the modes of chromosomal rearrangements in the evolution of the genus. Results

Materials and Methods The “spumarius” group Published data: P. spumarius: 2n = 23 (22 + Table 1 lists species, dates and collection X0) (Kuznetsova et al. 2003); P. arslani: localities, host plants, and number of 2n = 20 (18 + neo–XY) (Maryańska- specimens studied. were fixed in Nadachowska et al. 2008). Carnoy, a mixture of 96% alcohol and glacial acetic acid (3:1), and stored in fixative at 4 °C Philaenus tesselatus: 2n = 23 (22 + X0) until slides were made. Air–dried The male mitotic complement was composed chromosome preparations were made by of 23 chromosomes which gradually decrease squashing testicular follicles in 45% acetic in size, with the X chromosome close in size acid and freezing in dry ice. The coverslips to one of the longer pairs of autosomes were then taken off with a razor blade. The (probably AA3) (Figures 1a, 1b). All of the techniques of chromosome staining followed chromosomes had a well–defined holokinetic Kuznetsova et al. (2003) and Maryańska- structure without visible constrictions (the Nadachowska et al. (2008): conventional centromeres). In meiosis, 11 bivalents, one Feulgen-Giemsa staining for the visualisation clearly larger than the others, and a univalent of standard karyotypes, silver–staining for the X chromosome were observed (Figures 1c, visualization of the nucleolus organizing 1d). Noteworthy was the high level of regions (NORs), and C–banding for the condensation of the X in meiosis. The detection of the constitutive heterochromatic bivalents displayed one or occasionally two regions (C–heterochromatin). In order to (in larger bivalents; Figure 1c) reveal the molecular composition of C–bands, terminal/subterminal chiasmata. Apparently some slides were stained with base specific after C–banding, the karyotype was

Table 1. Species, collection localities, number of specimens, and host plants of six Philaenus species studied.

*Noteworthy that males of every species have 12 drop–shaped follicles per testis. However, in separate males the number of follicles varied from 11 to 13 in different testes. Journal of Insect Science | www.insectscience.org 4 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. characterized by a small amount of constitutive heterochromatin (C– Philaenus signatus: 2n = 24 (22 + neo–XY) heterochromatin) (Figures 1c, 1d). At In spermatogonial metaphases, 24 diakinesis and metaphase I (MI), small chromosomes were observed, among which subtelomeric C–bands were visible on larger were the very long X and five medium–sized bivalents and on the X (Figures 1c, 1d). In the ones (pairs AA1 and AA2, and the Y). All silver–stained mitotic (Figure 1e) and meiotic remaining chromosomes (pairs AA3-AA11) (Figure 1f) nuclei, masses of argyrophylic gradually decreased in size (Figures 3a-b). material (indicative of NORs) were more The sex determination system is suggested to often revealed on the largest (AA1) and on one be of a neo–XY type. The neo–X was much of the middle–sized autosomal pairs. The longer than AA1 and three times longer than CMA3 treatment revealed GC–rich regions the neo–Y. The neo–X chromosome was (probably corresponding to NORs) only on visible in the interphase nuclei as a long AA1 (Figure 1g). heteropycnotic body (Figure 3d). At diakinesis, there were 11 autosomal bivalents Philaenus loukasi: 2n = 20 (18 + neo–XY) with 1-2 chiasmata each, and an XY sex The male mitotic complement was composed bivalent, which was very large and highly of 20 chromosomes, including: 18 autosomes, heteromorphic. The neo–X (the original X + which more or less gradually decrease in size; fused autosome) and neo–Y (the other the Y chromosome, close in size to the homologue of the fused autosome) medium–sized autosomal pairs; and the X chromosomes were connected by a terminal chromosome, which was markedly longer chiasma. A part of the neo–X was than the AA1; the X was approximately three heteropycnotic at this stage, and the chiasma times longer than the Y (Figures 2a, 2b). This in the sex bivalent was always formed at a suggests a neo–XY sex determination. Silver point opposite to this part (Figures 3e-g). As impregnation showed that NOR sites were revealed by C–banding, the species displayed located on autosomes; however, we were a fairly large amount of heterochromatin, unable to detect the number of NOR–bearing located as prominent bands on one telomere autosomes (one or two pairs) at the mitotic (but more often on both telomeres) of every prometaphase (Figure 2c). At diakinesis, 10 chromosome (Figures 3c, 3f). Silver bivalents were observed (Figures 2d, 2e). All impregnation disclosed four argyrophilic areas the bivalents, including the heteromorphic XY in mitotic prometaphase nuclei (Figure 3h). bivalent, displayed one terminal chiasma each, Argyrophilic material was connected to except for AA1, which sometimes showed two chromosomes other than the neo–X (this latter or even three chiasmata in different locations is easily identified at this stage), suggesting (Figures 2d-2f). The marked heteromorphism the presence of two pairs of NOR–bearing of the XY pair and the presence of chiasma autosomes, though these failed to be were indicative of the neo–XY type. C– identified. banding induced prominent C–positive bands, both terminal and interstitial, in almost every Philaenus tarifa: 2n = 24 (22 + neo–XY) bivalent (Figure 2e). Male mitotic prometaphase showed 24 chromosomes including a very long X and The ”signatus“ group five medium–sized chromosomes (pairs AA1 Published data: absent. and AA2, and the Y). All remaining

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autosomes (pairs AA3-AA11) gradually were connected by a terminal chiasma decreased in size. The sex determination (Figures 5c, 5d). A part of the neo–X (the system was of the neo–XY type (as in P. original X) was heteropycnotic at this stage. signatus). However, the size difference Noteworthy was the chiasma, which was between the neo–X and AA1, as well as always formed in the sex bivalent at the point between the neo–X and the neo–Y, was not as opposite to the heteropycnotic part of the neo– marked as that in P. signatus. The neo–Y X. C–heterochromatin was visible as small but represented about 70% of the neo–X prominent terminal bands in the majority of chromosome length (Figures 4a, 4b). At bivalents (Figures 5c, 5d). In the diakinesis, there were 11 autosomal bivalents heteropycnotic part of the X chromosome, one with 1-2 chiasmata each, and the neo–XY sex telomere was marked with a large C–band bivalent, which was very large and (Figure 5d). heteromorphic. Neo–X and neo–Y chromosomes were connected by a terminal Philaenus italosignus: 2n = 23 (20 + neo– chiasma. A part of the neo–X (the original X) neo–X1X2Y) was heteropycnotic at this stage, and the Spermatogonial metaphase showed 23 chiasma in the sex bivalent was always chromosomes including 20 autosomes and formed at the point opposite to this part three sex chromosomes (Figures 6a-d). Based (Figure 4c, d). C–banding induced dot–like, on meiotic stages, sex chromosomes were faintly discernible bands on several bivalents, identified as X1, X2, and Y, and the sex indicative of a small amount of constitutive determination system of this species is heterochromatin in the complement (Figure suggested to be of a neo–neo–XY type. The 4d). Silver impregnation showed that two autosomes decreased in size from large to autosomal bivalents had NORs; however, small. Sex chromosomes were different in these bivalents failed to be identified (Figure size, with X1 and Y being the longest 4e). chromosomes of the set, and X2 was somewhat smaller than AA1. The X1 was Philaenus maghresignus: 2n = 24 (22 + neo– about twice as long as X2, and the latter was XY) about 1.5 times smaller than the Y. After Male mitotic prometaphase revealed 24 standard staining, two pairs only (AA1 and chromosomes including the very long X and AA10) could be easily distinguished among five medium–sized chromosomes (pairs AA1 autosomes; the remaining autosomes were of and AA2 and the Y). All remaining autosomes similar size and could be arranged in pairs (pairs AA3-AA11) showed a size gradient from only arbitrarily (Figure 6b). This species large to small (Figures 5a, 5b). The sex displayed a great deal of C–heterochromatin determination system is suggested to be of a (Figures 6c-g). Figures 6c and 6d (karyogram) neo–XY type. The neo–X was much longer show a C–banded early mitotic prometaphase, than the AA1 and approximately two times in which separate chromosomal pairs could be longer than the neo–Y, the latter being the identified based on combined analysis of sizes second largest chromosome of the set. In and C–banding patterns. The pair AA1 and diplotene/diakinesis, 11 autosomal bivalents each sex chromosome had numerous with one or two chiasmata and the very large prominent bands, both terminal and and heteromorphic neo–XY bivalent were interstitial. Each member of AA2 showed observed. Neo–X and neo–Y chromosomes terminal bands at ends, a subterminal band at

Journal of Insect Science | www.insectscience.org 6 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. one end, and a double band in the middle. The 2n = 22 + neo–XY (P. signatus, P. AA3 chromosomes displayed bands at ends, maghresignus, and P. tarifa), and 2n = 20 + one terminal and the other subterminal. The X1X2Y (P. italosignus). Thus, the three values remaining autosomes had three bands each, of autosome number (18, 20, 22) and the three two terminal and one interstitial. types of sex determination (X0, neo–XY, and X1X2Y) appear characteristic of as few as At diplotene/diakinesis, 10 autosomal eight Philaenus species. Such karyotypic bivalents and a trivalent of sex chromosomes diversity at the generic level is rare in were detected (Figures 6e-g). Bivalents Auchenorrhyncha (Kirillova 1986, 1988). generally had 1-2 chiasmata each; however, in larger bivalents three chiasmata sometimes Conventional opinion holds that holokinetic formed (Figure 6h). In the sex trivalent, the chromosomes contain a small amount of X1, X2, and Y were joined end–to–end constitutive heterochromatin, which is (probably by chiasmata) in the order: X1, Y, generally located on chromosome ends or in X2. In some diakinetic nuclei, sex their vicinities (Blackman 1987). However, chromosomes appeared as univalents (Figure Philaenus species showed both terminal and 6g). As expected, two daughter metaphase II interstitial C–bands on autosomes and sex (MII) cells formed with n = 10 + X1X2 and 10 chromosomes. The greatest amount of C– + Y, respectively (Figure 6i). Silver heterochromatin is found in P. italosignus,in impregnation of mitotic nuclei revealed a which prominent C–bands are numerous and variable number of chromosomes carrying variably located along the complement, argyrophilic material even in one male allowing the majority of homologous (Figures 6j, 6k). NOR–bearing chromosomes chromosomes to be identified. Thus, the were unable to be identified at this stage. present data agree with recent evidence from However, observation of silver–stained holokinetic animals (Kuznetsova et al. 1997, diplotene (Figure 6h) and MIs subjected to the 2009b; Maryańska-Nadachowska 1999; GC–specific fluorochrome CMA3 treatment Grozeva and Nokkala 2001; Golub et al. (Figure 6i) definitively showed the presence 2004; Angus et al. 2004; Perez et al. 2005; of an NOR on the sex trivalent. Franco et al. 2006; Bressa et al. 2005, 2008) and plants (Collet and Westerman 1984; Discussion Sheikh and Kondo 1995; Vanzela and Guerra 2000; Guerra and Garcia 2004), suggesting Characteristics of holokinetic chromosomes that the amount and distribution of C– of Philaenus heterochromatin in holokinetic chromosomes The six Mediterranean species in this study, are quite variable, as they are in monocentric together with data concerning the worldwide chromosomes (Guerra et al. 2010; Kuznetsova P. spumarius and the Mediterranean P. et al. 2010). arslani published earlier (Kuznetsova et al. 2003; Maryańska-Nadachowska et al. 2008), The low number of chiasmata (estimated to be represent an exhaustive taxonomic sampling 1-2 from cytogenetic analyses) is a common effort for Philaenus. In the genus Philaenus, pattern in the Auchenorrhyncha (Halkka four karyotype patterns have been described: 1964). It is suggested that this pattern 2n = 22 + X0 (P. tesselatus and P spumarius), represents one of the peculiar features of 2n = 18 + neo–XY (P. loukasi and P. arslani), holokinetic bivalents, and as such is

Journal of Insect Science | www.insectscience.org 7 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. irrespective of the group as a whole (Nokkala et al. 2004). This assessment was founded on In the Auchenorrhyncha, only single species the detailed observations of the behavior of a belonging to various genera are characterized three–chiasmatic bivalent during meiosis of by the neo–XY system. This type of sex Baeopelma foersteri. This bivalent was shown determination usually arises from the X0 to be incapable of completing anaphase I system as a result of fusion between the because of its inability to resolve the chiasma original X and an autosome, the homologue of located in its center. The authors attributed the latter playing the role of the neo–Y and this to a specific condensation process resulting in a lower number of autosomal pairs inherent in holokinetic chromosomes. (Halkka 1959; Blackman 1995; Maryańska- Inevitable elimination of cells with multiple Nadachowska et al. 2006; Kuznetsova et al. chiasmata thus creates strong selection against 2009a, 2010). The exceptions are species from the formation of more than two chiasmata in the tribe Almanini (Dictyopharidae) belonging holokinetic bivalents. In our study, three and to 11 genera characterized by the neo–XY even four chiasmata were observed in larger system (Kuznetsova 1986; Kuznetsova et al. bivalents of P. loukasi and P. italosignus, and 2009a). previously in larger bivalents of P. spumarius (Kuznetsova et al. 2003) and P. arslani (Maryańska-Nadachowska et al. 2008), as Within the genus Philaenus, a neo–XY system well as in several other auchenorrhynchan is found in five species: P. loukasi, P. arslani, species (Tian and Yuan 1997; Kuznetsova et P. signatus, P. maghresignus, and P. tarifa. al. 2009a, 2009b, 2010). Contrary to While these species share the same sex expectations, meiotic disturbances have never determination, they have a different number of been observed in any of these cases, autosomes: 18 in the first two (both from the suggesting that the question of the number of “spumarius” group) and 22 in the three others chiasmata that can be successfully resolved in (from the “signatus” group). However, the 2n a holokinetic bivalent is still unresolved. = 20 + XY chromosomal set was not found in the genus. This can be attributed either to the Sex chromosome evolution in Philaenus extinction of species with this karyotype or, XX/X0 sex determination is of common alternatively, to the existence of still occurrence in Auchenorrhyncha (Halkka unrecognized Philaenus species. 1959; Emeljanov and Kirillova 1990, 1992; Kuznetsova et al. 1998, 2009a, 2010; Clearly, the karyotype of 2n = 22 + neo–XY Kirillova 1986, 1988; Maryańska- Nadachowska et al. 2006), and almost (inherent in P. signatus, P. maghresignus, and ) could not have originated directly certainly represents the ancestral type of sex P. tarifa from 2n = 22 + X0. There are at least two determination in this group (Kuznetsova possible explanations for the origin of this 1986) and in as a whole (Blackman karyotype. It could gradually evolve through 1995). It is very probable that ancestral an autosomal fission resulting in 2n = 24 + X0 karyotype of the genus Philaenus is 2n = 24 + (with subsequent extinction of species with X0 (Figure 7), or even 2n = 28 + X0 as in this karyotype) followed by an X–autosome Neophilaenus lineatus, a representative of the fusion resulting in 2n = 22 + neo–XY. The most closely related genus (Halkka 1964; other possibility is that the closest ancestor of Kirillova 1986). the entire genus had already possessed 2n =

Journal of Insect Science | www.insectscience.org 8 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. 24 + X0, and this hypothesis appears more fusions. However, in these species, the plausible (see Figure 7). Philaenus italosignus multiple systems occur in terms of sex (2n = 20 + X1X2Y) demonstrates the next step chromosome polymorphism. Thus, the neo– of karyotype evolution within the “signatus” XnY system of P. italosignus is the first group. It is not difficult to explain the origin reported case of an autosomally–derived of the 2n = 20 + X1X2Y system of this species multiple sex chromosome system fixed at the based on the way in which sex chromosomes species level within Auchenorrhyncha. associate at metaphase I of spermatogenesis (Figure 6e). In this case, the original neo–Y There is no doubt that neo–XY systems have chromosome (in a complement with 2n = 22 + evolved several times independently in the neo–XY) probably fused with the homologue genus Philaenus, as evidenced by the of an autosomal pair resulting in the neo–neo– available results. The neo–X and neo–Y Y chromosome, the other homologue chromosomes differ in size among species. appeared as the X2 chromosome. The fused The observed size differences confirmed that autosomal pair was most probably that one the neo–sex chromosomes appeared as a result bearing an NOR, since the X2 in P. italosignus of fusion between ancestral X and one of the carries an NOR region. Interestingly, the neo– autosomes. The X is the longest chromosome neo–Y (presumably including the homologue in each species. In P. signatus and P. loukasi of the X2) did not carry an NOR in either (from the “signatus” and “spumarius” groups, silver impregnated or in CMA3 treated respectively), the neo–X is approximately preparations. Within the Hemiptera, multiple three times as long as the neo–Y, in P. tarifa sex chromosomes are very common in the and P. maghresignus (“signatus” group) it is (Ueshima 1979) and occasionally only 1.5 times as long as neo–Y, and in P. occur in , namely in arslani (“spumarius” group) neo–X and neo– Aphidoidea (Hales 1989) and Coccoidea Y chromosomes are approximately the same (Hughes-Schrader 1948). With a single size. Thus, at least four independent exception recently described in the translocation events have occurred in the Heteroptera (Jacobs 2004), all multiple sex evolution of the neo–XY in Philaenus species chromosome systems so far reported in these (Figure 7). These translocations clearly groups had arisen simply by fission of the involved various autosomes of the ancestral original sex chromosomes into two or more chromosomal complement and resulted in the pairs. Multiple X or Y chromosomes of this rise of the neo–X, which invariably exceeds kind are characteristically smaller than the the largest autosomes in size. In contrast, in original ones, and there is no accompanying the karyotype shared by P. spumarius and P. reduction in the number of autosomes tesselatus (2n = 22 + X0), the X chromosome (Blackman 1995). Until now only three is noticeably smaller than the largest hemipteran species, Cacopsylla sorbi and C. autosomes, supporting the occurrence of X– mali from (Sternorrhyncha) autosome fusions in the evolution of (Grozeva and Maryańska-Nadachowska 1995) Philaenus. Moreover, based on the and one Austragalloides sp. from the comparative size of neo–X and neo–Y auchenorrhynchan family Cicadellidae chromosomes of the recent neo–XY species, (Whitten 1968), were reported to show we can preliminarily infer the particular fused multiple sex chromosome systems of the autosomes in each translocation event. For X1X2Y type originated by X–autosome example, in P. arslani (relatively small size

Journal of Insect Science | www.insectscience.org 9 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. difference between sex chromosomes) fusion trees, the monophyly of the genus was well could have encompassed one of the larger (but supported, being congruent with not the largest, see below) autosomal pairs. In morphological, ecological, and chromosomal P. tarifa and P. maghresignus (larger data. The results confirmed the existence of difference in size between sex chromosomes) three lineages within the genus. The first it was probably one of the middle–sized pairs, lineage included P. maghresignus; the second whereas in P. signatus and P. loukasi (largest P. tarifa, P. italosignus, and P. signatus; and size difference between sex chromosomes) the the third P. loukasi, P. arslani, P. spumarius, fusion involved one of the smaller pairs. In all and P. tesselatus. The last clade, in turn, cases (data are available only for P. arslani, P. appeared to be subdivided into two further loukasi, P. tarifa, and P. signatus; Maryańska- groups, one with P. loukasi and P. arslani and Nadachowska et al. 2008; this paper) the the other with P. spumarius and P. tesselatus. fused autosome was not the NOR–bearing Taken together, molecular analyses and one. This inference is based on the evidence from host–plant relationships and observation that NORs reside on autosomes in distribution patterns suggested that the both X0 and neo–XY species. In two X0 ancestral Philaenus species may have used species (P. spumarius and P. tesselatus) these Asphodelus aestivus (the Mediterranean lily) autosomes are the largest and one of the as a host–plant, and this initial association medium–sized (probably 6th) pairs remains characteristic of P. maghresignus, P. (Kuznetsova et al. 2003; this paper). In all of tarifa, P. italosignus, and P. signatus. the neo–XY species, the sex determination Philaenus loukasi and P. arslani inhabit high system seems to be of quite recent origin, altitudes feeding mostly on xerophilic plants, since the derived neo–Y chromosome is still and this host–plant association is treated as a homologous with the autosomal part of the synapomorphic trait for these species, whereas neo–X, as evidenced by their chiasmatic the adaptation to a wide range of host–plants connections in meiotic prophases. is a synapomorphy of P. tesselatus and P. spumarius. Regarding P. spumarius, this has Conclusions been the leading factor promoting its postglacial expansion into temperate regions Numerous studies, each with a radically of Eurasia and other regions of the Palaearctic different approach, have been performed on (Maryańska-Nadachowska et al. 2010). the genus Philaenus to date. They have discussed a wide range of aspects such as We use the presumptions mentioned above to morphology, distribution, host plant formulate the hypothesis that the changes of associations (Drosopoulos and Remane 2000; host–plant associations of Philaenus species Drosopoulos 2003), molecular variation would have to be accompanied by karyotype (Maryańska-Nadachowska et al. 2010) and rearrangements (Figure 7). The lily species A. cytogenetic characters (Kuznetsova et al. aestivus was suggested to come from middle 2003; Maryańska-Nadachowska et al. 2008; Asia (Ayyad and Hilmy 1974; Rhizopoulou et this paper). For the reconstruction of al. 1997), and the nearest ancestor of phylogenetic relationships within Philaenus, Philaenus could have followed this plant into mitochondrial and nuclear genetic markers the Mediterranean region. This ancestor was were used (Maryańska-Nadachowska et al. likely to have had a karyotype of 2n = 24 + 2010). Based on the topologies of all obtained X0. An ancestral X0 sex determination system

Journal of Insect Science | www.insectscience.org 10 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. would allow the evolution of all other Genetic Diversity” and “Origin of the Philaenus karyotypes. There is a variety of Biosphere and Evolution of Geo–Biological ways in which this ancestral karyotype could Systems”. be transformed into other karyotypes. We suggest that X–autosome fusions (at least two, References each involving different autosomal pairs) first resulted in the rise of two different karyotypes Angus RB, Kemeny CK, Wood EL. 2004. The of 2n = 22 + neo–XY (that of P. C–banded karyotypes of the four British maghresignus and P. tarifa, on the one hand, species of Notonecta L. (Heteroptera: and that of P. signatus, on the other) followed ). Hereditas 140: 134-138. then by one further fusion between neo–Y and one more autosomal pair resulting in the Ayyad MA, Hilmy SH. 1974. The distribution karyotype of 2n = 20 + X1X2Y (inherent in P. of Asphodelus microcarpus and associated italosignus). The above rearrangements are species on the west Mediterranean coast of suggested to have occurred on A. aestivus. Egypt. Ecology 55: 511-524. The following rearrangements took place during the chromosome evolution of P. Blackman RL. 1987. Reproduction, loukasi and P. arslani: a fusion between two cytogenetics and development. In: Minks AK, autosomal pairs (producing 2n = 20 + X0 of Harrewijn P, Editors. , their Biology, an unknown species) followed by two Natural Enemies and Control. World Crop independent fusions, one between the original Pests. Vol. 2A. pp. 164-195. Elsevier. X and a small autosomal pair (giving the karyotype of P. loukasi) and the other Blackman RL. 1995. Sex determination in between the original X and a larger autosomal insects. In: Leather SR, Hardie J, Editors. pair (giving the karyotype of P. arslani). Insect Repreduction. pp. 57-94. CRC Press. Finally, P. tesselatus and P. spumarius possibly originated from the ancestor by Bressa MJ, Larramendy ML, Papeschi AG. means of fusions of two autosomal pairs. It 2005. Heterochromatin characterization in thus seems likely that at least eight autosome five species of Heteroptera. Genetica 124: and autosome–sex chromosome fusions have 307-317. occurred during the evolution of Philaenus. Bressa MJ, Franco MJ, Toscani MA, Papeschi Acknowledgements AG. 2008. Heterochromatin heteromorphism in Holhymenia rubiginosa (Heteroptera: Thanks are due to V. D’Urso and A. Coreidae). European Journal of Entomology Nadachowski for their help in collecting 105: 65-72. material. This research was partly supported by the Ministry of Science and Higher Collet C, Westerman M. 1984. Interspersed Education, Poland, grants no. 30301731/0639 distribution patterns of C–bands and satellite and N N303 579 839. VGK was also DNA in the holocentric chromosomes of supported by the Russian Foundation for Luzula flaccida (Juncaceae). Genetica 63: Basic Research (grant no.11-04-00734) and 175-179. programs of the Presidium of the Russian Academy of Sciences “Gene Pools and

Journal of Insect Science | www.insectscience.org 11 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. Drosopoulos S. 2003. New data on the nature chromosomes in Coreidae (Heteroptera). and origin of colour polymorphism in the European Journal of Entomology 103: 9-16. spittlebug genus Philaenus (Hemiptera: Aphrophoridae). Annales de la Societe Golub NV, Nokkala S, Kuznetsova VG. 2004. Entomologique de France 39: 31-42. Holocentric chromosomes of psocids (Insecta, Psocoptera) analysed by C–banding, silver Drosopoulos S, Quartau JA. 2002. The impregnation and sequence specific spittlebug Philaenus tesselatus Melichar, 1899 fluorochromes CMA3 and DAPI. Folia (Hemiptera, Auchenorrhyncha, ) is Biologica (Kraków) 52: 143-149. a distinct species. Zootaxa 68: 1-8. Grozeva S, Nokkala S. 2001. Chromosome Drosopoulos S, Remane R. 2000. numbers, sex determining systems, and Biogeographic studies on the spittlebug patterns of the C–heterochromatin distribution species group Philaenus signatus with the in 13 species de Lace Bugs (Heteroptera, description of two new allopatric species. Tingidae). Folia Biologica (Kraków) 49: 29- Annales de la Societe Entomologique de 41. France 36: 269-277. Grozeva S, Maryańska-Nadachowska A. 1995. Drosopoulos S, Maryańska-Nadachowska A, Meiosis of two species of Cacopsylla with Kuznetsova VG. 2010. The Mediterranean: polymorphic sex chromosomes in males area of origin of polymorphism and speciation (Homoptera, Psyllidae). Folia Biologica in the spittlebug Philaenus (Hemiptera, (Kraków) 43: 93-98. Aphrophoridae). Zoosystematics and Evolution 86: 125-128. Guerra M, Garcia MA. 2004: Heterochromatin and rDNA sites distribution Emeljanov AF, Kirillova VI. 1990. Trends in the holocentic chromosomes of Cuscuta and types of karyotype evolution in Cicadina approximata Bab. (Convolvulaceae). Genome (Homoptera). I. Karyotypic peculiarities and 47: 134-140. evolutionary changes in the karyotypes of of superfamily Cicadelloidea. Guerra M, Cabral G, Cuacos M, González- Entomological Review 69: 62-80. García M, González-Sánchez M, Vega J, Puertas MJ. 2010. Neocentrics and Emeljanov AF, Kirillova VI. 1992. Trends holokinetics (holocentrics): chromosomes out and types of karyotype evolution in Cicadina of the centromeric rules. Cytogenetic Genome (Homoptera). II. Peculiarities and Research 129: 82-96. evolutionary changes of the karyotypes in the superfamilies Cercopoidea, Cicadoidea, Hales DF. 1989. The chromosome Fulgoroidea and in the Cicadina as a whole. Schoutedenia lutea (Homoptera, , Entomological Review 71: 59-81. Greenideinae), with an account of meiosis in male. Chromosoma 98: 295-300. Franco MJ, Bressa MJ, Papeschi AG. 2006. Karyotype and male meiosis in Spartocera Halkka O. 1959. Chromosome studies on the batatas and meiotic behaviour of multiple sex Hemiptera, Homoptera, Auchenorrhyncha.

Journal of Insect Science | www.insectscience.org 12 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. Annales Academie Scientiarum Fennicae A IV Kuznetsova VG, Maryańska-Nadachowska A, 43: 1-71. Nokkala S. 1997. C banded karyotype of psyllid species Aphalara caltae (L.) Halkka O. 1964. Recombination in six (Psylloidea, Homoptera, Insecta). Cytologia homopterous families. Evolution 18: 81-88. 62: 237-239.

Halkka O, Halkka L. 1990. Population Kuznetsova VG, Maryańska-Nadachowska A, genetics of the polymorphic spittlebug, Yang Ch-T, O'Brien L. 1998. Karyotypes and Philaenus spumarius (L.). Evolutionary structure of testes in cicads belonging to Biology 24: 149-191. unsufficently known families of Fulgoroidea (Auchenorrhyncha, Homoptera). Folia Halkka O, Raatikainen MI, Vilbaste J. 1967. Biologica (Kraków) 46: 23-40. Ecology and ecological genetics of Philaenus spumarius (L.) (Homoptera). Annales Kuznetsova VG, Maryańska-Nadachowska A, Zoologici Fennici 4: 1-18. Nokkala S. 2003. A new approach to the Auchenorrhyncha (Hemiptera, Insecta) Hughes-Schrader S. 1948. Cytology of cytogenetics: Chromosomes of the meadow coccids (Coccoidea – Homoptera). Advances spittlebug Philaenus spumarius (L.) examined in Genetics 2: 127-203. using various chromosome banding techniques. Folia Biologica (Kraków), 51: 33- Jacobs HD. 2004. The evolution of a neo– 40. XY1Y2 sex chromosome system by autosome– sex chromosome fusion in Dundocoris Kuznetsova VG, Maryańska-Nadachowska A, nodulicarinus Jacobs (Heteroptera: Aradidae: Emeljanov A F. 2009a. A contribution to the Carventinae). Chromosome Research 12: 175- karyosystematics of the families 191. Dictyopharidae and Fulgoridae (Hemiptera: Auchenorrhyncha). European Journal of Kirillova VI. 1986. Chromosome numbers of Entomology 106: 159-170. world Homoptera Auchenorrhyncha. I. Fulgoroidea, Cercopoidea, and Cicadoidea. Kuznetsova VG, Maryańska-Nadachowska A, Entomological Review 65: 34-47. Nokkala S. 2009b. Karyotype characterization of planthopper species Hysteropterum Kirillova VI. 1988. Chromosome numbers of albaceticum Dlabola, 1983 and Agalmatium world Homoptera Auchenorrhyncha. II. bilobum (Fieber, 1877) (Homoptera: Cicadelloidea. Entomological Review 67: 80- Auchenorrhyncha: Issidae) using AgNOR-, C- 107. and DAPI/CMA3 –banding techniques. Comparative Cytogenetics 3: 111-123. Kuznetsova VG. 1986. Phylogenetical analysis of the chromosome variability and Kuznetsova VG, Maryańska-Nadachowska A, karyosystematics of Cicadina of the family Gnezdilov VM. 2010. Meiotic karyotypes and Dictyopharidae (Homoptera, testis structure of 14 species of the Auchenorrhyncha). Entomological Review 65: tribe Issini (Hemiptera: 88-106. Fulgoroidea, Issidae). European Journal of Entomology, 107: 465-480.

Journal of Insect Science | www.insectscience.org 13 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. Mediterranean species of Philaenus Mandrioli M, Borsatti F. 2007. Analysis of (Hemiptera: Auchenorrhyncha: heterochromatic epigenic markers in the Aphrophoridae) using mitochondrial and holocentric chromosomes of the nuclear DNA sequences. Systematic Acyrthosiphon pisum. Chromosome Research, Entomology, 35: 318-328. 15: 1015- 1022. Nast J. 1972. Palaearctic Auchenorrhyncha Mandrioli M, Manicardi GC, Marec F. 2003. (Homoptera) an Annotated Check List. PWN. Cytogenetic and molecular characterization of the MBSAT1 satelite DNA in holokinetic Noda H, Tatewaki R. 1990. Re–examination of chromosomes of the cabbage moth, Mamestra chromosomes of three species of rice brassicae (Lepidoptera). Chromosome planthoppers (Homoptera: ). Research 11: 51-56. Applied Entomology and Zoology 25: 538- 540. Marec F, Sahara K, Traut W. 2010. Rise and fall of the W chromosome in Lepidoptera. In: Nokkala S, Kuznetsova VG, Maryańska- Goldsmith MR, Marec F, Editors. Molecular Nadachowska A, Nokkala C. 2004. Biology and Genetics of the Lepidoptera, Holocentric chromosomes in meiosis. I. volume 3. pp. 49-63. CRC Press. Restriction of the number of chiasmata in bivalents. Chromosome Research 12: 733- Maryańska-Nadachowska A. 1999. B– 739. chromosome polymorphism in Rhinocola aceris (Psylloidea, Homoptera). Folia Perepelov EA, Bugrov AG, Maryańska- Biologica (Kraków) 47: 115-121. Nadachowska A. 2002. Constitutive heterohromatin in karyotypes of two Maryańska-Nadachowska A, Kuznetsova VG, Cicadidae species from Japan (Cicadoidea, Gnezdilov VM, Drosopoulos S. 2006. Hemoptera). Folia Biologica (Kraków) 50: Variability of the karyotypes, testes and 217-219. ovaries of planthoppers of the families Issidae, Caliscelidae and (Hemiptera: Pérez R, Rufas J, Suja J, Panzera F. 2005. Fulgoroidea). European Journal of Cytogenetic analysis of experimental hybrids Entomology 103: 505-513. in species of Triatominae (Hemiptera- Reduviidae). Genetica 125: 261-270. Maryańska-Nadachowska A, Kuznetsova VG, Abdul-Nour H. 2008. A chromosomal study Rhizopoulou S, Pantis JD, Triantafylli E, on the meadow spittlebug Philaenus arslani Vokou D. 1997. Ecophysiological adaptation Abdul-Nour & Lahoud, 1995 (Hemiptera, of Asphodelus aestivus to Mediterranean Auchenorrhyncha, Aphrophoridae) from climate periodicity: water relations and Lebanon. European Journal of Entomology energetic status. Ecography 20: 626-633. 105: 205-210. Sheikh AS, Kond K. 1995. Differential Maryańska-Nadachowska A, Drosopoulos S, staining with orcein, Giemsa, CMA and DAPI Lachowska D, Kajtoch Ł, Kuznetsova VG. for comparative chromosome study of 12 2010. Molecular phylogeny of the

Journal of Insect Science | www.insectscience.org 14 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al. species of Australian Drosera (Droseraceae). Zając MA, Wilson MC. 1984. The effect of American Journal of Botany 82: 1278-1286 nymphal feeding by the meadow spittlebug, Philaenus spumarius (L.) on strawberry yield Stewart AJA, Lees DR. 1996. The and quality. Crop Protection 3: 167-175. colour/pattern polymorphism of Philaenus spumarius (L.) (Homoptera: Cercopidae). Philosophical Transactions of the Royal Society of London B: Biological Sciences 351: 69-89.

Tian R, Yuan F. 1997. Chromosomes in twenty–five species of Chinese membracids (Homoptera: Membracidae). Entomologica Sinica 4: 150-158.

Ueshima N. 1979. Hemiptera II: Heteroptera. In: John B, Editor. Cytogenetics, Vol. 3: Insecta 6. Gebrüder Borntraeger.

Vanzela ALL, Guerra M. 2000. Heterochromatin differentiation in holocentric chromosomes of Rhynchospora (Cyperaceae). Genetics and Molecular Biology 23: 453-456.

Wagner W. 1959. Zoologische Studien in Figure 1a-g. Mitotic and meiotic chromosomes of Philaenus tesselatus. (a) mitotic prometaphase; (b) karyogram of mitotic Westgriechenland. IX. Tail Homoptera. prometaphase; (c) C–banded diakinesis (asterisks indicate two Sitzungsberichte der Sterreichisen Akademie chiasmata in the largest bivalent); (d) C–banded metaphase I; (e) Ag–stained diplotene (arrows indicate NORs); (f) incomplete der Wissenschaften, Mathematisch– mitotic prometaphase with NORs–bearing chromosomes Naturwissenschaftliche Klasse, Abteilung I (arrows); (g) CMA3–treated metaphase I with one positive signal on the largest bivalent (asterisk). Bar = 10 μm. Scale bar on (a) 168: 583-605. refers to (a), (b), and (f); scale bar on (c) refers to (c), (d), and (e). High quality figures are available online. White MJD. 1978. Modes of speciation. Freeman W. H. & Company, San Francisco.

Whitten MJ. 1968. An unusual chromosome system in a leafhopper (Homoptera: Auchenorrhyncha). Chromosoma 24: 37-41.

Wolf KW. 1996. The structure of condensed chromosomes in mitosis and meiosis of Figure 2a-f. Mitotic and meiotic chromosomes of Philaenus loukasi. (a) mitotic metaphase; (b) karyogram of mitotic metaphase; insects. International Journal of Insect (c) Ag–stained mitotic metaphase (arrows indicate two clusters of Morphology and Embryology 25: 37-62. argyrophilic material); (d) diakinesis; (e) C–banded diakinesis, in the larger autosomal bivalent two terminal chiasmata are visible; in small frame the largest autosomal bivalent with terminal and interstitial chiasmata from another plate is added; (f) diplotene, bivalent with three chiasmata (asterisks). Bar = 10 μm. Scale bar on (a) refers to (a) and (b); scale bar on (e) refers to (d), (e), and (f). High quality figures are available online. Journal of Insect Science | www.insectscience.org 15 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al.

Figure 4a-e. Mitotic and meiotic chromosomes of Philaenus tarifa. (a) mitotic metaphase; (b) karyogram of mitotic metaphase; (c) diakinesis, bivalents with one or two chiasmata, asterisks indicate two chiasmata in a large bivalent; (d) C–banded diakinesis; (e) Ag–stained diplotene, arrows indicate two bivalents bearing NORs. Bar = 10 μm. Scale bar on (a) refers to (a) and (b); scale Figure 3a-h. Mitotic and meiotic chromosomes of Philaenus bar on (c) refers to (c), (d), and (e). High quality figures are signatus. (a) mitotic metaphase; (b) karyogram of mitotic available online. metaphase; (c) C–banded mitotic prometaphase; (d) interphase, note the neo–X chromosome as a long heteropycnotic body; (e) diakinesis, bivalents are connected by terminal or interstitial chiasmata, asterik indicate two chiasmata in the largest bivalent; (f) C–banded diakinesis; (g) metaphase I; (h) silver–stained mitotic prometaphase (arrows indicate four clusters of argyrophilic material connected to chromosomes other than sex chromosomes). Bar = 10 μm. Scale bar on (c) refers to (a), (b), and (c); scale bar on (e) refers to (d-h). High quality figures are available online.

Figure 6a-l. Mitotic and meiotic chromosomes of Philaenus italosignus. (a) mitotic metaphase; (b) karyogram of mitotic metaphase; (c) C–banded mitotic prometaphase; (d) C–banded karyogram of mitotic prometaphase; (e) and (f) C–banded diplotene/diakinesis, sex trivalent with chromosomes joint end– Figureto–end in 2a-f. order:Mitotic X1-Y-X and2; (g)meiotic C–banded chromosomes diakinesis, of sex Philaenus loukasichromosomes. (a) mitotic appear metaphase; as univalents (b) karyogram (outlined); of (h) mitotic Ag–stained metaphase; Figure 5a-d. Mitotic and meiotic chromosomes of Philaenus (c)diplotene, Ag–stained asterisk mitotic indicates metaphase three (arrowschiasmata indicate in one twobivalent; clusters of maghresignus. (a) mitotic metaphase; (b) karyogram of mitotic argyrophilicheadarrow indicates material); NOR; (d) diakinesis; (i) two daughter (e) C–banded metaphases diakinesis, II with in n metaphase; (c) C–banded diplotene; (d) C–banded diakinesis, one the= 10 larger + X1 Xautosomal2 and n = bivalent10 + Y, respectively;two terminal (j)chiasmata and (k) Ag–stainedare visible; in telomere of the neo–X is marked with a large block of smallmitotic frame prometaphases the largest autosomalwith four andbivalent two NORs,with terminal respectively and heterochromatin (arrow), asterisks indicate two chiasmata in large interstitial(arrows); (l) chiasmata CMA3–treated from another metaphase plate I iswith added; one (f)positive diplotene, signal autosomal bivalents. Bar = 10 μm. Scale bar on (a) refers to (a) bivalenton the sex–chromosome with three chiasmata trivalent (asterisks). (asterisk). Bar Bar= 10 = μ 10m. μScalem. Scale bar and (b); scale bar on (c) refers to (c) and (d). High quality figures onbar (a) on refers(d) refers to (a) to and (a-d); (b); scale scale bar bar on on (e) (e) refers refers to to (e-k). (d), (e), High and are available online. (f).quality High figures quality are figures available are availableonline. online. Journal of Insect Science | www.insectscience.org 16 Journal of Insect Science: Vol. 12 | Article 54 Maryańska-Nadachowska et al.

Figure 7. Presumable directions of chromosome rearrangements during karyotype evolution and changes of host plant preferences of Philaenus species. Karyotype alterations occurred several times independently in the genus. High quality figures are available online.

Journal of Insect Science | www.insectscience.org 17