Journal of Biological Research 5: 3 – 21, 2006 J. Biol. Res. is available online at http://www.jbr.gr

— INVITED REVIEW — Evolutionary cytogenetics in

ALBA GRACIELA PAPESCHI* and MARI´A JOSE´ BRESSA

Laboratorio de Citogenética y Evolucio´ n, Departamento de EcologÈ´a, Genética y Evolucio´ n, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

Received: 2 September 2005 Accepted after revision: 29 December 2005

In this review, the principal mechanisms of karyotype evolution in bugs (Heteroptera) are dis- cussed. Bugs possess holokinetic chromosomes, i.e. chromosomes without primary constriction, the centromere; a pre-reductional type of meiosis for autosomes and m-chromosomes, i.e. they segregate reductionally at meiosis I; and an equational division of sex chromosomes at anaphase I. Diploid numbers range from 2n=4 to 2n=80, but about 70% of the have 12 to 34 chromosomes. The chromosome mechanism of sex determination is of the XY/XX type (males/females), but derived variants such as an X0/XX system or multiple sex chromosome sys- tems are common. On the other hand, neo-sex chromosomes are rare. Our results in het- eropteran species belonging to different families let us exemplify some of the principal chromo- some changes that usually take place during the evolution of species: autosomal fusions, fusions between autosomes and sex chromosomes, fragmentation of sex chromosomes, and variation in heterochromatin content. Other chromosome rearrangements, such as translocations or inver- sions are almost absent. Molecular cytogenetic techniques, recently employed in bugs, represent promising tools to further clarify the mechanisms of karyotype evolution in Heteroptera.

Key words: holokinetic chromosomes, karyotype evolution, molecular cytogenetics.

INTRODUCTION between 1897 and 1932, Wilson and Montgomery settled the foundations on Heteroptera cytogenetics. Cytogenetic studies in Heteroptera date from 1891 Some years later, between the 1930s and the 1960s, with Henking’s morphological study on the sper- Schrader and Hughes-Schrader contributed signifi- matogenesis of the bug Linnaeus cantly to a better knowledge on the organization and (). He described the presence of a function of heteropteran chromosomes and their chromatin body (the “X chromosome”) that showed behaviour during cell divisions (Ueshima, 1979; Man- an unusual staining during meiotic prophase and a na, 1984). peculiar behaviour during meiosis. The observations At present, although approximately 1600 het- of Henking and other cytologists, together with their eropteran species belonging to 46 families have been own observations in males and females of different cytogenetically analyzed, they are not a representa- Heteroptera species, were finally interpreted by Mc tive sample of the group (4.2% of approximately Clung (1901) and Wilson (1909b). They suggested a 38,000 described species) (Table 1) (Schuh & Slater, direct relationship between sex determination and 1995). Most studies have been made on male testes, presence of either one or two “X chromosomes” in because meiotic cells are more often encountered X0/XX systems, or an XY or XX chromosome pair than mitotic ones, and the earliest reports referred to in species with XY/XX systems. the male chromosome number and sex chromosome Through a series of publications that appeared system. No detailed analysis of heteropteran chro- mosome morphology is possible, since chromosomes * Corresponding author: tel.: +54 11 45763354, fax: +54 are holokinetic (a primary constriction, the centro- 11 45763384, e-mail: [email protected] mere, is lacking) and the chromosomes are generally

3 4 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera relatively small and of similar size. Although DNA regarded as telokinetic (Motzko & Ruthmann, 1984). measurement is the best means of testing fusion/frag- Both chromosome ends can show kinetic activity mentation hypothesis in karyotypic evolution of taxa during meiosis in such a way that the telomeric bearing holokinetic chromosomes, this technique has regions that were inactive during the first meiotic been rarely used in Heteroptera (Schrader & Hughes- division become active during the second one (Cama- Schrader, 1956, 1958; Maudlin, 1974; Papeschi, 1988, cho et al., 1985; Nokkala, 1985; Pérez et al., 1997; Cat- 1991; Panzera et al., 1992, 1995; Papeschi et al., 2001; tani et al., 2004). The holokinetic nature of het- Grozeva & Nokkala, 2003). Furthermore, cytogenet- eropteran chromosomes has led to the hypothesis ic studies at the population level in Heteroptera are that fusions and fragmentations would be the most still scarce. All these facts represent serious con- frequent mechanisms of karyotype evolution. The straints in comparative and evolutionary cytogenetics non-localized kinetic activity does not have the con- of this group. straints imposed by a localized centromere in species Recently, molecular approaches such as fluores- with monocentric chromosomes. In the latter, acen- cent banding and fluorescent in situ hybridization tric fragments or dicentrics are mitotically and meiot- with telomeric and ribosomal DNA probes (18S and ically unstable. 28S rDNA), have been carried out in bugs (Gonza´lez- The meiotic behaviour of autosomal bivalents, sex García et al., 1996; Sahara et al., 1999; Papeschi et al., chromosomes and m chromosomes is slightly differ- 2003; Rebagliati et al., 2003; Cattani & Papeschi, ent. As a rule, autosomal bivalents are chiasmatic, 2004; Cattani et al., 2004; Frydrychova´ et al., 2004; whereas sex chromosomes and m chromosomes are Grozeva et al., 2004; Hebsgaard et al., 2004; Ituarte & achiasmatic (Ueshima, 1979; Manna, 1984; Gonza´lez- Papeschi, 2004; Bressa et al., 2005; Vítkova´ et al., García et al., 1996; Suja et al., 2000). When autosomal 2005). These molecular techniques have substantial- bivalents form only one chiasma terminally located, ly contributed to a better understanding of the chro- they orientate at metaphase I with their long axes matin organization and composition of these holoki- parallel to the polar axis; they segregate reductional- netic chromosomes. However, there is still much to ly during meiosis I and equationally during meiosis II. be learnt before the mechanisms of karyotype evolu- On the other hand, bivalents with two terminal chias- tion and the role in the speciation of heteropterans is mata orientate equatorially and two different behav- completely clarified. iours have been described: a) one chiasma releases first and an axial orientation is finally achieved or b) CYTOGENETICS OF HETEROPTERA: alternative sites of kinetic activity become functional GENERAL FEATURES and no telokinetic activity is observed (Mola & Pa- peschi, 1993; Papeschi et al., 2003). This latter behav- The genetic system of Heteroptera has many charac- iour has been described in Pachylis argentinus Berg teristics that make it unique among most insect and Nezara viridula (Linnaeus); both species possess groups: the possession of holokinetic chromosomes, a pair of chromosomes carrying a secondary constric- a different meiotic behaviour for autosomes and sex tion, and when this particular pair presented two chi- chromosomes, a mean chiasma frequency of only one asmata, the alternative kinetic sites were located next chiasma per bivalent, and a pair of “m chromosomes” to the secondary constrictions (Papeschi et al., 2003). in some families. When chiasmata are medially located, the terms pre- All heteropteran species possess holokinetic chro- reduction and post-reduction lose their meaning. The mosomes (i.e. without a localized centromere). genetic information located at the chiasma to one During mitosis, microtubules attach to the entire pair of telomeric regions divides reductionally, while length of the sister chromatids and at anaphase, they the information located at the other half of the chro- migrate parallel to each other and perpendicular to mosome does so equationally. We consider meiosis in the polar spindle (Hughes-Schrader & Schrader, Heteroptera as pre-reductional taking into account 1961). At the ultrastructural level, kinetochore plates, the behaviour of bivalents with one terminal or sub- to which spindle microtubules are attached, cover terminal chiasma, in which most genetic information almost all the polar surface of the mitotic chromo- segregates reductionally at meiosis I; and the behav- somes (Buck, 1968; Comings & Okada, 1972). How- iour of fusion/fragmentation trivalents, in which the ever, kinetic activity during meiosis is restricted to the largest chromosome always segregates from the two telomeric regions and the chromosomes can be smaller ones at meiosis I (Ueshima, 1963; Papeschi, A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 5

1994). A post-reductional meiosis has been described rochromatic. The different pycnotic cycle of the m in other groups with holokinetic chromosomes (e.g. chromosomes reflects differences in chromatin pack- Juncaceae, Odonata) (Nordenskiöld, 1962; Mola, aging, and this pattern of chromatin condensation is 1995). probably related to the regulation of gene expression On the other hand, sex chromosomes are achias- (Bressa et al., 2005). However, there is still no infor- matic and behave as univalents in male meiosis I. mation on the genetic content of the m chromo- They divide equationally at anaphase I and associate somes. at meiosis II through the so-called “touch-and-go Further studies are required in order to clarify the pairing”. Nevertheless, pre-reduction of the sex chro- precise behaviour and function of the m chromo- mosomes has been described in three species of somes in different species. Are they always present as Anisops (A. fiebri Kirkaldy, A. niveus Fabricius, A. univalents or do they form a bivalent? Do they have sardea Kirkaldy) (), two species of different behaviour according to the genetic or envi- Ectrychotes (E. abbreviatus Reuter, E. dispar Reuter) ronmental milieu? Furthermore, which are the mech- (), two species of Archimerus (A. alterna- anisms that guarantee their correct segregation when tus Say, A. calcarator Fabricius) and four species of they are present as univalents? Do they represent Pachylis [P. argentinus Berg, P. gigas Barm., P. laticor- some kind of selfish DNA? Nothing can still be said nis (Fabricius), P. pharaonis (Herbst)] () and about their function in the genetic system of the het- in all the 29 species of (Ueshima, 1979; eropteran species possessing them. Grozeva & Nokkala, 2001; Papeschi et al., 2003). Finally, the m chromosomes are also achiasmatic, but SEX CHROMOSOME SYSTEMS associate at first meiotic division segregating pre- Sex chromosome systems described so far in reductionally. Heteroptera are simple systems of the types XY/XX (71.4% of approximately 1600 species cytogenetical- THE m CHROMOSOMES ly analyzed) and X0/XX (14.7%), and different mul-

Wilson (1905) introduced the term “m chromoso- tiple systems (XnY/XnXn, Xn0/XnXn, XYn/XX) mes” to describe the smallest chromosome pair in (13.5%). Neo-sex chromosome systems are very rare two species of Coreidae which behave differently and until present they have only been reported in from both the autosomes and the sex chromosomes seven species and subspecies (0.5%) (Table 1) (Ue- during male meiosis. Until present, the m chromo- shima, 1979; Bressa et al., 1999; Nokkala & Nokkala, somes have been reported in many heteropteran fam- 1999; Jacobs, 2004). ilies (Table 1) (Ueshima, 1979). Although the m Two hypotheses concerning the evolution of the chromosomes are generally of small size, they are sex-chromosome systems in Heteroptera have been actually defined by their meiotic behaviour: they are proposed. Ueshima (1979) suggested that the XY usually unpaired and thus achiasmatic during early system is derived from an ancestral X0 that is com- meiosis, and at late diakinesis they come close togeth- monly encountered in primitive taxa and in phyloge- er. At metaphase I, they are always associated end-to- netically related homopteran species (Halkka, 1959). end (touch-and-go pairing) forming a pseudobivalent By contrast, based on the presence of a Y chromo- which segregates reductionally at anaphase I. The some in very primitive heteropteran species, Nokkala second meiotic division is equational for the m chro- & Nokkala (1983, 1984) and Grozeva & Nokkala mosomes. (1996) suggested that the X0 system is a derived con- One exception to this meiotic behaviour has been dition from the ancestral XY that is present in the described in Coreus marginatus Linnaeus (Coreidae) majority of the species cytogenetically analyzed. by Nokkala (1986) and Suja et al. (2000), who report- Sex chromosome systems with multiple Xs or Ys ed that the m chromosomes were present as a biva- most probably originated through fragmentation of lent in some cells. the ancestral X or Y, respectively. The study of one The m chromosomes generally show allocycly population of Belostoma orbiculatum Estévez & Pol- with respect to both the autosomes and the sex chro- hemus (), polymorphic for the sex mosomes during male meiosis, but the heterochro- chromosome system, provided evidence that supports matin characterization in this chromosome pair this hypothesis (Papeschi, 1996). The analysis of two showed that they are by no means completely hete- samples from one population of Punta Lara (Buenos 6 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera

TABLE 1. Number of species cytogenetically analyzed and principal cytogenetic features of heteropteran families

Taxa No of 2n m Sex Chromosome species Range Mode chromosomes Systems 4 (21-22) present/absent X0, XY, XnY 2 30 present X0 Hebroidea Hebridae 4 (19-27) absent X0, XY, neo-system HydrometroideaHydrometridae 3 (19-20) absent X0, XY 21 (19-31) 21/23 absent X0, XY 5 (21-25) absent X0, XY Mesoveloidea 1 35 absent XnY Nepoidea Belostomatidae 27 (4-30) 29 absent XY, XnY, neo-system 11 (22-46) 43 absent X0, XY, XnY Ochteroidea 1 35 absent XnY 1 50 ? Not determined Corixoidea 28 (24-26) 24 present XY Micronectidae 2 (24-30) absent XY Naucoroidea 10 (20-51) present X0 Notonectoidea Notonectidae 14 (24-26) 24 present XY, Xn0 3 23 present X0 Saldoidea 9 (19-36) 35 present X0, XY Leptopodoidea Leptopodidae 1 28 absent XY Reduvioidea Reduviidae 125 (12-34) 22 absent X0, XY, XnY Microphysoidea Mycophysidae 3 14 absent XY Joppeicoidea Joppeicidae 1 24 absent XY Miroidea 168 (14-80) 34 absent X0, XY, Xn0, XnY Tingidae 28 (12-14) 14 absent X0, XY Naboidea 30 (18-40) 18 absent XY 5 (24-32) absent XY 44 (10-44) 31 absent XY, XnY 3 (6-12) absent XY 33 (7-40) 14/27 absent XY, XnY, neo-system 10 (12-16) 12 absent XY 12 (12-31) 12 absent XY, XnY 7 (14-21) 14 absent XY, XnY 303 (6-27) 14 absent XY, XnY, neo-system 15 (10-14) 12 absent XY 21 (12-14) 12 absent XY 1 12 absent XY Urostylidae 3 (14-16) absent XY 14 (16-42) 16 absent XY 1 14 present XY 402 (10-30) 14/16 present/absent X0, XY, XnY, XYn 4 (22-24) absent XY 11 (11-17) 13/17 present/absent X0, XnY Pyrrhocoridae 21 (12-33) 16 absent X0, Xn0, neo-system 22 (13-17) 13 present X0, Xn0 Coreidae 108 (13-28) 21 present X0, XY, Xn0 25 (13-15) 13 present X0 Stenocephalidae 3 14 present XY, Xn0 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 7

Aires province, Argentina) showed that 24 male indi- organization of single copy and repeated sequences in viduals had the simple system XY, but five specimens comes mainly from extensive studies on from both samples had a multiple system X1X2Y. The Drosophila and early observations in other dipteran comparison of area measurements of the sex chro- species (Blanchelot, 1991), and more recent studies in mosomes in individuals with 2n=16 and 2n=17 homopteran and lepidopteran species (Bizzaro et al., revealed that the relative size of the X1 plus the X2 1996; Manicardi et al., 1996; Spence et al., 1998; Man- did not differ significantly from the relative size of the drioli et al., 1999a, b, c; Mandrioli & Volpi, 2003; single X. This pointed out that the X chromosome of Mandrioli et al., 2003). the simple system has fragmented into two chromo- Despite the general presumption that constitutive somes of unequal size, one of them a little larger than heterochromatin is an inert material, there is abun- the Y chromosome (Papeschi, 1996). dant and increasing evidence that constitutive hete- To date, neo-sex chromosomes in Heteroptera rochromatin can have important functions in chro- have been only found in two species of Lethocerus (L. mosome pairing and segregation, position effect var- indicus Lep. et Servielle, Lethocerus sp. Mayr) (Belo- iegation and can even contain genes and other func- stomatidae) (Chickering & Bacorn, 1933; Jande, tional DNA sequences (Sumner, 2003). 1959), and in Rhytidolomia senilis (Say) (Pentatomi- Early reports on C positive heterochromatin in dae) (Schrader, 1940). In these species, both the heteropterans showed that C bands are terminally ancestral X and Y chromosomes fused each with one located. This led to the suggestion that the principle chromosome of an autosomal pair, resulting in a neo- of equilocal heterochromatin distribution of Heitz X/neo-Y system. The neo-system described in Hebrus (1933, 1935) (i.e. the tendency of heterochromatin of pusillus Fallén (Hebridae) originated through the non-homologous chromosomes to be located at sim- fusion of the ancestral X chromosome with one auto- ilar positions) is also applied to Heteroptera (Solari some (Nokkala & Nokkala, 1999); but the neo-sex & Agopian, 1987; Papeschi, 1991; Panzera et al., chromosome systems of Dysdercus albofasciatus Berg 1995; Cattani et al., 2004; Bressa et al., 2005). Howev- (Pyrrhocoridae) (Bressa et al., 1999) and of Dundo- er, recent reports describe a different distribution coris nodulicarinus noveni Jacobs and D. nodulicarinus pattern in some species. In Tenagobia fuscata (Sta˚l) septeni Jacobs (Aradidae) (Jacobs, 2004) have a more (Corixidae), Triatoma patagonica Del Ponte (Reduvi- complex origin. idae), Petillia patullicollis Walk. (Coreidae) and In Dysdercus albofasciatus the origin of the neo- Nezara viridula (Linnaeus) (Pentatomidae), while XY system involved, most probably, a subterminal most bands are terminally located, one autosomal insertion of the ancestral X chromosome in an auto- pair presents a conspicuous C positive band in an some followed by a large inversion, which included interstitial position (corresponding at least in the part of the original X chromosome (Bressa et al., latter to a nucleolus organizer region) (Camacho et 1999). In Dundocoris nodulicarinus novenus the neo- al., 1985; Dey & Wangdi, 1990; Panzera et al., 1997; Papeschi et al., 2003; Ituarte & Papeschi, 2004). The XY1Y2 system probably originated by the fusion of a large autosome with the X chromosome (neo-X), analysis of 13 species of Tingidae and four species of while the homologue of the fused autosome formed a Nabis (Nabidae) using C banding revealed the pres- neo-Y (=Y ) and the ancestral Y chromosome, the ence of very scarce C positive heterochromatin locat- 1 ed either at telomeric positions or, in some chromo- Y2. The neo-XY1/neo-Y2 sex chromosome system of D. nodulicarinus septeni probably originated by a later somes, at interstitial ones (Grozeva & Nokkala, 2003; et al fusion of one pair of autosomes with the neo-X and Grozeva ., 2004). Finally, a completely different pattern has been described in batatas the original Y chromosome (Y ) (Jacobs, 2004). 2 (Fabricius) (Coreidae), in which large heterochro- matic blocks are located interstitially in all autosomal HETEROCHROMATIN pairs (Franco, 2005). Repeated DNA sequences in insect genomes are Heterochromatin could be an active component organized according to different patterns. They occur of heteropteran chromosomes. Its accumulation in either as families of repeated elements interspersed the karyotype of the species is not a random process throughout the genome or as large arrays usually rep- and some constraints would regulate its acquisition resenting satellite DNA sequences (Brutlag, 1980; and/or accumulation in different karyotypes. Blanchelot, 1991). The current knowledge on the There is still little information about heterochro- 8 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera matin base composition in Heteroptera, i.e. whether tatomomorpha, the families Coreidae, Lygaeidae and the heterochromatin is enriched in AT or GC bases Pentatomidae are the best represented (108, 402 and as revealed by fluorescent banding techniques (DAPI 303 species, respectively). and CMA3 staining, respectively). Most reports refer- Most discussions on karyotype evolution in ring to heterochromatin characterization describe C Heteroptera use the concept of modal chromosome bands as DAPI-bright/CMA3-dull. The presence of number, that is, the commonest number present in a CMA3 bright bands has been reported in a few group. Sometimes this concept can be applied at a species at interstitial or terminal position, either on family level; more often, it is applicable to lower cat- autosomes or sex chromosomes, and they are gener- egories such as tribes or genera. Many times, the ally associated to nucleolus organizer regions (Gonza´- modal number is considered the ancestral one for the lez-García et al., 1996; Papeschi et al., 2001; Papeschi group under analysis (Ueshima, 1979). & Bressa, 2002; Papeschi et al., 2003; Rebagliati et al., Among the principal mechanisms of karyotype 2003; Cattani et al., 2004; Ituarte & Papeschi, 2004; evolution, autosomal fusions and both autosomal and Bressa et al., 2005). sex chromosome fragmentations can be included Sex chromosomes stain brightly with both fluo- (Ueshima, 1979; Manna, 1984; Thomas, 1987; Pape- rochromes, but their fluorescence probably repre- schi, 1994, 1996). Other chromosome rearrange- sents differences in chromatin packaging between the ments, such as inversions and reciprocal transloca- autosomes and the sex chromosome rather than dif- tions have been rarely reported (Papeschi & Mola, ferences in base composition (Rebagliati et al., 2003). 1990; Bressa et al., 1998; Pérez et al., 2004). When Very striking differences in the amount and distri- analyzing karyotype evolution at the family level, bution of C positive heterochromatin have been some karyotypes are highly homogeneous, while observed in species of Triatoma Laporte (Reduviidae). others show intensive processes of karyotype alter- According to Panzera and colleagues (1995), despite ations. the fact that heterochromatin variations represent the The Pentatomidae are one of the largest families main source of karyological differentiation among and of Heteroptera with approximately 760 genera and within species, they do not appear to affect homolo- 4100 species. The family is worldwide in distribution gous chromosome pairing and then they should not and well represented in all of the major faunal play a direct role in the speciation process (Panzera et regions (Schuh & Slater, 1995). Within this family, al., 1992; Pérez et al., 1992; Panzera et al., 1995). 303 species and subspecies belonging to 126 genera have been cytogenetically analyzed. The male diploid CHROMOSOME NUMBER AND numbers are between 6 and 27 with a modal number KARYOTYPE EVOLUTION of 14 chromosomes, which is present in 85% of the The male diploid chromosome number of Heteropte- species. The sex chromosome system is XY/XX, ra ranges from 2n=4 (Lethocerus sp., Belostomati- except for only three species: Macropygium reticulare Rhytidolomia senilis dae) to 2n=80 (four species of Lopidea Uhler, Miri- (Fabricius) (X1X2Y), (neo-X/ dae) (Fig. 1). However, the male diploid number of neo-Y), and Thyanta calceata (Say) (X1X2Y) (Reba- 14 is the most represented (460 species), followed by gliati et al., 2005). This family is cytogenetically highly the diploid numbers 2n=16 (186 species), 2n=34 homogeneous and karyotype changes probably played (92 species), 2n=12 (89 species), 2n=13 (80 spe- a minor role during speciation. cies), 2n=21 (70 species), 2n=22 (76 species), and In Aradidae, at least 211 genera containing about 2n=24 (76 species). The Heteroptera comprise eight 1800 species are currently classified in eight subfami- major groups, and cytogenetic reports are unequally lies. Aradids are represented in all major faunal distributed not only among these groups but also regions with five out of the eight subfamilies being within them: Dipsocoromorpha (6 species), Gerro- essentially cosmopolitan, but with three being re- morpha (34 species), Nepomorpha (97 species), Lep- stricted to the Australian-New Zealand-South Amer- topodomorpha (10 species), Cimicomorpha (407 ican arc (Schuh & Slater, 1995). The known chromo- species) and Pentatomomorpha (1016 species) (Table some numbers of the Aradidae (33 taxa) range from 1). Within the Cimicomorpha, the families Miridae 2n=7 to 2n=48 with two modal numbers of 27 and and Reduviidae are the more extensively studied (168 14, and sex chromosome systems including XY, and 125 species, respectively) and within the Pen- X1X2Y, X1X2X3Y, X1X2X3X4Y and neo-sex chromo- A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 9

FIG. 1. Male diploid chromosome numbers and sex chromosome systems in Heteroptera.

somes (neo-XY1Y2 and neo-XY1/neo-Y2) (Jacobs & (Ueshima, 1979; Franco, 2005). The most common Groeneveld, 2002; Jacobs, 2004). The diversity in sex chromosome system of the family is X0/XX chromosome number and sex chromosome systems (male/female) (64.3%) followed by the multiple found in this family illustrates an intensive process of system X1X20/X1X1X2X2 (32.1%). A multiple system karyotype evolution. Although it is risky to speculate X1X2X30 in males has been ascribed to one popula- on the ancestral chromosome number of the Aradi- tion of Coreus marginatus Linnaeus (0.9%); and three dae, Jacobs & Liebenberg (2001) suggested that since species of Acantocephala (as Metapodius), namely A. 2n=12+XY has been encountered in several genera femorata Fabricius, A. granulosa (Dallas), and A. ter- in three of the six subfamilies thus far studied, it minalis (Dallas), have been reported as having an would probably represent the ancestral number of the XY/XX system (2.7%). However, the three latter family. should be considered as a special situation, since the The Coreidae include at least 250 genera and presence of a variable number of supernumeraries (B 1800 species that are worldwide in distribution, but chromosomes) that associate with the X chromosome are more abundant in the tropics and subtropics complicates the picture (Ueshima, 1979; Papeschi et (Schuh & Slater, 1995). The diploid number of the al., 2003; Cattani & Papeschi, 2004; Cattani et al., family ranges from 13 to 28, with a mode of 21, which 2004; Bressa et al., 2005). is present in 47 out of the 108 species cytogenetically A striking feature of the Xn0 system in this family analyzed (43.5%). The Coreidae are also character- is that the X chromosomes are generally intimately ized by the possession of a pair of m chromosomes associated during male meiosis, and in most species that has been described in 81.9% of the species the tightly conjoined Xs were positioned outside the 10 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera ring of autosomes on both metaphase plates. Manna some system has been described in several species of

(1984) pointed out that in species with the Xn0 sex Pyrrhocoridae (10 out of 19 species), Notonectidae (3 chromosome system the number of X chromosomes out of 14 species), Miridae (1 out of 168 species) and should be confirmed in the spermatogonial comple- Stenocephalidae (1 out of 3 species) (Ueshima, 1979; ment, because during meiosis the multiple Xs tend to Manna, 1984; Grozeva & Nokkala, 1996; Grozeva, fuse and then appear as a single element. Similar 1997; Bressa et al., 1999; Nokkala & Nokkala, 1999; observations have been made by different authors Bressa et al., 2003; Angus et al., 2004). (Manna, 1951; Dutt, 1957; Parshad, 1957; Fossey & Two heteropteran families are included in the Liebenberg, 1995). superfamily Pyrrhocoroidea: Largidae and Pyrrho- Besides the coreids, similar multiple sex chromo- coridae. The former is represented in all major zoo-

TABLE 2. Diploid chromosome numbers in species of Pyrrhocoroidea

Taxa 2n 2n (male) References Largidae Larginae Largus cinctus (Herrich-Schaeffer) 11 10+X0 (Wilson, 1909a) L. fasciatus Blanchard 13 12+X0 (Vidal & Lacau, 1985) L. humilis (Drury) 13 12+X0 (Piza, 1953) L. rufipennis Laporte 13 12+X0 (Piza, 1946; Mola & Papeschi, 1993; Bressa et al., 1998) L. succintus Herrich-Schaeffer 13 12+X0 (Wilson, 1909a) Macrocheraia grandis Gray 15 14+X0 Banerjee, 1958;, Banerjee, 1959; [= Lohita grandis (Gray)] Manna & Deb-Mallick, 1981) Physopeltinae Physopelta cinticollis Sta˚l 15 12+2m+X0 (Ueshima, 1979; Manna et al., 1985) 2 P. gutta Burmeister 17 12+2m+X1X Y (Manna et al., 1985) P. quadrigutta Bergs. 17 12+2m+X1X2Y (Manna et al., 1985) P. schlanbuschi (Fabricius) 17 14+X1X2Y (Ray-Chaudhuri & Manna, 1955)

Pyrrhocoridae Antilochus conqueberti (Fabricius) 27 26+X0 (Parshad, 1957; Banerjee, 1958) Cenaeus abortivus Gerstäcker 33 32+X0 (Ueshima, 1979) Dysdercus albofasciatus Berg 12 10+neo-XY (Bressa et al., 1999) D. chaquensis Freiberg 13 12+X0 (Mola & Papeschi, 1997) D. cingulatus (Fabricius) 16 14+X1X20 (Sharma et al., 1957; Banerjee, 1958; Manna & Deb-Mallick, 1981) D. evanescens Distant 16 14+X1X20 (Manna & Deb-Mallick, 1981) D. fasciatus Signoret 16 14+X1X20 (Banerjee, 1958) D. honestus Bloete 15 14+X0 (Piza, 1947b) D. imitator Bloete 13 12+X0 (Bressa, 2003)

D. intermedius Distant 16 14+X1X20 (Ruthmann & Dahlberg, 1976) D. koenigii (Fabricius) 16 14+X1X20 (Ray-Chaudhuri & Banerjee, 1959) D. peruvianus Guérin-Menéville 16 14+X1X20 (Mendes, 1949; Piza, 1951) D. ruficollis Linnaeus 13 12+X0 (Piza, 1947b)

Dysdercus sp. 16 14+X1X20 (Manna, 1951) D. superstitiosus (Fabricius) 16 14+X1X20 (Banerjee, 1958; Kuznetsova, 1988) Iphita limbata Sta˚l 20 14+X1X2X3X4X5X60 (Banerjee, 1958) Odontopus sexpunctatus Laporte 27 26+X0 (Banerjee, 1958) Pyrrhocoris apterus (Linnaeus) 23 22+X0 (Henking, 1891; Wilson, 1909b) P. tibialis Sta˚l 23 22+X0 (Takenouchi & Muramoto, 1967; Ueshima, 1979)

Pyrrhopeplus posthumus Horvath 24 22+X1X20 (Parshad, 1957) Scantius volucris (Gerstäcker) 19 18+X0 (Parshad, 1957) A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 11 geographic regions, but is most abundant and diverse m chromosomes and have an X0/XX sex chromo- in the tropics and subtropics. Approximately 15 some mechanism. The number of autosomes varies genera and over 100 species are known. The Pyrrho- between 10 and 14. All the studied species that coridae are chiefly tropical and subtropical, with only belong to the other subfamily (Physopeltinae) possess a very few species reaching into temperate Holarctic. 12 autosomes plus two m chromosomes, but different

However, they are found in all major zoogeographic sex chromosome mechanisms (X0 or X1X2Y). regions. Approximately 30 genera and 300 species are Cytologically, Pyrrhocoridae are a heterogeneous known (Schuh & Slater, 1995). family and 21 species belonging to eight genera have From the cytogenetic point of view, 10 species of been cytogenetically analyzed. Chromosome numbers Largidae and 21 species of Pyrrhocoridae are known range from 12 to 33. Dysdercus Guérin-Ménéville, the (Table 2). Six species of the subfamily Larginae lack most studied within this family, is present both

FIG. 2. Dysdercus chaquensis: cells at diakinesis (A) and metaphase I (B); D. ruficollis: cells at diakinesis (C) and metaphase I (D); D. albofasciatus: cells at diakinesis (E) and metaphase I (F); D. imitator: cells at diakinesis (G) and metaphase I (H). Arrows point sex chromosome in A-D, G, H and neo-XY bivalent in E, F. Bar=10 Ìm. Haematoxylin staining. 12 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera in the Old and the New Worlds. So far, from the 13 Hydrocyrius Spinola, and Limnogeton Mayr). Cytoge- cytogenetically analyzed species, seven belong to the netic reports on Belostomatidae refer to the male Old World, while the remaining six are American chromosome complement and meiosis of seventeen species (Ueshima, 1979; Manna & Deb-Mallick, Belostoma species, three Dyplonychus species and 1981; Mola & Papeschi, 1997; Bressa et al., 1999, seven Lethocerus species (Table 3). The modal di-

2002, 2003). The Old World species [(Dysdercus cin- ploid chromosome number is 2n=26+X1X2Y (pre- gulatus (Fabricius), D. evanescens Distant, D. fascia- sent in 11 species of Belostoma), although in both tus Signoret, D. intermedius Distant, D. koenigii (Fa- subfamilies Belostomatinae and Lethocerinae, spe- bricius)], Dysdercus sp. and D. superstitiosus (Fabri- cies with a noticeable reduction in diploid number cius) share a common male diploid chromosome are encountered: 2n=14+XY (three species of Be- number of 16 and an X1X20/X1X1X2X2 sex chromo- lostoma), 2n=6+XY [B. oxyurum (Dufour) and L. some system (Table 2) (Manna, 1951; Banerjee, 1958; americanus Leidy], and 2n=2+neo-X/neo-Y (Le- Ray-Chaudhuri & Banerjee, 1959; Ruthmann & thocerus sp.) (Chickering, 1927b; Chickering & Ba- Dahlberg, 1976; Ueshima, 1979; Manna & Deb-Mal- corn, 1933; Papeschi, 1992; Papeschi & Bressa, 2002). lick, 1981; Kuznetsova, 1988). The six neotropical Our results, together with previous cytogenetic infor- species are much more distinct not only in the male mation on the family, led us to propose an ancestral diploid chromosome number, i.e. from 12 to 16, but karyotype 2n=26+XY, from which the present also in the sex chromosome system (X0, X1X20 and karyotypes 2n=26+X1X2Y should have derived neo-XY, males) (Table 2) (Ueshima, 1979; Mola & through the fragmentation of the ancestral X chro- Papeschi, 1997; Bressa et al., 1999, 2003). Consider- mosome. The analysis of a population of Belostoma ing 2n=15=14+X0 (male) as the ancestral chro- orbiculatum, polymorphic for the sex chromosome mosome complement from Dysdercus, the multiple system, lend support to this hypothesis (Papeschi, sex chromosome system should have originated 1996). The DNA content has been determined in through fragmentation of the original X chromosome nine Argentine species of Belostoma and there are giving rise to a diploid chromosome number 2n=16 significant differences in the DNA content among

=14+X1X20. Both situations are found in Dysdercus them (Table 3). Heterochromatic C positive bands honestus (Mendes, 1947, 1949; Piza, 1947b, 1951) and are terminally located, and since they are DAPI-

D. peruvianus (Piza, 1947a, 1951; Mendes, 1949), bright and CMA3-dull, they probably represent AT- respectively. On the other hand, an autosomal fusion rich DNA sequences (Figs 3, 4). The comparison of between two non-homologous chromosomes could the DNA and heterochromatin content among the have led to a reduction in the diploid number (2n= species suggests that both the amount of C positive 13=12+X0), which has been reported in D. chaque- heterochromatin and C negative chromatin changed nsis (Mola & Papeschi, 1997) and D. ruficollis (Piza, during evolution. The species with reduced chromo- 1947b), as well as in D. imitator (Bressa et al., 2003). some complements are also characterized by a low Finally, a later fusion between the original X chro- DNA content and very scarce C positive heterochro- mosome and an autosome took place during kary- matin. We have suggested that these karyotypes otype evolution, resulting in the neo-XY sex chromo- probably originated from the ancestral chromosome some system in D. albofasciatus, which possesses the complement through chromosome fusions. Besides, lowest diploid chromosome number of the genus the scarce C positive heterochromatin implies either reported so far (2n=12=10+neo-XY) (Fig. 2) that the ancestral karyotype was devoid of this kind of (Bressa et al., 1999). heterochromatin, or else, that it became lost during The Belostomatidae, often referred to as giant the fusion events. water bugs, are distributed worldwide, although their Finally, fluorescence in situ hybridization with an greatest diversity is in the tropics. According to Lauck rDNA probe (Fig. 5) and DAPI and CMA3 banding & Menke (1961), the 146 species of the family can be (Fig. 4) in B. oxyurum (2n=6+XY), B. micantulum grouped into three subfamilies: Lethocerinae (includ- (Sta˚l) (2n=14+XY), and B. elegans (Mayr) (2n=26 ing the cosmopolitan genus Lethocerus Mayr), Hor- +X1X2Y) revealed the presence of a NOR at the vathininae (represented only by the genus Hor- telomeric region of the X and Y chromosomes in the vathinia Montandon), and Belostomatinae (with the first two species, but at the telomeric region of an genera Belostoma Latreille, Diplonychus Laporte, autosomal pair in the latter. In the three species, the A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 13 ) 3 3 9 1

, n r o c a B

&

g n i r e k c i h C

; 2 3 9 1

, a 7 2 idau, 1985; Papeschi, 1988, 1991) 9 Bidau, 1985; Papeschi, 1991) Bidau, 1985; Papeschi, 1988) Bressa, 2004) B 1

, g n i r e k c i h C (Papeschi & (Papeschi & (Jande, 1959) (Papeschi, 1996) (Papeschi, 1994) (Banerjee, 1958; Bagga, 1959; Jande, 1959) (Chickering, 1927b) (Papeschi & Bressa, 2004) (Jande, 1959) (Papeschi, 1988) (Papeschi, 1991) (Papeschi, 1992) (Papeschi & (Papeschi, 1992) (Papeschi, 1992) (Papeschi, 1991) (Bawa, 1953; Jande 1959) (Chickering, 1932) (Mongomery, 1901, 1906) (Papeschi & (Papeschi & Bidau, 1985) (Papeschi, 1992) (Ueshima, 1979) ( (Chickering, 1927b) (Papeschi, 1992) (Chickering, 1916, 1927a) References – – – – – – – – – – – – – – – – – – 58 30 15 60 38 60 59 37 25 % Het. – – – – – – – – – – – – – – – – – – 0.88 1.21 1.93 1.02 1.55 1.74 1.13 1.11 0.53 (pg) DNA (1C) Y Y Y Y Y Y Y Y Y Y Y 2 2 2 2 2 2 2 2 2 2 2 X X X X X X X X X X X 1 1 1 1 1 1 1 1 1 1 1 6+XY 6+XY 26+XY 14+XY 14+XY 26+XY 26+XY 14+XY 22+XY 26+XY 26+XY 22+XY 26+XY 2n (male) 26+X 26+X 26+X 26+X 26+X 26+X 26+X 26+X 26+X 26+X 26+X 2+neoX-neoY 24+neoX-neoY 6 9 8 4 8 2 28 16 16 28 16 24 28 28 2 29 28 29 29 24 29 29 29 29 28 29 29 29 2n ca. 30 Spinola Leidy (Fabricius) (Herrich-Schaeffer) l) ˚ (Mayr) l) ˚ (Sta Estévez & Polhemus De Carlo (Haldeman) (Say) De Carlo Montandon Montandon (Dufour) (Mayr) (Sta (Dufour) Lep. et Ser. sp. (Fabricius) sp. (Montandon) (Mayr) (Say) Montandon (Montandon) (Montandon) B. oxyurum B. indentatum B. orbiculatum B. plebejum D. subrhombeus Lethocerus annulipes Lethocerus americanus L. griseus L. indicum L. melloleitaoi Lethocerus L. uhleri D. rusticus B. bergi B. cummingsi B. dentatum B. dilatatum B. discretum B. elegans B. elongatum B. gestroi B. martini B. micantulum B. flumineum Belostoma Diplonychus annulatus Belostoma bifoveolatum Lethocerinae TABLE 3. Diploid chromosome numbers, DNA and heterochromatin content in Belostomatidae Taxa Belostomatinae 14 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera

FIG. 3. C-banding of cells at diakinesis of Belostoma oxyurum (A), B. micantulum (B), B. elegans (C), B. dilatatum (D), B. elongatum (E), and B. dentatum (F). Arrows point sex chromosomes. Bar=10 Ìm. A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 15

FIG. 4. DAPI (A, C, E) and CMA (B, D, F) banding in: spermatogonial prometaphase (A, B) of Belostoma oxyurum; diffuse stage (C, D) of B. micantulum; diakinesis (E, F) of B. elegans. Arrowheads point CMA3-bright bands corresponding to NOR regions. Arrows point sex chromosomes. Bar=10 Ìm. 16 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera

FIG. 5. Fluorescence in situ hybridization with an 808 bp 18S rDNA probe in: spermatogonial prometaphase (A) and diffuse stage (B) of Belostoma oxyurum; spermatogonial prometaphase (C) and diffuse stage (D) of B. micantulum; diakinesis (E, F) of B. elegans. Asterisks point rDNA regions. Bar=10 Ìm. A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera 17

cutta, 12: 19-22. NOR region is also detected as a CMA3-bright/ DAPI-dull band. These observations suggest that the Banerjee MK, 1958. A study of the chromosomes during reduction in the diploid number probably involved meiosis in twenty-eight species of the fusion of the ancestral sex chromosome pair with (Heteroptera, Homoptera). Proceedings of the zoo- the autosomes carrying the NOR. Alternatively, the logical society of Calcutta, 11: 9-37. Banerjee MK, 1959. Chromosome elimination during rDNA gene cluster could have been translocated meiosis in the males of Macroceroea (Lohita) grandis from an autosomal location (as in B. elegans) to the X (Gray). Proceedings of the zoological society of Calcut- B. micantulum and Y chromosomes (as detected in ta, 12: 1-8. and B. oxyurum). Further studies in other species of Bawa SR, 1953. Studies on insect spermatogenesis. I. He- Belostoma will shed light on the karyotype evolution miptera-Heteroptera. The sex chromosomes and within the genus. cytoplasmic inclusions in the male germ cells of Lac- cotrephes maculatus Fabr. and Sphaerodema rusticum CONCLUDING REMARKS Fabr. Research bulletin east of the Panjab university, 39: 181-192. More than a hundred years have gone since Henk- Bizzaro D, Manicardi GC, Bianchi U, 1996. Chromosomal ing’s first report on Pyrrhocoris apterus, but chromo- localization of a highly repeated EcoRI DNA frag- some organization, function and evolution in ment in Megoura viciae (Homoptera, ) by Heteroptera is far from being fully understood. nick translation and fluorescence in situ hybridiza- Molecular cytogenetic techniques are promising tools tion. Chromosome research, 4: 392-396. that will help uncover the mechanisms governing Blanchelot A, 1991. A Musca domestica satellite sequence chromatin organization of holokinetic chromosomes, detects individual polymorphic regions in insect their behaviour during mitosis and meiosis, and the genome. Nucleic acids research, 19: 929-932. importance of chromosome change during the speci- Bressa MJ, 2003. Citogenética ba´ sica y evolutiva de He- ation of heteropterans. tero´ pteros fito´ fagos de interés agroecono´ mico de la Fusions and fragmentations, along with variations Argentina. Tesis Doctoral, Universidad Nacional de La Plata. in the amount of heterochromatin, location and con- Bressa MJ, Papeschi AG, Mola LM, Larramendy ML, stitution are the principal mechanisms of karyotype 1998. Meiotic studies in Largus rufipennis (Castelnau) evolution so far reported in Heteroptera. However, (Largidae, Heteroptera). II. Reciprocal translocation different genera and even families are karyotypically heterozygosity. Heredity, 51: 253-264. more variable than others, a fact that probably Bressa MJ, Papeschi AG, Mola LM, Larramendy ML, reflects differences in the biology of the species, and 1999. Meiotic studies in Dysdercus Guérin Méneville their environmental relationships. The coming years 1831 (Heteroptera: Pyrrhocoridae). I. Neo-XY in will probably outline some answers, but also bring Dysdercus albofasciatus Berg 1878, a new sex chro- about more new questions. mosome determining system in Heteroptera. Chro- mosome research, 7: 503-508. ACKNOWLEDGEMENTS Bressa MJ, Fumagalli E, Ituarte S, Frassa MV, Larramendy ML, 2002. Meiotic studies in Dysdercus Guérin Mé- The authors wish to thank Dra. Lidia Poggio, Dr. neville 1831 (Heteroptera: Pyrrhocoridae). II. Evi- Carlos Naranjo, and Dr. Axel Bachmann for their dence on variations of the diffuse stage between wild teachings and guidance. Different grants from and laboratory-inbred populations of Dysdercus cha- CONICET, CIC, ANPCyT, Fundacio´n Antorchas, quensis Freiberg, 1948. Hereditas, 137: 125-131. UNLP and UBA made possible to carry out our Bressa MJ, Papeschi AG, Fumagalli E, van Doesburg PH, investigations. Larramendy M, 2003. Cytogenetic and nucleolar meiotic cycle analyses in Dysdercus imitator Blöte, 1931 (Heteroptera, Pyrrhocoridae) from Argentina. REFERENCES Folia biologica, 51: 135-141. Angus RB, Kemeny CK, Wood EL, 2004. The C-banded Bressa MJ, Larramendy M, Papeschi AG, 2005. Hete- karyotypes of the four British species of Notonecta L. rochromatin characterization in five species of (Heteroptera: Notonectidae). Hereditas, 140: 134- Heteroptera. Genetica, 124: 307-317. 138. Brutlag DL, 1980. Molecular arrangement and evolution of Bagga S, 1959. On the pre-reduction of the sex chromo- heterochromatic DNA. Annual review of genetics, 14: somes during meiosis in a belostomatid Lethocerus 121-144. indicum. Proceedings of the zoological society of Cal- Buck RC, 1968. Mitosis and meiosis in Rhodnius prolixus: 18 A. G. Papeschi and M. J. Bressa — Evolutionary cytogenetics in Heteroptera

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