Karyotype and sex chromosome differentiation in two Nalassus species (Coleoptera, Tenebrionidae)

Dirim Şendoğan; Nurşen Alpagut-Keskin

doi:10.3897/CompCytogen.v10i3.9504

Abstract

Cytogenetic features of Nalassus bozdagus Nabozhenko & Keskin, 2010 and Nalassus plebejus Küster, 1850 were analysed using conventional and differential staining. Mitotic and meiotic chromosomal analysis revealed the diploid number as 2n = 20 (9+Xy_p) in both species. Besides the general resemblance of two Nalassus Mulsant, 1854 karyotypes, important differences related to variations in the number of metacentric/submetacentric chromosomes, localization of highly impregnated regions which are considered as NOR and heterochromatin distribution are clearly observed. The most prominent difference between two species is found related to the X chromosome which is clearly larger in N. bozdagus and has a conspicuous secondary constriction on the long arm. As a result of silver staining, the existence of highly impregnated areas associated with Xy_p of N. bozdagus in both prophase I and metaphase I, suggests that NORs are seemingly located on sex chromosomes. On the other hand, the potential NORs of N. plebejus were observed only in prophase I nuclei. With the application of fluorescence dye DAPI, the AT rich chromosome regions and Xy_p which forms the parachute configuration were shown in both species.

Keywords

Karyotype, Tenebrionidae , Helopini , Nalassus bozdagus , Nalassus plebejus , sex chromosomes, heterochromatin, NOR, DAPI

Introduction

In the light of fossil and molecular dating analysis, the darkling beetles are dispersed and diversified over the last 180 million years prior to Gondwanan fragmentation. Tenebrionids represent a hyperdiverse family of Coleoptera with ca. 20000 recognized species worldwide. In consequence of undergoing multiple evolutionary radiations, tenebrionids show considerable morphological variations and several adaptations in life history traits such as feeding behaviour, habitat preferences, flight ability etc. Although higher level of tenebrionid phylogeny based on sequences from seven out of nine subfamilies shows well supported monophyly, the subfamilies Diaperinae, Pimeliinae and Tenebrioninae were recovered as paraphyletic or polyphyletic (Kergoat et al. 2014a, b).

The karyotypes of more than 250 darkling beetle species have been determined (Holecová et al. 2008a, Juan and Petitpierre 1991a, Blackmon and Jeffery 2015, Gregory 2016). Although most species present a karyotype with 2n = 20, the diploid number ranges from 2n = 14 to 2n = 38 (Juan and Petitpierre 1991a, Pons 2004, Holecová et al. 2008a, Lira-Neto et al. 2012). Chromosomal data are only available for several representatives of subfamilies Lagriinae, Tenebrioninae, Pimeliinae, Alleculinae and Diaperinae mostly distributed in Mediterranean (Juan and Petitpierre 1991a).

The genus Nalassus Mulsant, 1854 (Tenebrioninae: Helopini) comprises 71 described taxa distributed mainly in the Western Palearctic, but with disjunctively isolated species in the Russian Far East and Northern China (Medvedev 1987, Nabozhenko 2001). Even though a significant part of the species is found in alpine and subalpine mountainous belts with high level of local endemism, some species that are adapted to lowlands have wider distribution. In the recent reviews of Nalassus species from the European part of CIS (Commonwealth of Independent States), Caucasus, Iran, Georgia, China and Turkey, several new species and combinations were also noted (Nabozhenko 2001, Nabozhenko 2007, Nabozhenko 2008, Keskin and Nabozhenko 2010, Nabozhenko 2011, Nabozhenko 2013, Nabozhenko 2014, Nabozhenko and Ivanov 2015). Therefore, the actual diversity is certainly higher than previously estimated and the monophyly of the genus Nalassus needs to be tested with several new additional characters. The chromosomes of Nalassus have not yet been studied. Furthermore, cytogenetic data concerning the tribe Helopini which provide no more than chromosome numbers and sex determination systems are only known for some Nesotes and Probaticus species (Juan and Petitpierre 1986, 1989, 1991a, 1991b, Palmer and Petitpierre 1997).

In this study, with the aim of providing first cytogenetic information about Nalassus, the mitotic and meiotic chromosomes of endemic N. bozdagus and widespread N. plebejus were analysed using conventional, DAPI fluorochrome staining and silver impregnation.

Material and methods

The meiotic and mitotic chromosomes of 12 male N. plebejus and 4 male N. bozdagus individuals from Western Anatolia were analysed. The specimens of Nalassus plebejus were retrieved from Ballıkayalar Natural Park, Gebze (40°50'22.96"N / 29°30'56.11"E, 120m) and the specimens of Nalassus bozdagus were collected from Bozdağ, İzmir (38°15'17.49"N / 27°57'44.72"E, 2300m). Adult beetles were collected on the trunks of trees and on the ground at night when they are active.

The chromosome preparations were obtained from the gonads of male specimens using Murakami and Imai’s (1974) splashing method with some modifications. Briefly, testes were carefully dissected and macerated with sterilized needles. Testes were treated with hypotonic solution (0.65% KCl) for 5 minutes and fixed in 3:1 ethanol: acetic acid at least for 1 h on ice.

We also applied a microspreading method (Chandley 1994) for obtaining prophase I nuclei. The slides were stained with 4% Giemsa in phosphate buffer pH 6.8, for 20 minutes for standard staining. The silver impregnation technique of Patkin and Sorokin (1984) was performed to determine the possible NOR regions. Briefly, slides were incubated in distilled water for 30 min. at room temperature and stained with AgNO₃ working solution (2:1, 50% AgNO₃: 2% gelatin containing 0.5% formic acid) in a humid chamber at 60 °C for 3-10 minutes. After a golden-brown color has developed, the reaction was stopped by rinsing with distilled water. Slides were then dehydrated, counterstained with 4% Giemsa in phosphate buffer pH 6.8.

For determining of heterochromatin distribution, the slides were mounted with antifade mounting medium with fluorochrome DAPI (Vectashield) specific to AT-rich chromosomal regions. The visualization of DAPI stained plates were carried out with Olympus BX50 fluorescent microscope.

The mitotic and meiotic plates were analysed and photographed with Zeiss Axio Scope light microscope using ZEN software. The chromosomal measurements were made with the LEVAN plugin (Sakamoto and Zacaro 2009) and the karyotypes and idiograms were created with the CHIAS plugin (Kato et al. 2011) of the programme IMAGE J (Rasband 1997-2015).

Results

Conventional Giemsa staining

Analysis of spermatogonial cells of Nalassus bozdagus and Nalassus plebejus revealed the diploid chromosome number as 2n = 20 (9+Xy_p) (Fig. 1). In both species, most of the autosomes showed metacentric morphology, the X chromosomes were submetacentric and the y chromosomes were subtelocentric. In N. bozdagus the autosomal pairs 8 and 9 were submetacentric while in N. plebejus autosomal pairs 1, 5 and 8 were submetacentric. The smallest chromosome in both species was determined to be the y chromosome (~1 µm). The biggest chromosome of Nalassus bozdagus was the X chromosome (~4.315 µm), in Nalassus plebejus the biggest chromosome was the 1. chromosome with the length of 4.442 µm (Table 1).

Figure 1.

a–b N. bozdagus 2n = 20. a karyotype b idiogram c–d N. plebejus 2n = 20 c karyotype d idiogram. Bar = 5 µm.

Table 1.

Download as

CSV

XLSX

Chromosome morphologies and measurements of N. bozdagus and N. plebejus. CI: centromere index, RL: relative length, AR: arm ratio, *secondary constriction.

	N. bozdagus					N. plebejus
Chromosome	Length (µ)	CI	%RL	AR	Morphology	Length (µ)	CI	%RL	AR	Morphology
1	3.895	46	12.4	1.20	m	4.442	35	14.26	1.8	sm
2	3.420	47	10.89	1.23	m	4.207	48	13.5	1.04	m
3	3.375	46	10.74	1.29	m	3.316	43	10.64	1.4	m
4	3.204	46	10.2	1.10	m	3.117	43	10	1.28	m
5	2.876	47	9.16	1.20	m	3.222	28	10.34	2.52	sm
6	2.715	48	8.64	1.06	m	3.040	44	9.76	1.27	m
7	2.204	48	7.02	1.07	m	2.439	45	7.83	1.18	m
8	2.162	33	6.88	2.13	sm	2.476	29	7.95	2.38	sm
9	2.149	32	6.84	2.01	sm	1.853	46	5.95	1.17	m
X	4.315	28	13.74	2.47	sm*	2.04	30	6.55	2.31	sm
y	1.097	20	3.5	3.88	st	1.010	18	3.24	1.17	st

In prophase I nuclei, all chromosomes of Nalassus bozdagus showed dark heterochromatic blocks mainly located in centromeric regions (Fig. 2a–b). But in Nalassus plebejus, while most of the chromosomes have relatively small amounts of heterochromatin dispersed throughout the whole length (Fig. 2c–d), only 2 chromosomes with distinctive heterochromatic blocks were observed.

Figure 2.

a–b N. bozdagus with dark heterochromatic blocks on all chromosomes. a leptotene–zygotene b pachytene c–d N. plebejus with two distinctive heterochromatic blocks (black arrows); c leptotene–zygotene d pachytene. Orange arrow indicates Xy_p sex bivalent, Bar = 5 µm.

In diplotene/diakinesis of N. plebejus, 5-6 rod-shaped (terminal chiasma), 2-3 ring-shaped (two terminal chiasmata) and 1-2 cross-shaped (interstitial chiasma) bivalents were observed (Fig. 3a). In diakinesis/metaphase I; most of the homologous chromosomes of both species formed rod shaped bivalents due to being monochiasmatic and 2-3 ring shaped bivalents due to being bichiasmatic (Fig. 3b–c). In metaphase I plates, the parachute formation of sex bivalents was clearly observed for both Nalassus species (Fig. 4a–b). In metaphase II plates, relatively small sized 10 chromosomes (Fig. 4c–d) were observed. However, the plates which possessed the minute y chromosome were seemed to have only 9 chromosomes in their haploid sets. (Fig. 4d).

Figure 3.

a diplotene–diakinesis in N. plebejus b–c diakinesis–metaphase I b N. bozdagus c N. plebejus. Bar = 5 µm.

Figure 4.

a–b metaphase I a N. bozdagus b N. plebejus c–d metaphase II c N. bozdagus d N. plebejus. Arrows show Xy_p sex bivalents, Bar = 5 µm.

Sex chromosomes of two species were differed from each other by the length of X chromosome. The X chromosome of N. bozdagus was determined to be almost twice the size of the X chromosome of N. plebejus (Fig. 1, Table 1).

Differential staining

Silver nitrate staining of the chromosomes of Nalassus bozdagus revealed the presence of a highly impregnated nucleolus (NOR) associated with one of the long chromosomes in prophase I nuclei (Fig. 5a–b) and that Xy_p sex bivalent is strongly argyrophilic in metaphase I (Fig. 5c). In Nalassus plebejus, these strongly argyrophilic regions were observed only in pachytene nuclei (Fig. 5d). With base-specific (A-T) DAPI staining; metaphase I plates and prophase I nuclei were observed. In metaphase I stages there were no significant difference between species (Fig. 6a–b). Prophase I nuclei of N. bozdagus showed strong signals on pericentromeric heterochromatic blocks compared to other chromosomal regions (Fig. 6c). On the other hand, in N. plebejus only some centromeric regions showed slightly stronger fluorescence signals (Fig. 6d).

Figure 5.

Silver nitrate staining a–c N. bozdagus d N. plebejus. Black arrows indicate NOR, red arrows indicate argyrophilic sex bivalents, Bar = 5 µm.

Figure 6.

DAPI staining a metaphase I of N. plebejus b metaphase I of N. bozdagus c prophase I nucleus of N. bozdagus d prophase I nucleus of N. plebejus. Arrows show Xy_p sex bivalents and arrowheads indicate heterochromatic regions, Bars = 5 µm.

Discussion

The family Tenebrionidae is considered a karyologically conservative group due to the frequent occurrence of 2n = 20 formula (Juan and Petitpierre 1991a, Palmer and Petitpierre 1997). Heretofore, variation in the diploid chromosome numbers between 14–38 within the family are also noted (Juan and Petitpierre 1991a, Pons 2004, Holecová et al. 2008a, Lira-Neto et al. 2012). Although, tenebrionid karyotypes characterized with predominant presence of metacentric chromosomes (Guenin 1950, 1951a, b; Smith 1952, Yadav and Pillai 1974, Yadav et al. 1980, Juan and Petitpierre 1988, 1989, 1990, Juan et al. 1989), several species from different subfamilies have mostly subtelocentric/acrocentric sets (e.g. Laena reiteri Weise 1877, 2n = 18, Holecová et al. 2008a, Palembus dermestoides Fairmaire 1893, 2n = 20, Almeida et al. 2000). Furthermore, many tenebrionid beetles possess similar chromosome number but differ in karyotype structure, which reveal additional evidence for karyotype divergence through the intra-chromosomal rearrangements. The major patterns of karyological variations in tenebrionid beetles are mainly observed in sex determining systems, chromosome morphology and distribution of heterochromatin (Juan and Petitpierre 1990, Petitpierre et al. 1991, Juan and Petitpierre 1991a-b, Juan et al. 1993, Bruvo-Madaric et al. 2007).

Tenebrionidae comprises 9 subfamilies but most of the cytogenetically studied species (96%) belong to the Pimeliinae and Tenebrioninae (Bouchard et al. 2005, Holecová et al. 2008a). The diploid number in Pimeliinae shows a decrease from 2n = 20 to 2n = 18 caused by fusion of an autosomal pair while in Tenebrioninae there is a tendency of increased diploid number probably caused by centric fissions (Juan and Petitpierre 1991a).

We showed here that the karyotypes of Nalassus bozdagus and Nalassus plebejus consist of 10 pairs of chromosomes (2n = 20) (Fig. 1), which is considered as modal chromosome number for Tenebrionidae (Juan and Petitpierre 1991a, Pons 2004, Holecová et al. 2008a, Lira-Neto et al. 2012). The presence of heteromorphic sex chromosomes for both species is confirmed by occurrence of a Xy_p configuration in both conventionally (Fig. 4a-b) and differentially (Fig. 5c, 6a–b) stained metaphase I plates. The Xy_p sex determining system is the most frequent type among Tenebrionidae as well as order Coleoptera (Smith and Virkki 1978, Juan and Petitpierre 1991a). However, sex chromosomes or sex determining systems are mentioned as one of the major chromosomal changes involved in tenebrionid divergence.

Besides the general resemblance of two Nalassus karyotypes, important differences related to X chromosomes, variations in the number of metacentric/submetacentric chromosomes (Fig. 1, Table 1), localization of highly impregnated regions which are considered as NOR (Fig. 5a–d) and heterochromatin distribution (Fig. 6c-d) are clearly observed. The most prominent difference between two species is found related to X chromosome which is clearly larger (13.74% of total complement) in N. bozdagus and has a conspicuous secondary constriction on the long arm (Fig. 1). It was also observed that metaphase I plates of N. bozdagus have relatively larger Xy_p (Fig. 4a–b). The increase in relative length of X which does not alter parachute configuration is named as giant Xy_p and generally thought to be derived from either heterochromatin amplification or translocation (Dutrillaux and Dutrillaux 2009). Difference in size and heterochromatin content of X chromosomes also observed in two closely related tenebrionid species of Gonocephalum Solier 1834 (Tenebrioninae) (Juan and Petitpierre 1989).

The differences found in chromosome morphology (1., 5. and 9. pairs) between these two Nalassus species are thought to be related to pericentromeric inversions that resulted in centromeric shift. Pericentromeric rearrangements are already known within several Coleopteran families such as Cicindelidae, Chrysomelidae, Meloidae, Scarabaeidae and Tenebrionidae (Serrano 1981, Petitpierre 1983, Juan et al. 1990, Almeida et al. 2000, Petitpierre and Garneria 2003, Wilson and Angus 2005, De Julio et al 2010, Petitpierre 2011).

The karyotypes of N. bozdagus and N. plebejus also show obvious differences, especially in distribution of heterochromatin. The presence of strong signals on pericentromeric heterochromatin blocks on all chromosomes of N. bozdagus (Fig. 2a–b) and only few chromosomes in N. plebejus (Fig. 2c–d) was supported with both conventionally and differentially stained prophase I nuclei (Fig. 4g–h). Although, occurrence of heterochromatin observed mainly in the pericentromeric areas of the tenebrionid chromosomes, variability of heterochromatin localization and composition were also reported (Juan and Petitpierre 1989, 1991, Pons 2004, Rozek et al. 2004, Cabral-de-Mello et al. 2010, Schneider et al. 2007).

As a result of silver staining, the existence of highly impregnated areas associated with Xy_p of N. bozdagus in both prophase I and metaphase I, suggests that NORs are seemingly located on sex chromosomes (Fig. 5a–c). On the other hand, the potential NORs of N. plebejus were observed only in prophase I nuclei (Fig. 5d). Similar findings on argyrophilic Xy_p in metaphase I as well as prophase I were previously reported for several beetles such as Zophobas aff. confusus Gebien 1906 (Tenebrionidae) (Lira- Neto et al. 2012), Lagria villosa Fabricius 1781 (Tenebrionidae) (Goll et al. 2013), Palembus dermestoides (Tenebrionidae) and Epicauta atomaria Germar 1821 (Meloidae) (Almeida et al. 2000). Although, rDNA-FISH studies has shown that these strongly argyrophilic areas in prophase I bivalents are associated with NOR (Juan et al. 1993, Bruvo-Madaric et al. 2007), the existence of highly impregnated areas on sex chromosomes until metaphase I thought to be related to association and segregation of sex bivalents due to nucleolar material or distinctive heterochromatin (Juan et al. 1993, John and Lewis 1960). The association between sex chromosomes and nucleolar material is widely known for several animal groups from mammalians to insects (Smith and Virkki 1978, Virkki et al. 1991, Tres 2005), although autosomal localization of NORs by FISH using 18S rDNA probes were also reported for some tenebrionid species (Goll et al. 2003, Juan et al. 1993).

It was observed that bichiasmatic autosomes form ring bivalent while monochiasmatics form rod bivalents due to terminal chiasmata (Fig. 3b–c). Ring bivalents are frequent among Tenebrionidae, Scarabaeidae, Meloidae, Buprestidae, Curculionidae, Chrysomelidae and Cerambycidae (Petitpierre 1985, Bisoi and Patnaik 1988, Petitpierre and Garneria 2003, Karagyan et al. 2004, 2012, Lachowska et al. 2004, 2006a, 2006b, Rozek et al. 2004, Angus et al. 2007, Holecová et al. 2008b). During diploten-diakinesis of N. plebejus, in addition to ring and rod bivalents we also observed cross shaped bivalents (Fig. 3a) due to interstitial chiasmata.

The information acquired from metaphase I plates of only few Nesotes Allard 1876 species (Juan and Petitpierre 1986, 1989, 1991a, 1991b) and Probaticus ebeninus A. Villa and J. B. Villa, 1838 (Palmer and Petitpierre 1997) are the only cytogenetic data concerning the tribe Helopini. On the basis of metaphase I plates, it was only briefly noted that 5 Nesotes species have similar 2n = 20, 9+ Xy_p formula (Juan and Petitpierre 1986, 1989, 1991a) and P. ebeninus have 2n = 20, 9+XY (Palmer and Petitpierre 1997). Although, our findings for chromosome numbers correspond to previous cytogenetic data, comparative genomic analyses of Helopini require detailed descriptions of chromosome morphologies.

In conclusion, this study revealed that the cytogenetic features differed between endemic N. bozdagus and widespread N. plebejus. But, in the absence of molecular cytogenetic and phylogenetic approaches, it is not possible to make a strong conclusion about the major forces underlying these chromosomal variations. For definitive testing of the general trends in both Nalassus and tenebrionid karyotype evolution, it is necessary to increase the taxa sampling for major tenebrionid lineages.

Acknowledgements

We are sincerely grateful to Dr. Maxim Nabozhenko and Dr Bekir Keskin for sharing their experience about Helopini. We also wish to thank to Dr Ersen Aydın Yağmur and Beril Gündoğan, for valuable help with collection of the specimens, Molecular Cytogenetic Lab members in Faculty of Medicine-Ege University for their help and Prof. Dr. R. Angus for his suggestions on earlier versions of this paper. Specimens included in this study are part of collected and sequenced material of a molecular systematic research project (TBAG-Project No:112T445).

References

Almeida MC, Zacaro AA, Cella DM (2000) Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xy sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques. Hereditas 133: 147–157. doi: 10.1163/22119434-900000220

Angus RB, Wilson CJ, Mann DJ (2007) A chromosomal analysis of 15 species of Gymnopleurini, Scarabaeini and Coprini (Coleoptera: Scarabaeidae). Tijdschrift Voor Entomologie 150: 201–211.

Bisoi MR, Patnaik SC (1988) A Chromosome Study of Seven Species of Indian Coleoptera (Meloidae, Tenebrionidae and Coccinellidae). Caryologia 41(3-4): 309–321. doi: 10.1080/00087114.1988.10797871

Blackmon H, Jeffery PD (2015) Coleoptera Karyotype Database. The Coleopterists Bulletin 69.1: 174–175. doi: 10.1649/0010-065X-69.1.174

Bouchard P, Lawrence JF, Davies AE, Newton AF (2005) Synoptic classification of the world Tenebrionidae (Insecta: Coleoptera) with a review of family-group names. Annales Zoologici 55(4): 499–530.

Bruvo-Madaric B, Plohl M, Ugarkovic D (2007) Wide distribution of related satellite DNA families within the genus Pimelia (Tenebrionidae). Genetica 130: 35–42. doi: 10.1007/s10709-006-0017-2

Cabral-de-Mello DC, Moura RC, Carvalho R, Souza MJ (2010) Cytogenetic analysis of two related Deltochilum (Coleoptera, Scarabaeidae) species: diploid number reduction, extensive heterochromatin addition and differentiation. Micron 41: 112–117. doi: 10.1016/j.micron.2009.10.005

Chandley AC, Speed RM, Ma K (1994) Meiotic Chromosome Preparation. In: Gosden JR (Ed.) Chromosome Analysis Protocols.Methods in Molecular Biology 29: 27–40. doi: 10.1385/0-89603-289-2:27

De Julio M, Rodrigues-Fernandes F, Costa C, Almeida MC, Cella DM (2010) Mechanisms of karyotype differentiation in Cassidinae sensu lato (Coleoptera, Polyphaga, Chrysomelidae) based on seven species of the Brazilian fauna and an overview of the cytogenetic data. Micron 41: 26–38. doi: 10.1016/j.micron.2009.07.013

Dutrillaux AM, Dutrillaux B (2009) Sex chromosome rearrangements in Polyphaga beetles. Sexual Development 3(1): 43–54. doi: 10.1159/000200081

Dutrillaux AM, Xie H, Dutrillaux B (2007) Nucleolus and chromosome relationships at pachynema in four Scarabaeoidea (Coleoptera) species with various combinations of NOR and sex chromosomes. Chromosome Research 15: 417–427. doi: 10.1007/s10577-007-1133-2

Goll LG, Artoni RF, Vicari MR, Nogaroto V, Petitpierre E, Almeida MC (2013) Cytogenetic analysis of Lagria villosa (Coleoptera, Tenebrionidae): Emphasis on the mechanism of association of the Xy(p) Sex Chromosomes. Cytogenetic and Genome Research 139(1): 29–35. doi: 10.1159/000341674

Gregory TR (2016) Animal Genome Size Database. http://www.genomesize.com

Guenin HA (1950) Chromosomes et Hétérochromosomes de Ténébrionidés. Genetica 25: 157–182. doi: 10.1007/BF01784829

Guenin HA (1951a) La formule chromosomiale de Coleopteres tenebrionides nordafricains, I. Pimeliines et Tentyriines. Bulletin de la Société Vaudoise des Sciences Naturelles 65: 7–18.

Guenin HA (1951b) La formule chromosomiale de Coleopteres tenebrionides nordafricains, II. Erodiines. Revue Suisse de Zoologie 58: 471–475.

Holecová M, Rozek M, Lachowska D (2008a) The First Cytogenetic Report on Laena reitteri Weise, 1877 with Notes on Karyotypes of Darkling Beetles. Folia Biologica (Kraków) 56(3-4): 213–217. doi: 10.3409/fb.56

Holecová M, Rozek M, Lachowska D (2008b) Karyotype Analysis of Three Weevil Species from the Genera Brachysomus (Boheman, 1845) and Strophosoma Billberg, 1820 (Coleoptera, Curculionidae). Folia Biologica (Kraków) 56(1): 103–110. doi: 10.3409/fb56_1-2.25-29

John B, Lewis KR (1960) Nucleolar controlled segregation of the sex chromosomes in beetles. Heredity 15: 431–439. doi: 10.1038/hdy.1960.107

Juan C, Petitpierre E (1986) Karyological Analyses on Tenebrionid beetles from Balearic Islands. Génética Ibérica 38: 231–244.

Juan C, Petitpierre E (1988) A chromosome survey of North African and Eastern Mediterranean tenebrionids (Coleoptera). Cytobios 54: 85–94.

Juan C, Petitpierre E (1989) C-banding and DNA content in seven species of Tenebrionidae (Coleoptera). Genome 32: 834–839. doi: 10.1139/g89-519

Juan C, Petitpierre E (1990) Karyological differences among Tenebrionidae (Coleoptera). Genetica 80: 101–108. doi: 10.1007/BF00127130

Juan C, Petitpierre E (1991a) Chromosome numbers and sex determining systems in Tenebrionidae. Advances in Coleopterology167–176.

Juan C, Petitpierre E (1991b) Evolution of Genome Size in Darkling Beetles. Genome 34: 169–73. doi: 10.1139/g91-026

Juan C, Petitpierre E, Oromi P (1989) Chromosomal Analyses on Tenebrionids from Canary Islands. Cytobios 57: 33–41.

Juan C, Pons J, Petitpierre E (1993) Localization of tandemly repeated DNA sequences in beetle chromosomes by fluorescent in situ hybridization. Chromosome Research 1: 167–174. doi: 10.1007/BF00710770

Karagyan G, Kuznetsova VG, Lachowska DL (2004) New Cytogenetic Data on Armenian Buprestids (Coleoptera, Buprestidae) with a Discussion of Karyotype Variation within the Family. Folia Biologica (Kraków) 52: 151–158. doi: 10.3409/1734916044527601

Kato S, Ohmido N, Fukui K (2011) CHIAS 4 ver 1.02.

Kergoat GJ, Bouchard P, Clamens AL, Abbate JL, Jourdan H, Jabbour-Zahab R, Genson G, Soldati L, Condamine FL (2014a) Cretaceous environmental changes led to high extinction rates in a hyperdiverse beetle family. BMC Evolutionary Biology 14(220): 1–13. doi: 10.1186/s12862-014-0220-1

Kergoat GJ, Soldati L, Clamens AL, Jourdan H, Jabbour-Zahab R, Genson G, Bouchard P, Condamine FL (2014b) Higher level molecular phylogeny of darkling beetles (Coleoptera: Tenebrionidae). Systematic Entomology 39(3): 486–499. doi: 10.1111/syen.12065

Keskin B, Nabozhenko MV (2010) A New Species and New Records of the Genus Nalassus Mulsant, 1854 (Coleoptera : Tenebrionidae : Helopini) from Turkey. Annales Zoologici 60(1): 23–28. doi: 10.3161/000345410X499489

Lachowska D, Holecová M, Rozek M (2004) Notes on chromosome numbers and C-banding patterns in karyotypes of some weevils from Central Europe (Coleoptera, Curculionoidea: Apionidae, Nanophyidae, Curculionidae). Folia Biologica 52(1): 61–66.

Lachowska D, Rozek M, Holecová M (2006a) Karyotypic characterization of three weevil species (Coleoptera: Curculionidae, Brachyderini). Folia Biologica 54(1): 13–17. doi: 10.3409/173491606777919139

Lachowska D, Rozek M, Holecová M, Kajtoch L (2006b) Cytogenetic differences between Peritelus familiaris and Centrinemus leucogrammus (Coleoptera: Curculionidae: Entiminae: Peritelini). European Journal of Entomology 103: 687–690. doi: 10.14411/eje.2006.089

Lira-Neto AC, Silva GM, Moura RC, Souza MJ (2012) Cytogenetics of the Darkling Beetles Zophobas aff. confusus and Nyctobates gigas (Coleoptera, Tenebrionidae). Genetics and Molecular Research 11(3): 2432–2440. doi: 10.4238/2012

Medvedev GS (1987) Review of Tenebrionid Beetles of the Genus Cylindronotus Fald. (Coleoptera, Tenebrionidae) of Kazakhstan and Middle Asia. Trudy Zoologicheskogo instituta AN SSSR 170: 99–104. [In Russian]

Murakami A, Imai H (1974) Cytological evidence for holocentric chromosomes of the silkworms, Bombyx mori and B. mandarina, (Bombycidae, Lepidoptera). Chromosoma 47: 167–178. doi: 10.1007/BF00331804

Nabozhenko MV (2001) On the Classification of the Tenebrionid Tribe Helopini, with a Review of the Genera Nalassus Mulsant and Odocnemis Allard (Coleoptera, Tenebrionidae) of the European Part of CIS and the Caucasus. Entomological Review 81(8): 909–942.

Nabozhenko MV (2007) Review of the subgenus Helopondrus Reitter, 1922 of the genus Nalassus Mulsant, 1854 (Coleoptera: Tenebrionidae) of Turkey. Russian Entomological Journal 16(4): 453–56.

Nabozhenko MV (2008) Nalassus glorificus (Seidlitz, 1896) – New Synonym of Nalassus pharnaces Allard, 1876 (Coleoptera, Tenebrionidae). Caucasian Entomological Bulletin 4(1): 85.

Nabozhenko MV (2011) Two New Species of the Genus Nalassus Mulsant, Subgenus Helopondrus Reitter (Coleoptera: Tenebrionidae) from Turkey. Stuttgarter Beitrage Zur Naturkunde A 4: 263–67.

Nabozhenko MV (2013) Two New Taxa of the Genus Nalassus Mulsant , 1854 (Coleoptera: Tenebrionidae) from Georgia. Caucasian Entomological Bulletin 9(2): 261–64.

Nabozhenko MV (2014) New Darkling Beetles of the Tribe Helopini (Coleoptera: Tenebrionidae) from Iran. Caucasian Entomological Bulletin 10(2): 237–41.

Nabozhenko MV, Ivanov SN (2015) A New Nalassus Mulsant, 1854 (Coleoptera: Tenebrionidae: Helopini), the First Representative of the Genus from the Russian Far East. Zootaxa 3955(1): 137–41. doi: 10.11646/zootaxa.3955.1.9

Palmer M, Petitpierre E (1997) New Chromosomal Findings on Tenebrionidae from Western Mediterranean. Caryologia 50(2): 117–23. doi: 10.1080/00087114.1997.10797391

Patkin EL, Sorokin AV (1984) Nucleolus-Organizing Regions Chromosomes in Early Embryogenesis of Laboratory Mice. Bulletin of Experimental Biology and Medicine (USSR) 96: 92–94.

Petitpierre E (1983) Karyometric Differences among Nine Species of the Genus Chrysolina Mots. (Coleoptera, Chrysomelidae). Canadian Journal of Genetics and Cytology 25: 33–39. doi: 10.1139/g83-006

Petitpierre E (1985) Chromosome number and sex determining mechanism in four species of Galerucinae beetles with particular reference to the meiotic system of Monolepta erythrocephala (Ol.) (Chrysomelidae). Actas do II Congresso Ibérico de Entomologia (Lisboa) 1: 47–54.

Petitpierre E (2011) Cytogenetics, cytotaxonomy and chromosomal evolution of Chrysomelinae revisited (Coleoptera, Chrysomelidae). ZooKeys 157: 67–79. doi: 10.3897/zookeys.157.1339

Petitpierre E, Garnería I (2003) A cytogenetic study of the leaf beetle genus Cyrtonus (Coleoptera, Chrysomelidae). Genetica 119: 193–199. doi: 10.1023/A:1026010102779

Petitpierre E, Juan C, Alvarez-Fuster A (1991) Evolution of Chromosomes and Genome Size in Chrysomelidae and Tenebrionidae. In: Belles X, Blas M, Zunino M (Eds) Advances in Coleopterolgy.European association of coleopterology, Barcelona, 129–44.

Pons J (2004) Evolution of diploid chromosome number, sex-determining systems and heterochromatin in Western Mediterranean and Canarian species of the genus Pimelia (Coleoptera: Tenebrionidae). Journal of Zoological Systematics and Evolutionary Research 42: 81–85. doi: 10.1046/j.1439-0469.2003.00247.x

Rasband WS (1997-2015) ImageJ. U.S. National Institutes of Health, Bethesda, Maryland, USA. http://imagej.nih.gov/ij/

Rozek M, Lachowska D, Petitpierre E, Holecová M (2004) C-bands on chromosomes of 32 beetle species (Coleoptera: Elateridae, Cantharidae, Oedemeridae, Cerambycidae, Anthicidae, Chrysomelidae, Attelabidae and Curculionidae). Hereditas 140: 161–170. doi: 10.1111/j.1601-5223.2004.01810.x

Sakamoto Y, Zacaro AA (2009) LEVAN, an ImajeJ plugin for morphological cytogenetic analysis of mitotic and meiotic chromosomes, Initial version. http://rsbweb.nih.gov/ij/

Schneider MC, Rosa SP, Almeida MC, Costa C, Cella DM (2007) Chromosomal similarities and differences among four Neotropical Elateridae (Conoderini and Pyrophorini) and other related species, with comments on the NOR patterns in Coleoptera. Journal of Zoological Systematics and Evolutionary Research 45: 308–316. doi: 10.1111/j.1439-0469.2006.00398.x

Serrano J (1981) Chromosome numbers and karyotype evolution of Caraboidea. Genetica 55: 51–60. doi: 10.1007/BF00134005

Smith SG (1952) The cytology of some tenebrionid beetles (Coleoptera). Journal of Morphology 91: 325–364. doi: 10.1002/jmor.1050910206

Smith SG, Virkki N (1978) Coleoptera. In: Animal Cytogenetics Vol. 3 Insecta 5. Borntraeger, Berlin.

Tres L (2005) XY chromosomal bivalent: nucleolar attaction. Molecular Reproduction and Development 72: 1–6. doi: 10.1002/mrd.20334

Virkki N, Mazzella C, Denton A (1991) Silver staining of the Xy_p sex bivalent. Cytobios 67: 47–63.

Wilson CJ, Angus RB (2005) A Chromosomal Analysis of 21 Species of Oniticellini and Onthophagini (Coleoptera: Scarabaeidae). Tijdschrift Voor Entomologie 148: 63–76. doi: 10.1163/22119434-900000167

Yadav JS, Pillai RK (1974) Cytology of two species of Australian leafminers (HispinaeChrysomelidae). Cytobios 11: 75–79.

Yadav JS, Pillai RK, Karamjeet (1980) Chromosome numbers of Tenebrionidae (Polyphaga: Coleoptera). Biologia 26: 31–41.