Karyotype evolution in progress: A new diploid number in Belostoma candidulum (Heteroptera: Belostomatidae) from Argentina leading to new insights into its ecology and evolution

A novel chromosome complement (2n = 14 = 12 + XY/XX; male/female sex chromosomes), male meiosis behaviour, het­ erochromatin characterization, and frequency and distribution of chiasmata are described for the first time in specimens from a natural population of the giant water bug, Belostoma candidulum Montandon, 1903 (Heteroptera: Belostomatidae) from Argentina. To date, specimens of B. candidulum have been reported by other authors in a sample from a natural population from Brazil. Our results dem­ onstrate that Argentinean and Brazilian populations have different diploid numbers and chromosomal features. During male meiosis, autosomal bivalents generally show a single chiasma, behave as telokinetic chromosomes (i.e. kinetic activity is restricted to terminal regions), and divide reductionally at anaphase I; in contrast, the sex chromosomes are achiasmatic, behave as univalents and segregate equationally at anaphase I. Among autosomal bivalents of B. candidulum, one is remarkably larger and may present one or two termi­ nal chiasmata, showing rod, V-shaped and ring configurations. Here we propose a new mode of segregation for ring bivalents, since it is not essential that one of the chiasmata is released during anaphase I because alternative sites for microtubule attachment become functional for the normal chromosome segregation to the poles. Heterochromatin content is very scarce in specimens from Argentinean B. candidulum populations, revealing C­positive interstitial and terminal dots in three pairs of autosomes and C­blocks at both ends of X chromosome, whereas the Y chromosome is mainly C­positive. One of the C­positive bands from X and Y chromosomes is DAPI­ dull/CMA-bright, which could represent the nucleolus organizing region (NOR) detected by fluorescent in situ hybridization (FISH). The location of the NORs in both sex chromosomes allowed us to use them as a cytological marker to describe their behaviour during meiosis. Despite the fact that specimens from the Argentinean and Brazilian populations have been classified as a single species due to their morphological similarity, our results suggest that both populations are chromosomal races or even morphologically­identical cryptic species. The results obtained support the hypothesis that karyotype of B. candidulum originated through autosomal fusions and the fusion of the X and Y chromosomes with the ancestral NOR­autosomal pair. Lastly, the genus Belostoma represents an excellent model for assessing the main mechanisms involved in the karyotype evolution in organisms with holokinetic chromosomes, from which inferences may be made concerning its broader ecology and evolution.

All Belostoma species studied share similar cytogenetic characteristics: (a) presence of holokinetic chromosomes, i.e. without a primary constriction; (b) occurrence of syn izesis (meiotic chromatin condenses eccentrically on one side of the nucleous; McClung, 1905) and diffuse stage; (c) a telokinetic activity in meiosis and a holokinetic ac tivity in mitosis; (d) autosomal bivalents are synaptic and chiasmatic, whereas sex chromosomes are asynaptic and achiasmatic; (e) smallest bivalents have a single chiasma and the largest possess one or two chiasmata; (f) autoso mal bivalents segregate reductionally and sex chromo somes divide equationally in the first meiotic division; (g) an inverse relationship between chromosome number and chromosome size, with autosomal bivalents decreasing in size; (h) an inverse relationship between total chromosome length (TCL) and DNA content and Cbanding pattern; and (i) in species with reduced chromosome numbers and a simple sex chromosome system (XY/XX), the nucleolus organizing region (NOR) is located in both sex chromo somes, whereas in species with the modal diploid number of autosomes (26) and a multiple sex chromosome system (X 1 X 2 Y/X 1 X 1 X 2 X 2 ), it is located in a pair of homologous and slides were prepared by the squash technique in a drop of 2% acetic-haematoxylin (Sáez, 1960).For C-and fluorescent band ing and the FISH technique, spread chromosome preparations were made from the testes and ovaries of male and female adults.Cells were dissociated in a drop of 60% acetic acid with the help of tungsten needles and spread on the slide using a heating plate at 45°C as described in Traut (1976).The preparations were dehy drated in an ethanol series (70, 80 and 96%, 30 s each) and stored at -20°C until use.

chromosome bandings
Heterochromatin content, distribution and nucleotide compo sition were analysed by means of C-and sequential fluorescent DAPI (4',6diamidino2phenylindole) and CMA 3 (chromomycin A3) banding.Details of the methods used are discussed in greater depth and broadly follow those as detailed in Papeschi (1988) and Poggio et al. (2011).

Statistical analysis
Suitable cells at the meiotic metaphase were selected and the mean descriptive values of the karyotype calculated using in formation obtained from 88 cells.The total chromosome length measurements (TCL), relative average length (µm; length of an autosomal bivalent as a percentage of the TCL) and relative chro mosomal percentage (%) were performed with Micro Measure for Windows, version 3.3 (Reeves & Tear, 2000).Measurements of autosomal bivalents and sex chromosomes were performed in metaphase I. Differences in relative average length and rela tive chromosomal percentage were analysed using the Kruskall Wallis ANOVA test on ranks for global comparisons (P < 0.05), followed by MannWhitney U tests for contrasts between treat ments, since the data were not normally distributed (Daniel, 1990).The presence of one ring, rod or Vshaped bivalent (of the largest autosomal pair) was frequently observed in 153 cells from three males.Configuration frequencies of the largest biva lent were analysed by means of Chi-squared goodness of fit to be distributed at diplotene, diakinesis and metaphase I stages.The location of NORs in the XY pseudobivalent at metaphase II, in which the kinetically active or inactive, was tested in 46 cells from three males using a Chi-squared goodness of fit test to de termine whether or not the kinetic behaviour is a random process.Statistical analyses were done using Statistix for Windows, ver sion 2.0 (Analytic Software, 1998).
The last cytogenetic characteristic described above al lowed us to propose an ancestral male karyotype 2n = 26 + XY sex chromosomes for Belostoma, in which the NOR is located in one pair of autosomes, and involving two paths of karyotype evolution: (i) a fragmentation of the single ancestral X chromosome derived in a multiple sex chromo some system which led to a karyotype 2n = 26 + X 1 X 2 Y, with retention of the ancestral NORautosome pair; and (ii) several autosomal fusions plus the fusion of the ancestral sex chromosome pair with the NORautosome pair result ing in reduced chromosome numbers (2n = 14 + XY, 2n = 6 + XY), and increase of chromosome size, and which led to the presence of ribosomal DNA (rDNA) clusters in both X and Y chromosomes (Papeschi & Bressa, 2006;Chirino et al., 2013).
In the present study, a novel chromosome complement found in specimens taken from a natural population of Be lostoma candidulum Montandon, 1903 from Argentina is described for the first time.The male and female karyo types, the male meiosis behaviour, and frequency and dis tribution of chiasmata were studied.The content, location and distribution of heterochromatin were analysed by C and fluorescent banding.Using fluorescence in situ hybrid ization (FISH) with the 18S rDNA probe, we localized the position of the NORs and showed that the presence of these in both sex chromosomes allowed us to use them as a cyto logical marker to describe their behaviour during meiosis.
Belostoma represents an excellent model for evolution ary cytogenetic studies since this genus shows great in terespecific variation in the diploid chromosome number, many chromosome changes and interespecific differences in the DNA content.The presence of a novel chromosome complement allows increased insight into the evolution of the karyotypes including the role of chromosome fusions in the genus Belostoma.

chromosome preparations
The specimens were brought alive to the laboratory and their gonads dissected out in a physiological saline solution as earlier described for the pyralid moth, Ephestia sp.Guenée, 1845 (Gla ser, 1917: cited by Lockwood, 1961), swollen in a hypotonic so lution and fixed as described in Chirino et al. (2013).For mitotic and meiotic analysis, gonads were kept at 4°C in 70% ethanol for CMA 3 , and red for Cy3), and processed with the appropriate software.

reSultS chromosome complement and meiosis
The chromosome complement of B. candidulum is 2n = 14 = 12 + XY/XX (male/female sex chromosmes) and has a total chromosome length (TCL) of 28.5 ± 5.2 µm.An autosomal pair is easily identified due to its large size among the remaining five pairs of autosomes that gradually decrease in size (Table 1).The X chromosome is medium sized and the Y is the smallest of the complement (Figs 1, 2a-b; Table 1).In mitotic prometaphase in both sexes, the X chromosomes have a secondary constriction (Fig. 2a-b).During mitotic anaphase, the sister chromatids migrate in parallel to the spindle poles, in contrast to meiosis where the kinetic activity is restricted to the chromosome ends, and the chromosomes can be regarded as telokinetic (Fig. 2c).
The meiotic behaviour was similar to that previously described for other Belostoma species.Pairing occurred at synizesis, and at pachytene both sex chromosomes, close to each other, were condensed and the 6 autosomal biva lents were joined through their terminal regions (Fig. 2d).At the diffuse stage, all bivalents decondensed completely and the sex chromosomes were seen to be positively het eropyknotic, almost always associated but usually indi vidually recognisable (Fig. 2e).From diplotene onwards, 6 bivalents and two sex chromosomes univalents were clearly discernible (Fig. 2f).At early diakinesis, both sex chromosomes were negatively heteropyknotic (data not shown).During late diakinesis, the 6 autosomal bivalents became isopyknotic and the X and Y sex univalents were negatively heteropyknotic (Fig. 2g-i).At metaphase I, au tosomal bivalents were arranged in a ring, but the X and Y univalents did not show a defined position (Fig. 2j, k).Anaphase I was reductional for autosomes and equational for sex chromosomes.All telophase I nuclei exhibited 8 chromosomes in each pole (6 + XY; data not shown).The second meiotic division followed without an interkinesis stage.In metaphase II, the XY pseudobivalent was local ized in the middle of the autosomal ring (Fig. 2l).Both sex chromosomes were negatively heteropyknotic and they were oriented towards the opposite spindle pole.At ana phase II, 7 chromosomes migrated to one pole (6 + Y and 6 + X; data not shown).
The largest autosomal bivalent showed one or two chi asmata at terminal position (Fig. 2f-k).Three kinds of bi valents were frequently observed from diplotene to meta phase I: rod (autosomal bivalent with one chiasma; Fig. 2f, g), Vshaped (autosomal bivalent that one of the two chiasmata releases first; Fig. 2h) and ring bivalents (auto somal bivalent with two chiasmata; Fig. 2f, i).When the largest bivalent had two chiasmata, one of them released first, one pair of terminal regions became free to attach to the spindle, and the bivalent remained linked by the second chiasma, and took a V-shaped configuration (Fig. 2j).In other cells, the ring bivalent was stabilized at the meta phase plate with the longitudinal axes perpendicular to the polar axis and released the homologous chromosomes without liberating the two chiasmata at anaphase I, i.e. the ring bivalent separated as half rings, and kinetic activity in volved sites other than the chromosome ends (Fig. 2k).The observed frequencies were significantly different from ex pected assuming random segregation because the presence of one rod or ring bivalent was mainly observed, while the Vshaped bivalent was even rarer (Table 2).Rod and ring arrangements showed very little variation from diplotene to metaphase I, since the largest bivalent had one or two terminal chiasmata at the same frequency.Thus in general, it does not appear essential that one of the chiasmata is  2.5 ± 0.6 ef 8.9 ± 1.7 ef X chromosome 1.6 ± 0.6 fg 6.4 ± 0.9 fg Y chromosome 0.8 ± 0.3 h 2.9 ± 0.6 h 1 The comparisons of chromosomal lengths (mean ± SE, μm) and the relative chromosomal percentage (mean ± SE, %) were made using a oneway KruskallWallis test (H = 680.21,df = 7, 696, P < 0.0001 for chromosomal lengths measurements; H = 659.44,df = 7, 696, P < 0.0001 for relative chromosomal lengths meas urements).Different letters indicate significant differences (P < 0.05).released previously in order to stabilize the ring bivalent at the metaphase plate (Table 2).

chromosome bandings
Heterochromatin content was very scarce in all speci mens of B. candidulum and Cbanding revealed very little interstitial and terminal dots in three autosomal bivalents tested (Fig. 3a-c).A single nucleolus was present, visible as a negative heteropyknotic body, which was invariably associated with one of the sex chromosomes (Fig. 3d).The X chromosome showed two terminally located blocks, whereas the Y chromosome was mainly Cpositive (Fig. 3d-e).After diakinesis, the Cpositive dots and blocks were not observed (Fig. 3f).Heterochromatic bands were neither AT nor GC rich as revealed by DAPI and CMA 3 sequential fluorescent banding for any chromosome pair in both mitotic and meiotic cells (Fig. 4), except for the X and Y chromosomes, which showed a DAPIdull/CMAbright band at one terminal position (Fig. 4).

location of rdnA
FISH involving 18S rDNA probes revealed two clusters of rDNA genes located at one end of both sex chromo somes on all phases of meiosis in a similar location to the DAPIdull/CMAbright band (Fig. 5a-d).This molecular marker was a sequence used to spot a particular location on both sex chromosomes during the second meiotic division.
The presence in both sex chromosomes of one rDNA clus ter at only one chromosomal end allowed determination of whether the kinetic behaviour occurring at both ends (and whether carrying the NOR or not) was a random process at metaphase II.At the second metaphase, the XY pseudo bivalent showed four distinctive orientations: Type XY1 (kinetic activity located at both ends with rDNA signal in both sex chromosomes; Fig. 5d), Type XY2 (kinetic activ ity located at both ends without rDNA signal in both sex chromosomes; Fig. 5e), Type XY3 (kinetic activity locat  ed at end with rDNA signal in X chromosome and at end without rDNA signal in Y chromosome; Fig. 5f), and Type XY4 (kinetic activity located at end without rDNA signal in X chromosome and at end with rDNA signal in Y chro mosome; Fig. 5g).The frequencies observed in the second metaphase revealed that the kinetic activity was indeed a random process (15 cells for Type XY1; 10 cells for Type XY-2; 16 cells for Type XY-3; 5 cells for Type XY-4; χ 2 = 6,696, df = 3, P = 0.0823).

dIScuSSIon
The most remarkable cytological features observed in specimens of B. candidulum from Argentina as here stud ied were a lower novel chromosome complement 2n = 12 + XY/XX (male/female sex chromosomes) and the large size of one pair of autosomes.The specimens examined were also seen to show heterochromatic blocks in both autoso mal and X chromosomes, a Y chromosome with mainly Cpositive banding, and a secondary constriction in the X chromosome.Until now, all Brazilian male specimens of this giant water bug species showed 2n = 16 = 14 + XY, with the karyotype composed of four large, nine medium, and one small chromosomes (Bardella et al., 2012).Our results, in contrast, revealed that Argentinean and Brazilian populations have different diploid numbers and chromo somal features.
Argentinean and Brazilian samples are geographically separated by long distances (from 900 km to 2,805 km) and restricted to small geographic areas showing a patchy distribution (Ribeiro, 2007;Ribeiro & Estévez, 2009).In the present study, the specimens of B. candidulum were collected outside the regional distribution reported for this species in Argentina (Ribeiro, 2007;Ribeiro & Estévez, 2009).As far as is known, the distribution areas of these two populations do not overlap, i.e. are allopatric.Given the isolation of their habitats, the limited dispersal poten tial, the holokinetic nature of the chromosomes, and the presence of fixed chromosomal variants, Argentinean and Brazilian B. candidulum populations should be considered as two chromosomal races or two cryptic species.
Autosomal fusions and autosomal and sex chromosome fragmentations have seemingly played the most important role in chromosomal evolution in Heteroptera (Ueshima, 1979;Manna, 1984;Thomas, 1987;Papeschi 1994Papeschi , 1996;;Pérez et al., 2004).The cytogenetic data available for Be lostoma suggest that the karyotype evolution within this genus has proceeded through fragmentation of the atavic X chromosome, increase in heterochromatin content and au tosomal and/or sex chromosome fusions (Papeschi, 1988(Papeschi, , 1994(Papeschi, , 1996;;Papeschi & Bressa 2006;Bardella et al., 2012;Chirino et al., 2013).Autosomal fusions may be favoured from an evolutionary point of view as a mechanism for re taining supergenes.Since each autosomal bivalent has a single chiasma, the fusion of two nonhomologous auto somal pairs and the further restriction to a single crossing over in the fused autosomal pair, implies a reduction in the overall frequency of recombination.Thus, segregation of coadapted and selectively favoured alleles is prevented.Consequently, it is highly likely that the karyotype of B. candidulum originated from the ancestral complement through several fusions among pairs of autosomes, as well as by the fusion of the X and Y chromosomes with the ancestral NORautosomal pair, making that rDNA genes were present in both X and Y sex chromosomes (Fig. 6).This hypothesis is supported by the existence of an inverse relationship between chromosome size and chromosome number (Papeschi, 1988(Papeschi, , 1992)).
This present study also revealed a direct relationship between chromosome size and frequency of chiasmata, in which the largest autosomal bivalent showed one or two terminal chiasmata at the same frequency.The presence of one or two chiasmata resulted in three interesting cytoge netic characteristics in meiosis, namely: (i) the presence of rod, ring and Vshaped bivalents; (ii) rod bivalents orien tated axially but ring bivalents orientated equatorially at metaphase I; and (iii) when the ring bivalent released one of their chiasmata, it adopted an axial orientation.
The presence of ring and Vshaped bivalents at diakine sismetaphase I have previously been shown in giant wa Fig. 6.Hypothetical proposal of evolution in genus Belostoma, following two ways of karyotype evolution (modified from Chirino et al., 2013).
ter bugs of the genus Belostoma (Papeschi & Bidau, 1985;Papeschi, 1988Papeschi, , 1994Papeschi, , 1996;;Papeschi & Bressa, 2006;Bardella et al., 2012) as well as other heteropteran species with reduced chromosome complements (Camacho et al., 1985;Mola & Papeschi, 1993;Bressa et al., 1998Bressa et al., , 1999Bressa et al., , 2001Bressa et al., , 2002aBressa et al., , b, 2005;;Jacobs & Liebenberg, 2001;Reba gliati et al., 2001;Jacobs & Groeneveld, 2002;Papeschi et al., 2003;Franco et al., 2006;Rebagliati & Mola, 2010;Poggio et al., 2011).Therefore, all reports obtained from bivalents with two chiasmata stated that ringbivalents congressed to the metaphase I plate showing holokinetic behaviour.In many cases, the ring bivalent separates at one chromosomal end and the homologous chromosomes remain associated by the second chiasma, maintaining an endtoend association.Afterwards, the kinetic activity in volved the terminal regions attaching to the spindle, and the bivalent showed a Vshaped morphology followed by rod-like configuration.Conversely, in a few cases, when the ring bivalent had a repetitive DNA sequences adja cent to the NORs in a medial position in both homologous chromosomes, microtubule bundles interact with the entire length of them and no chiasma was released, the NORs ap peared at opposite poles and attached to the spindle fibres, and the bivalent maintained a ring-shape configuration (Papeschi et al., 2003).
In the Argentinean male specimens of B. candidulum, when the largest autosomal bivalent showed two chiasmata but had no NORs, half of the ring bivalents released one chiasma and the holokinetic interaction with the spindle became telokinetic at late metaphase.On the other hand, when the ring bivalent was stabilized at the metaphase plate maintaining both chiasmata, the bivalent did not open, nor did it adopt the Vshaped morphology; thus, the ring bivalent kept a holokinetic interaction with the spin dle.This being so, the ringshaped bivalent should have some mechanism/s to ensure its attachment to the spindle fibres; other alternative sites, not only terminal regions and secondary constrictions or sites next to them, would be able to acquire kinetic activity.It cannot be discounted that these alternative sites for microtubule attachment are also functional in rod bivalents.In these bivalents, the in teraction between the alternative sites and the microtubules would be weaker and surpassed by the terminal regions (Papeschi et al., 2003).
In B. candidulum from Argentina, characterization of heterochromatin revealed very scarce Cpositive dots at terminal and interstitial positions along three autosomal bivalents.The Cbanding pattern was in agreement with previous observations made for other species of the genus Belostoma, with respect to an inverse relationship between the size of the chromosomes and the amount and size of C positive bands (Papeschi, 1988(Papeschi, , 1992)).Species with high chromosome numbers (2n = 26 + X 1 X 2 Y) have several and/or conspicuous Cpositive bands, whereas those with reduced diploid numbers (2n = 6 + XY, 2n = 12 + XY, 2n = 14 + XY) exhibit few and very small Cpositive dots and bands (Papeschi, 1988(Papeschi, , 1991(Papeschi, , 1992(Papeschi, , 1994(Papeschi, , 1995;;Papeschi & Bressa, 2006;Chirino et al., 2013;this study).The use of Cbanding to identify content, location and distribution of constitutive heterochromatin, to detect heteromorphisms and to distinguish homologues is noteworthy, since it pro vides information about the chromosome organization, which is highly valuable when studying karyotype evolu tion.Thus, we hypothesize that the ancestral male karyo type, 2n = 26 + XY, was characterized by its scarce Cpos itive heterochromatin and, from this karyotype, two events occurred, namely: (a) an increase in heterochromatin con tent in species with multiple sex chromosome systems af ter the fragmentation of the original X chromosome; and (b), a preservation of scarce heterochromatin content in species with reduced diploid numbers, since the increase of Cpositive heterochromatin was restricted through the several fusions produced between autosomes and/or sex chromosomes.
FISH experiments using 18S rDNA probes revealed a single rDNA cluster located at one terminal region of both sex chromosomes.In addition, fluorescent banding revealed a DAPIdull/CMAbright band at one terminal position on the X and Y chromosomes, indicating the pres ence of CGrich sequences.Since CGrich constitutive het erochromatin often occurs in the NOR regions (Papeschi et al., 2003;Rebagliati et al., 2003;Cattani et al., 2004;Cat tani & Papeschi, 2004;Papeschi & Bressa, 2006;Criniti et al., 2009;Poggio et al., 2011;Chirino et al., 2013), we sug gest that these two bands on both sex chromosomes could be associated with two rDNA clusters revealed by rDNA FISH.These results support our hypothesis that species with simple sex chromosome systems possesses the NOR regions in both X and Y chromosomes, whereas in spe cies with multiple sex chromosome systems, the NOR is positioned in one autosomal pair only (Fig. 6).Moreover, this study provides further evidence that kinetic activity in X and Y chromosomes is randomly located on one of the two sex chromosome ends, and that it is independent of the end selected in the other sex chromosome; therefore both terminal regions of each of them could be kinetically active at the second meiotic division.FISH experiments hence provide a consistent marker to analyse the behav iour of chromosomes during meiosis (Papeschi et al., 2003;Poggio et al., 2011;present study).
Lastly, our results as presented here showed that the determination of the number and location of Cpositive, DAPI and CMAbands, and the number and location of the NORs make them essential cytological markers since the lack of a primary constriction and/or secondary con strictions in organisms with holokinetic chromosomes has hampered cytogenetic studies and a properly chromosome identification.As a result, these techniques are clearly very useful tools for the study of the karyotype structure, meiotic behaviour and chromosome evolution in groups with holokinetic chromosomes; use of such approaches may also contribute to the analysis of changes in karyo type related to the evolutionary process and to understand taxonomic relationships.The genus Belostoma surely con stitutes a very interesting group from a cytogenetic point of view, since it exhibits a great variety of chromosome complements with simple and multiple sex chromosome systems, and the comparative study of the karyological features of this genus (Paspeschi & Bressa, 2006;Chirino et al., 2013) could well be used for systematic and ecologi cal/evolutionary studies of considerable interest.

Fig. 5 .
Fig. 5. Location of rDNA genes in male meiotic chromosomes of B. candidulum using FISH involving 18S rDNA probes (red sig nals, arrows).Chromosomes were counterstained with DAPI (blue).a -diffuse stage; b -diplotene; c -diakinesis; d-e -metaphase II; d -type XY1 pseudobivalent (kinetic activity located at both ends with rDNA signal in both sex chromosomes); e -type XY2 pseudobivalent (kinetic activity located at both ends without rDNA signal in both sex chromosomes); f -type XY3 pseudobivalent (kinetic activity located at end with rDNA signal in X chromosome and at end without rDNA signal in Y chromosome); g -type XY4 pseudobivalent (kinetic activity located at end without rDNA signal in X chromosome and at end with rDNA signal in Y chromosome).pII = pseudobivalent.Bar = 10 µm.

Table 1 .
Comparison of the male meiotic size of chromosomes (2n = 12 + XY) during metaphase I of B. candidulum.

Table 2 .
Frequency of cells at the diplotene, diakinesis and metaphase I stages in which the first bivalent exhibited rod, V-shaped and ring configurations.