Chromosome mapping of 28 S ribosomal genes in 11 species of Cassidinae ( Coleoptera : Chrysomelidae )

In this study, we examined for the fi rst time the distribution of the 28S ribosomal genes in beetles of the subfamily Cassidinae. More than 55% of the species in this subfamily have a similar karyotype, 2n = 16 + Xyp. For this work, we selected species belonging to the tribes Cassidini and Mesomphaliini, which have, respectively, the most conserved and diversifi ed karyotype characteristics within the Cassidinae. An analysis of 11 species revealed that rDNA sites on one pair of autosomes is the most frequent pattern, occurring in 10 species. This condition occurs in the seven genera examined and in species of both of the tribes, Cassidini and Mesomphaliini. Nevertheless, the differences in the locations of 28S rDNA were more pronounced in the tribe Cassidini and among species with similar karyotype characteristics. On the other hand, in Mesomphaliini, the increase in the diploid number was not accompanied by an increase in the number of ribosomal sites. Moreover, the comparison of the number and localization of major rDNA sites with the distribution of constitutive heterochromatin indicates that there is no direct correlation between the dispersion of constitutive heterochromatin and 28S rDNA genes in Cassidinae.


INTRODUCTION
The repetitive DNA sequences constitute a large fraction of the eukaryote genomes and include satellite, microsatellite and minisatellite regions, multigene families and transposable elements (Charlesworth et al., 1994).The major ribosomal gene is one of these multigene families and is formed by a transcription unit that encodes the 18S, 5.8S and 28S rRNA genes.In the genome, these genes occur as multiple copies organized in tandem, which are located in one or more nucleolar organizer regions (NORs) (Long & Dawid, 1980;Cabrero & Camacho, 2008;Nguyen et al., 2010).The rDNA is considered to be a useful chromosomal marker in cytogenetic studies because it can reveal synapomorphies and provide information about the mechanisms of chromosomal evolution in related groups of species (Bombarová et al., 2007;Cabrero & Camacho, 2008;Nguyen et al., 2010).
The location of the NORs is widely used in cytogenetic studies, which are mainly revealed by silver impregnation (Ag-NOR) (Howell & Black, 1980), because it is a simple and low cost technique (Rufas et al., 1982).However, the Ag-NOR technique stains only active rDNA sites.On the

MATERIALS AND METHODS
The species analyzed in this work and their respective collection localities are listed in Table 1.The voucher specimens were deposited in the entomological collection of the Museu Paraense Emilio Goeldi (MPEG, curator O.T. Silveira), Belém, state of Pará, Brazil.
Most species of Coleoptera have two rDNA clusters located on one autosomal pair.This pattern occurs in approximately 80% and 65% of the species of Adephaga and Polyphaga, respectively, including those with very divergent diploid numbers and/or sex chromosome systems, for example, 2n = 56 + X0, 2n = 18 + X 1 X 2 X 3 Y, 2n = 18 + Xy p , 2n = 14 + neoXY and 2n = 10 + XY (see revision in Schneider et al., 2007).Among the Polyphaga, most of the data on the distribution of rDNA is for the family Scarabaeidae, with more than 120 species characterized to date.The occurrence of two autosomal NORs is also widespread within this family, but closely related species do differ in the presence of a ribosomal cluster on more than one autosomal pair and/or on sex chromosomes (Colomba et al., 2006;Silva et al., 2009;Cabral-Mello et al., 2010, 2011a, b;Oliveira et al., 2010Oliveira et al., , 2012b)).
Within Cassidinae, only two species of Mesomphaliini are investigated regarding the location of Ag-NOR.In Chelymorpha varians (Blanchard, 1851) (2n = 20 + Xy p ), the NOR is coincident with the secondary constriction on the short arm of the 5th pair of autosomes.In Botanochara angulata (Germar, 1824) (2n = 51 = 48 + X p neoXneoY p ), NORs can be seen on some autosomal chromosomes, which are not identifi ed (Yadav & Pillai, 1975;Postiglioni et al., 1990).In this study, we mapped the 28S rDNA gene in 11 species of the tribes Cassidini and Mesomphaliini, with the aim of understanding the role of the variation in the number and location of this gene in the evolution of chromosomes in these groups.

RESULTS
The cytogenetic analysis of fi ve Cassidini species revealed the following diploid numbers and sex chromosome systems: 2n = 38 + Xy p in Agroiconota inedita, 2n = 20 + Xy p in Cha.immaculata and Cha.sexpunctata, and 2n = 16 + Xy p in Deloyala cruciata and Microctenochira optata.In the Mesomphaliini, it revealed 2n = 20 + Xy p in Che.cribraria and 2n = 40 + Xy p in Paraselenis fl ava.In addition, we examined for the fi rst time, the karyotypes of two species of Cassidini: M. gnata with 2n = 18 and M. stigmatica with 2n = 20 + Xy p , and two species of Mesomphaliini: Cyr.infl ata with 2n = 20 + Xy p and Cyr.cyanea with 2n = 38 + Xy p .All these species have biarmed chromosomes.Due to the absence of diplotene cells, it was not possible to determine the sex chromosome system of M. gnata.
In the species of Cassidini, three patterns in the distribution of the ribosomal clusters were revealed by FISH (Fig. 1): terminal/subterminal region of one autosomal pair in A. inedita, Cha.immaculata, Cha.sexpuctata and M. stigmatica (Fig. 1a-c, h-i); interstitial region of one autosomal pair in D. cruciata and M. gnata (Fig. 1d-f); terminal region of two autosomal pairs and interstitial region of one pair in M. optata (Fig. 1g).In M. gnata, the hybridization signals in early prophase I cells indicate an out of sex vesicle, confi rming the presence of autosomal rDNA.
The hybridization signals in species with one terminal rDNA clusters are located in chromosomes of different size, i.e., in A. inedita, Cha.immaculata and M. stigmatica, the 28S rDNA is located in medium-sized metacentric chromosomes, while in Cha.sexpuctata this gene occurs In all species in the tribe Mesomphaliini, rDNA-FISH revealed 28S sites located in the terminal/subterminal region of only one autosomal pair.However, in Che.cribraria and Che.infl ata, the rDNA clusters occur in submetacentric chromosomes of large and medium size, respectively (Fig. 2a-b); in Cyr.cyanea and P. fl ava, the chromosomes with hybridization signals have a metacentric morphology and are small (Fig. 2c-d).

DISCUSSION
The analyses of male mitotic and meiotic cells in fi ve species of Cassidini: A. inedita, Cha.immaculata, Cha.sexpunctata, D. cruciata and M. optata, and two Mesomphaliini species: Che.cribraria and P. fl ava, confi rmed the diploid number and the sex chromosome system previously described for these species (Lopes et al., 2016).For the two species of Cassidini, characterized here for the fi rst time, only M. stigmatica (2n = 20 + Xy p ) had a diploid number greater than those recorded for the other species in this genus: M. aciculata, M. gnata, M. optata and M. quadrata (Lopes et al., 2016;this work).This increase in the diploid number may correspond to a derived condition within the genus Microctenochira and even in the tribe Cassidini, taking into account that approximately 60% of the species in this tribe, belonging to 11 distinct genera, have a diploid number 2n = 18 (De Julio et al., 2010;Lopes et al., 2016).
In the tribe Mesomphiliini, the diploid number is 2n = 22 and an Xy p sex chromosome system, verifi ed here for Che.infl ata, are similar to those recorded for Che.cassidea, Che.cribraria, Che.indigesta, Che.nigricolis and Che.varians (Stevens, 1906;De Vaio & Postiglioni, 1974;Vidal, 1984;Postiglioni et al., 1990Postiglioni et al., , 1991;;Virkki et al., 1992;Lopes et al., 2016).On the other hand, Cyr.cyanea has the karyotype 2n = 38 + Xy p .Among the species of Mesomphaliini, diploid numbers similar to or greater than 2n = 40, occur predominantly in the genera Botanochara, whose species also invariably have multiple sex chromosome systems (for review see De Julio et al., 2010).In this tribe, the only exception is P. fl ava with 2n = 40 + Xy p .Thus, only a phylogenetic analysis of the Mesomphaliini can clarify if the large diploid number is a shared characteristic of this tribe or appeared independently in some genera.
Physical mapping of the 28S rDNA gene in Cassidinae revealed that 10 of the 11 species studied have a conserved pattern regarding the number of ribosomal sites.In addi- tion, it revealed the presence of rDNA on one autosomal pair in seven distinct genera and in species of both the tribes Cassidini and Mesomphaliini.All these species have the Xy p sex chromosome system type and the constitutive heterochromatin is predominantly located in the pericentromeric region, but they differ in terms of their diploid number, chromosome sizes and number of chromosomal pairs with constitutive heterochromatin (for details see Lopes et al., 2016).These results corroborate the data obtained for the Coleoptera as a whole, in which the presence of rDNA sites on a pair of autosomes seems to be the most stable evolutionary pattern and may be the ancestral condition in this order.The occurrence of one pair of autosomal rDNA clusters in beetles remains unchanged in species with a conserved diploid number and same sex chromosome system as well as in those with derived karyotypes (Schneider et al., 2007).
Among the 11 species examined in this study, only in Cha.immaculata the ribosomal cistrons are co-localized with constitutive heterochromatin on the terminal region of the chromosomes.The co-localization of these two chromosomal markers is reported in other coleopteran families, such as Curculionidae, Elateridae, Geotrupidae, Lucanidae, Scarabaeidae and Tenebrionidae (Virkki & Sepúlveda, 1990;Virkki et al., 1990;Colomba et al., 1996Colomba et al., , 2000aColomba et al., , b, 2004;;Vitturi et al., 1999Vitturi et al., , 2003;;Moura et al., 2003;Bione et al., 2005;Schneider et al., 2006;Dutrillaux et al., 2007;Cabral-Mello et al., 2010;Lira-Neto et al., 2012).Cabralde Mello et al. (2011a), in their comparison of the number and localization of major rDNA sites with the distribution of constitutive heterochromatin in 22 beetles in the subfamily Scarabaeinae, verifi ed two patterns: (1) a stable number of rDNA sites (one autosomal pair) in species with constitutive heterochromatin predominantly located in the centromeric/pericentromeric region and (2) an increased number of rDNA cistrons in species with constitutive heterochromatin dispersed in the chromosomes.Conse-quently, these authors suggest that the same mechanism of chromosomal alteration (ectopic recombination) could be associated with the dispersion of these two chromosomal regions in the genome.Among the species studied by us, Cha.immaculata differs from other species of Cassidini in having rDNA genes located only in the terminal region of one autosomal pair, but constitutive heterochromatin dispersed among pericentromeric, telomeric and interstitial regions on autosomes (Lopes et al., 2016).In contrast, D. cruciata and M. optata has a different number and/or location of the ribosomal sites, compared to other species of Cassidini, D. cruciata with two interstitial rDNA sites and M. optata with three sites of rDNA, two terminal and one interstitial, but constitutive heterochromatin located only in the pericentromeric region of the chromosomes.These results seem to indicate that there is no direct correlation between the dispersion of constitutive heterochromatin and the 28S rDNA gene in Cassidini, and other mechanisms, such as inversion or transposition, may have been involved in the movement of the rDNA sites in these species.However, it is now necessary to use additional techniques to confi rm the presence and identify the heterochromatin in species of Cassidinae.
In all the species studied here, the 28S rDNA is located in terminal/subterminal region of the chromosomes, with the exception of D. cruciata, M. gnata and M. optata, in which there are signals of hybridization in the interstitial regions.It is important to emphasize that these three species have diploid numbers and sex chromosome systems similar to most Cassidini, i.e., 2n = 16 + Xy p .In Coleoptera, the location of the major rDNA genes (or NOR) is only established in around 30% of the species investigated, that is 22% of the species with terminal rDNA, 1.6% with interstitial and 6.4% with centromeric rDNA.The species with interstitial and/or centromeric rDNA cistrons are mainly members of the families Scarabaeidae and Chrysomelidae (subfamily Alticinae).As recorded in the present study, the changes in the location of the sites of rDNA are described in species with similar karyotype characteristics (Virkki, 1983;Yadav et al., 1992;Bione et al., 2005;Almeida et al., 2006Almeida et al., , 2010;;Arcanjo et al., 2009Arcanjo et al., , 2013;;Silva et al., 2009;Cabral de Mello et al., 2010, 2011b;Oliveira et al., 2010Oliveira et al., , 2012b)).The differences in the location of rDNA in closely related species with similar chromosomal characteristics may be a result of small chromosomal rearrangements, which change the position of the ribosomal site without modifying the metacentric chromosomal morphology.
In summary, the results obtained in this study reveal that the changes involving the 28S rDNA are pronounced in the tribe Cassidini, which has the most uniform karyotype within the subfamily Cassidinae.In the tribe Mesomphaliini, the increase in the diploid number is not accompanied by an increase in the number of ribosomal genes.Finally, we show that the changes in the number and location of the ribosomal genes occur between species with similar karyotypes, indicating that the rearrangements in these specifi c genes may have resulted in the chromosomal evolution in this group.
ACKNOWLEGEMENTS.This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (2011/21643-1; 2012/12619-2) and Conselho Nacional de Desenvolvimento Científi co e Tecnológico, CNPq (302416/2013-7).The authors are grateful to M.C. de Almeida of Universidade Estadual de Ponta Grossa, UEPG, Brazil, for her helpful with improving the FISH technique, to anonymous referees and the Associate Editor for his critical reading of this manuscript.

Table 1 .
The species of Cassidinae analyzed in the present study, including the number of individuals and the locality where collected in the state of São Paulo, Brazil.