8urn:lsid:arphahub.com:pub:A71ED5FC-60ED-5DA3-AC8E-F6D2BB5B3573urn:lsid:zoobank.org:pub:C8FA3ADA-5585-4F26-9215-A520EE683979Comparative CytogeneticsCCG1993-07711993-078XPensoft Publishers10.3897/CompCytogen.v10i4.1039210392Research ArticleHemipteraHeteropteraInsectaEvolutionary biologyGeneticsSystematicsBrazilSouth AmericaAnalysis of the karyotype structure in Ricollaquadrispinosa (Linneus, 1767): inferences about the chromosomal evolution of the tribes of Harpactorinae (Heteroptera, Reduviidae)TiepoAngélica Nunes1PezentiLarissa Forim1Ferraz LopesThayná Bisson1da SilvaCarlos Roberto Maximiano1DionisioJaqueline Fernanda1FernandesJosé Antônio Marin2Da RosaRenata1renata-darosa@uel.brhttps://orcid.org/0000-0003-4258-7244Departamento de Biologia Geral, CCB, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445, Km 380, Caixa Postal 6001, CEP 86051-970, Londrina, PR, BrasilUniversidade Estadual de LondrinaLondrinaBrazilInstituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Universidade Federal do Pará, 66075-110; PA, BrasilFederal University of ParáParáBrazil
Corresponding author: Renata da Rosa (renata-darosa@uel.br)
Academic editor: N. Golub
201609122016104719729374AE00D-FFED-5748-153E-1167FFCD773AB7D2A6DB-D7B8-4A37-9DD2-57F5296861361985660509201601112016Angélica Nunes Tiepo, Larissa Forim Pezenti, Thayná Bisson Ferraz Lopes, Carlos Roberto Maximiano da Silva, Jaqueline Fernanda Dionísio, José Antônio Marin Fernandes, Renata Da RosaThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/B7D2A6DB-D7B8-4A37-9DD2-57F529686136
The subfamily Harpactorinae is composed of six tribes. Phylogenetic studies bring together some of Harpactorinae tribes, but by and large the data on evolutionary relationships of the subfamily are scarce. Chromosome studies are of great importance for understanding the systematics of different groups of insects. For Harpactorinae, these studies are restricted to some subfamilies and involved only conventional chromosome analysis. This work analyzed cytogenetically Ricollaquadrispinosa (Linneus, 1767). The chromosome number was determined as 2n = 24 + X1X2Y in males. In metaphase II the autosomal chromosomes were organized in a ring with the pseudo-trivalent of sex chromosomes in its center. After C-banding followed by staining with DAPI, AT-rich blocks in autosomes were observed and the negatively heteropycnotic sex chromosomes. The data obtained, together with existing data for other species of the group, indicated that different chromosomal rearrangements are involved in the evolution of the species. In addition, a proposal of karyotype evolution for the subfamily, based on existing phylogenetic studies for the group is presented.
Tiepo AN, Pezenti LF, Lopes TBF, da Silva CRM, Dionisio JF, Fernandes JAM, Da Rosa R (2016) Analysis of the karyotype structure in Ricolla quadrispinosa (Linneus, 1767): inferences about the chromosomal evolution of the tribes of Harpactorinae (Heteroptera, Reduviidae). Comparative Cytogenetics 10(4): 719–729. https://doi.org/10.3897/CompCytogen.v10i4.10392
Introduction
Reduviidae are the largest family of predatory insects of the suborder Heteroptera, consisting of approximately 7000 species (Kaur et al. 2009, Weirauch et al. 2014). Harpactorinae is the largest subfamily of Reduviidae, and is composed of six tribes: Apiomerini, Diaspidiini, Ectinoderini, Harpactorini, Tegeini and Rhaphidosomatini (Schuh and Slater 1995, Zhang et al. 2015). However, some authors consider Dicrotelini as a tribe (Miller 1954, Tomokuni and Cai 2002, Weirauch et al. 2014). Phylogenetic studies suggest that the first three tribes form a separate clade from the last three tribes (Davis 1969, Coscaron and Melo 2003, Zhang and Weirauch 2014, Zhang et al. 2015).
In Harpactorinae cytogenetic studies are restricted to only three of the six tribes: Apiomerini, Dicrotelini, and Harpactorini (Table 1), showing diploid numbers ranging from 12 to 30, a predominance of 24 autosomes and several sex systems (XY, XnY) (Table 1) (Kuznetsova et al. 2011, Kaur and Kaur 2013). Probable, the cytogenetical variations result from chromosomal rearrangements in autosomes and sex chromosomes. This type of alteration is an important factor in the speciation process, since causing dramatic effects on fertility (Spirito 1998, Rieseberg 2001, Livingstone and Rieseberg 2003, Nosil et al. 2009, Macaya-Sanz et al. 2011).
Cytogenetic studies in Harpactorinae.
Tribe
Species
Diploid number (♂)*
Reference
Apiomerini
Apiomeruslanipes
22A+XY
Poggio et al. 2007
Apiomeruscrassipes
22A+XY
Payne 1912
Apiomerusflaviventris
22A+XY
Ueshima 1979
Apiomerusspissipes
22A+XY
Ueshima 1979
Apiomerus sp.
22A+XY
Ueshima 1979
Heniarteshuacapistana
22A+XY
Ueshima 1979
Dicrotelini
Henricohahniatypica
24A+X1X2X3Y
Kaur and Kaur 2013
Harpactorini
Achollaampliata
24A+X1X2X3Y
Payne 1909
Achollamultispinosus
20A+X1X2X3X4X5Y
Troedsson 1944
Ariluscristatus
22A+X1X2X3Y
Payne 1909
Coranusfuscipennis
24A+X1X2Y
Jande 1959
Coranus sp.
24A+X1X2Y
Kaur and Kaur 2013
Cosmoclopiusnigroannulatus
24A+X1X2X3Y
Poggio et al. 2007
Cosmoclopiuspoecilus
24A+X1X2X3Y
Poggio et al. 2007
Cydnocoriscrocatus
24A+X1X2Y
Dey and Wangdi 1988
Euagoraserythrocephala
24A+X1X2Y
Kaur and Kaur 2013
Euagorasplagiatos
24A+X1X2Y
Kaur and Kaur 2013
Fitchiaspinulosa
24A+ X1X2Y
Payne 1909
Harpactorfuscipes
24A+X1X2X3Y
Ueshima 1979
Iranthaarmipes
24A+X1X2X3Y
Kaur and Kaur 2013
Lophocephalaguerini
24A+X1X2Y
Satapathy and Patnaik 1989
Montinaconfusa
12+XY
Bardella et al. 2014
Polididusarmatissimus
10A+XY
Banerjee 1958
Polididus sp.
10A+XY
Manna and Deb-Mallick 1981
Pselliopuscinctus
24A+ X1X2X3Y
Payne 1912
Repiptaflavicans
18A+XY
Bardella et al. 2014
Repiptataurus
24A+ X1X2X3Y
Kaur et al. 2012
Ricollaquadrispinosa
24+X1X2Y
Present study
Rhynocoriscostalis
24A+X1X2X3Y
Kaur and Kaur 2013
Rhynocorisfusicipes
24A+ X1X2X3Y
Dey and Wangdi 1988
Rhynocoriskumarii
24A+X1X2X3Y
Kaur and Kaur 2013
Rhynocorismarginatus
24A+ X1X2X3Y
Satapathy and Patnaik 1989
Rhynocoris sp.
24A+X1X2X3Y
Kaur and Kaur 2013
Rocconotaannulicornis
24A+X1X2Y
Payne 1909
Sineacomplexa
24A+X1X2X3Y
Payne 1909
Sineaconfusa
24A+X1X2X3Y
Payne 1909
Sinearileyi
24A+X1X2X3 X4X5Y
Payne 1912
Sineaspinipes
24A+X1X2X3Y
Payne 1909
Sphedanolesteshimalayensis
24A+X1X2X3Y
Kaur and Kaur 2013
Sycanuscollaris
24A+X1X2X3Y
Jande 1959
Sycanuscroceovittatus
24A+X1X2X3Y
Kaur and Kaur 2013
Sycanus sp.
24A+X1X2X3Y
Manna 1951
Velinusnodipes
24A+X1X2X3Y
Toshioka 1936
Velinusannulatus
24A+X1X2X3Y
Kaur and Kaur 2013
Vesbiuspurpureus
24A+XY
Manna and Deb-Mallick 1981
Zelusexsanguis
24A+XY
Payne 1909
Zelus sp. close to Z.leucogrammus
24A+XY
Poggio et al. 2007
Zeluslaticornis
24A+XY
Bardella et al. 2014
♂ – males; A – autosomes; XY – sex system XY
Evolutionary relationships related to karyotype changes are poorly known for Harpactorinae, and the majority of karyological reports in Harpactorinae are restricted to conventional analysis without the application of banding techniques (Cai and Tomokuni 2003). The present study analyzed cytogenetically, for the first time, Ricollaquadrispinosa (Linneus, 1767) (Harpactorini) in order to elucidate its karyotype structure and relate this to existing data on Harpactorinae. In addition, we presented different proposals for the phylogenetic relationships of this group based on the chromosomal data available so far.
Material and methodsSamples and collection sites
Fifteen male specimens of R.quadrispinosa were collected from Iguaçu National Park - Foz do Iguaçu - Brazil - 25°37'40.67"S; 54°27'45.29"W (DDM). Each individual was identified and deposited at the
Federal University of Pará
(UFPA).
Chromosome preparations and conventional staining
The gonads of the adult specimens were dissected in physiological solution for insects (7.5g NaCl, 2.38g Na2HPO4, 2.72g KH2PO4 in 1L of distilled water). The testes were treated with tap water for 3 min and fixed in methanol:acetic acid (3:1) for 30 min. Chromosome preparations were performed through cellular suspension by maceration in a drop of 60% acetic acid, with each gonad previously treated with 45% acetic acid. These preparations were submitted to conventional staining with Giemsa 3% and also to chromosome banding techniques. Chromosome measurements were carried out using the computer application MicroMeasure version 3.2 (Reeves and Tear 2000).
Chromosome banding
The distribution of heterochromatin was analyzed by Giemsa C-banding according to Sumner (1972), after treatment with 0.2M HCl for 10 min at room temperature, Ba (OH)2 for 1 min and 40 s at 60 °C, and 2× SSC for 1 hour at 60 °C. The AT-rich bands were detected with
4’-6-diamino-2-phenylindole
(DAPI), respectively, according to Schweizer et al. (1983). The slides were stained with 2µg/mL DAPI for 30 min. Slides were mounted with a medium composed of glycerol/McIlvaine buffer (pH 7.0) 1:1, plus 2.5mM MgCl2. All images were acquired with a Leica DM 4500 B microscope, equipped with a DFC 300FX camera and Leica IM50 4.0 software, and optimized for best contrast and brightness with iGrafx Image software.
Results
The males of R.quadrispinosa presented 2n = 24 + X1X2Y. In metaphase II, the autosomes are arranged in ring while the three sex chromosomes form a pseudo-trivalent in the center (Fig. 1a, b, d, e). After C-banding, sex chromosomes were shown to be negatively heteropycnotic at all stages (Fig. 1a, b). The C-banding followed by conventional staining highlighted positive heteropycnotic blocks in the interphase nuclei (Fig. 1c). It was also possible to observe positive heteropycnotic blocks in terminal regions of the majority of autosomes and in interstitial region of one pair of chromosomes (Fig. 1d).
192B0674-00F8-5DE9-9F1F-DF8FD4A2EA1E
Meiocytes of Ricollaquadrispinosa. A, B conventional staining: metaphase II C conventional staining: interphase nucleus D Giemsa C-banding: metaphase II EDAPI staining: metaphase II. Sex chromosomes indicated by arrows. Interstitial heterochromatic block indicated by asterisk. Scale bar: 5µm.
https://binary.pensoft.net/fig/114273
The fluorochrome staining with DAPI performed after the C-banding revealed several AT-rich blocks in the autosomes, which were located in both the terminal and interstitial regions of the autosomes while the sex chromosomes were shown to be negatively heteropycnotic (Fig. 1e).
Discussion
The number of autosomes observed in R.quadrispinosa (24) was similar to that revealed in the most species of the tribe Harpactorini (Table 1), and represents the karyotype conservation regarding the number of autosomes in this group. On the contrary, the multiple sex system observed in R.quadrispinosa (X1X2Y) has only been reported in another eight species (Table 1) (Kaur and Kaur 2013, Jande 1959, Dey and Wangdi 1988, Payne 1909, Satapathy and Patnaik 1989). Cytogenetic data exist only for three tribes: Apiomerini, Dicrotelini, and Harpactorini (Table 1). These studies are scarce considering the great diversity of the subfamily, with approximately 2800 species (Weirauch et al. 2014).
Within Harpactorinae, there is a very striking karyotype conservation in the Apiomerini tribe, where all species studied so far have presented 2n = 22 + XY (Table 1). As of now, these data support the proposed phylogeny for the group (Tomokuni and Cai 2002, Weirauch 2008, Hwang and Weirauch 2012, Zhang et al. 2015), where Apiomerini form a clade separate from Harpactorini. Analyzing the existing cytogenetic data and those obtained by us, a large karyotype variation within Harpactorini can be seen with 2n = 12 to 2n = 30 and different sex systems (Table 1), which reinforces its phylogenetic distance from Apiomerini.
According to Poggio et al. (2007), the ancestral chromosome number in Reduviidae is 2n = 28, with XY system, while the karyotypes with 22 autosomes are more common in Reduviidae (Ueshima 1979). Considering this, two evolutionary trends may be proposed for Harpactorinae: (i) reduction in the number of autosomes through episodes of chromosomic fusion, and (ii) increase in the number of sex chromosomes due to chromosomic fission events. Thus the occurrence of fissions and fusions probably gave rise to the karyotype R.quadrispinosa, and put the Apiomerini species in a condition closer to an ancestral karyotype
In the Harpactorini, twenty-one species present 2n = 24 + X1X2X3Y and 9 species present 2n = 24 + X1X2Y (Table 1). Karyotypes with multiple systems with a larger number of X chromosomes are observed only in two species, Achollamultispinosus (De Geer, 1773) (2n = 20 + X1X2X3X4X5Y) and Sinearileyi Montandon, 1893 (2n = 24 + X1X2X3X4X5Y). Although the variation in the sex chromosome systems is large, the number of autosomes is the same in the majority of species. In Heteroptera, the most common sex mechanism is XX/XY (Papeschi and Bressa 2006). Two hypotheses regarding the evolution of sex systems in Heteroptera have been proposed. The first hypothesis suggests that advanced Heteroptera, derived from the extinct group Gerromorpha, still have the plesiomorphic condition X0; thus, the XX/XY system is derived (Ueshima 1979). In contrast, the second hypothesis suggested that the X0 system is derived from the ancestral system XY (Nokkala and Nokkala 1983, 1984, Grozeva and Nokkala 1996). This last hypothesis appears to be plausible, since studies by Grozeva et al. (2014) in Xenophyescacus Bergroth, 1924 (Peloridiidae, the sister group of Heteroptera) show a tendency to lose the Y chromosome during evolution. Regarding the origin of multiple sex systems, Ueshima (1979) and Papeschi and Bressa (2006) state that they are probably the result of fragmentations of the original sex chromosomes. This would likely be the origin of the multiple sex systems of R.quadrispinosa, which have originated by breaks in the XY sex systems of ancestors.
For Dicrotelini, the only species have been cytogenetically studied, Henricohahniatypical Breddin, 1900 with 2n = 28 (Kaur and Kaur 2013). If consider only the diploid number, this species would be closer to the species of the Harpactorini. However, according to the phylogeny proposed by Zhang et al. (2015), the Dicrotelini form a separate clade, closer to Apiomerini than Harpactorini. In this way, due to lack of cytogenetic studies in the group, it is not possible to trace an evolutionary line within the tribe. The analysis of more species of Dicrotelini could help to elucidate this hypothesis.
Even taking into account the phylogenetic studies for the group proposed by Zhang et al. (2015), cytogenetic analyzes corroborate the differentiation of Apiomerini from Harpactorini, the former being the more conserved tribe. It is possible to group the species with similar karyotypes within Harpactorini, where those with low diploid numbers and simple sex system form separate branches from those with a higher diploid number and multiple sex systems. Considering the above, coupled with the chromosomal number found in the sister group and most of the species of the subfamilies of Reduviidae, we propose an ancestor with 2n = 24 (22 + XY) for Harpactorinae (Fig. 2). Apiomerini would have remained closer to the ancestral karyotype. Observing Figure 2, it is possible to note that an autosome fusion event (event A) would have given rise to the karyotypes of the species with 2n = 12 + XY, and then a second fusion event (event B) would have originated the karyotypes of the species with 2n = 10 + XY. These karyotypes are observed in Montinaconfusa (Stål, 1859) (Bardella et al. 2014) and two species of the genus Polididus Stål, 1858 (Banerjee 1958, Manna and Deb-Mallick 1981), respectively. This result can be confirmed by molecular analysis, where these genera are grouped forming a separate clade within Harpactorinae (Zhang and Weirauch 2014).
E7F23A3F-C06B-531A-BA1D-9C2B2CC1A594
Chromosomal evolution in Apiomerini and Harpactorini. Evolutionary events marked by caps: A fusion of autosomes B fusion of autosomes C fission of autosomes D fusion of autosomes E fission of sex chromosomes F fission of sex chromosomes. Chromosomal formulae represent the diploid number. The tribe Dicrotelini was not included in scheme because only one species has been studied cytogenetically in this tribe.
https://binary.pensoft.net/fig/114274
Another chromosomal alteration, the fission of autosomal chromosomes (C event) would have led to a new branch within the Harpactorini, originating 2n = 24 + XY (Zelus Fabricius, 1803 and Vesbius Stål, 1866 species) (Table 1). Also in this branch, chromosomal fusion events (D event) would originate the karyotypes with 2n = 18 + XY (Fig. 2), observed in Repiptaflavicans (Amyot and Serville, 1843) (Bardella et al. 2014). Phylogenetically Zelus and Repipta are close (Zhang and Weirauch 2014) and occupy a separate branch within the Harpactorini distant from other species with simple sex systems, which allows us to put them in this position with respect to the karyotypic evolution. Variations in this karyotype were observed in R.taurus (Fabricius, 1803) (Kaur et al. 2012).
The multiple sex systems would have arisen by the fission of the X chromosomes of the ancestral XY system (event E) to give the karyotype 2n = 24 + X1X2Y, observed in nine species of the tribe (Table 1). A second fission event of sex chromosomes (F event) would have led to the most common karyotype observed in Harpactorini, 2n = 24 + X1X2X3Y, with maintenance of the number of autosomes revealed in twenty-one species of the tribe (Table 1). This explains the intermediate position of the species with these karyotypes in the phylogeny of Harpactorini (Zhang and Weirauch 2014). So far only the one species of Harpactorini, Sinearileyi (Payne 1912), has a different karyotype with 2n = 24 + X1X2X3X4X5Y, that probably represents an isolated event of karyotypic variation, since all other species of the genus have 28 chromosomes (Table 1).
In addition to differences in the number of autosomes and sex chromosomes, in R.quadrispinosa the sex chromosomes are presented as negatively heteropycnotic. Also in metaphase II it is possible to notice several AT rich blocks occupying the terminal and, rarely, interstitial regions of the autosomes. Different patterns of heterochromatin have been reported in other 5 Harpactorinae species (Bardella et al. 2014). Thus in Apiomeruslanipes (Fabricius, 1803) the presence of terminal C-DAPI+/CMA3+ bands in the terminal region was shown, and the heterochromatic sex chromosomes of this species exhibit different florescent patterns. In Montinaconfusa (Stål, 1859) C-DAPI+/CMA3+ bands were observed in both terminal regions of the two largest autosomes and sex chromosomes. M.confusa also showed the third autosomal pair with a C-DAPI+/CMA3+ band in only one terminal region, whereas the three smaller pairs were totally C-DAPI+/CMA3+. In Cosmoclopiusnigroannulatus Stål, 1860 and Zeluslaticornis (Herrich-Schaeffer, 1853) only one of the sex chromosomes in each species was totally DAPI+ and CMA3+ in each species. Repiptaflavicans (Amyot & Serville, 1843) has not demonstrated fluorescent bands in autosomes and sex chromosomes (Bardella et al. 2014). Thus, in Harpactorinae a wide variety of different patterns of C-heterochromatin distribution between the chromosomes was revealed, that implying a large divergence in the karyotypic evolution of species of this subfamily.
Considering the influence of the chromosomal rearrangements in the speciation processes, particularly those involved in the differentiation of sex chromosomes, we can suggest that these alterations were fundamental as mechanisms of pre-zygotic reproductive isolation. It is probable that these chromosomal alterations caused the separation of groups, as different species are observed in the same geographical region, leading to a process of sympatric speciation.
Acknowledgments
The authors are grateful to the National Iguaçu Park staff members for their technical assistance; to Edson Mendes Francisco for his help with the sample collection. This work was supported by CNPq, Fundação Araucária and FAEPE/UEL-PUBLIC 2016. The researchers received permission from the Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio to collect specimens.
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