Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 169-175, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1120 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Cynthia Aparecida Valiati Barreto, Marco Antônio Peixoto, Kés- sia Leite de Souza, Natália Martins Travenzoli, Renato Neves Feio, Jorge Abdala Dergam (2021) Further insights into chromosomal evolution of the genus Enyalius with karyotype description of Enyalius boulengeri Etheridge, 1969 (Squamata, Leiosauridae). Caryologia 74(3): 169-175. doi: 10.36253/caryolo- gia-1120 Received: October 21, 2020 Accepted: July 22, 2021 Published: December 21, 2021 Copyright: © 2021 Cynthia Aparecida Valiati Barreto, Marco Antônio Pei- xoto, Késsia Leite de Souza, Natália Martins Travenzoli, Renato Neves Feio, Jorge Abdala Dergam. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID MAP: 0000-0003-0564-7068 Further insights into chromosomal evolution of the genus Enyalius with karyotype description of Enyalius boulengeri Etheridge, 1969 (Squamata, Leiosauridae) Cynthia Aparecida Valiati Barreto1,*, Marco Antônio Peixoto1,2, Kés- sia Leite de Souza1, Natália Martins Travenzoli1, Renato Neves Feio3, Jorge Abdala Dergam1 1Laboratório de Sistemática Molecular – Beagle, Av. PH Holfs, S/N, Departamento de Biologia Animal, Universidade Federal de Viçosa (UFV), CEP 36570-900, Viçosa, Minas Gerais, Brazil 2Laboratório de Biometria – LABIO, Av. PH Holfs, S/N, Departamento de Biologia Geral, Universidade Federal de Viçosa (UFV), CEP 36570-900, Viçosa, Minas Gerais, Brazil 3Museu de Zoologia João Moojen – MZUFV, Vila Gianetti, n° 32, Departamento de Bio- logia Animal, Universidade Federal de Viçosa (UFV), CEP 36570-900, Viçosa, Minas Gerais, Brazil *Corresponding author. E-mail: cynthiavaliatibarreto@gmail.com Abstract. The genus Enyalius is composed of 11 described species inhabiting forest areas in Amazônia, Cerrado and Atlantic forest biomes. Currently, eight species with high levels of chromosome variation have been karyotyped. The study aims to char- acterize the karyotype of Enyalius boulengeri, with classical and molecular techniques, and improve knowledge about the karyotype evolution of the lizard genus Enyalius. The species has 2n = 36 chromosomes (8m + 4sm + 24mc), FN = 24; NORs and 18S rDNA were subtelomeric and located on chromosome pair 2. Repetitive DNA probes (CAT)10 accumulated on centromeric and terminal regions of some macrochromo- somes. (GA)15 probe showed conspicuous accumulation on the pericentromeric region of chromosome pairs 1 and 6. Repetitive FISH patterns obtained with (GC)15 probe marked the pericentromeric region of the first chromosome pair. All probes showed accumulation in the microchromosomes. The chromosomal formula found in E. bou- lengeri has been considered the ancestral karyotype for pleurodont Iguania. The genus Enyalius is characterized by two distinctive chromosomal groups; one with highly con- served karyotypes, whereas the other is karyotypically diverse. Our molecular cytoge- netics data are promising and will increase knowledge about the genus Enyalius chro- mosome evolution. Keywords: Ag-NOR banding; Cytogenetics; FISH; Lizards; rDNA 18S; Repetitive DNA probes. 170 Cynthia Aparecida Valiati Barreto et al. INTRODUCTION Cytogenetic studies on lizards of pleurodont Igua- nia infraorder suggest that there are two distinct trends on chromosome evolution in this taxon: some genera present chromosome variability, such as supernumerary chromosomes, sexual elements and large differences in chromosomal number and size (e. g. Liolaemus, Norops, and Sceloporus); on the other hand, many families show a conserved karyotype (Gorman & Atkins 1967; Pinna- Senn et al. 1987; Pellegrino et al. 1999; Bertolotto et al. 2002). Based on these results, the karyotype with 2n = 36 chromosomes and distinction between macrochromo- somes (M) and microchromosomes (mc) (12M + 24mc) has been proposed as the ancestral karyotype for Igua- nia (Paull et al. 1976). However, chromosome banding reveals that these conservative karyotypes present some polymorphisms, such as different positions of nucleolar organizing region (NOR) in some chromosome pairs, varying patterns of heterochromatin and different mc morphology (Bertolotto et al. 1996; Kasahara et al. 2004). The advent of techniques banding heterochromatin regions in DNA is promising for the advance of the com- prehension of the genome structure and evolution (Mar- tins et al., 2011). Microsatellite regions apparently accu- mulate on regions with low levels of replication, such as telomeric and centromeric ones, and are easily detected by Fluorescence in situ Hybridization (FISH) techniques, as indicated in plants, anurans and fishes (Soares-Scott et al. 2005; Peixoto et al. 2015; Cunha et al. 2016). Although cytogenetic studies using molecular tools are still scarce on reptiles, they allow to understand relations between populations or/and species, and identify sexual elements at karyotypic components of species (Martins et al. 2011). The genus Enyalius (Wied, 1821) is composed of 11 described species inhabiting forest areas in Amazônia, Cerrado and Atlantic forest biomes (Rodrigues et al. 2014; Costa & Bérnils 2018). Moreover, cryptic species are indicated to occur along the Atlantic forest (Rod- rigues et al. 2014). Currently, eight species of the genus have been karyotyped (Bertolotto, 2006; Rodrigues et al., 2014), showing a certain degree of karyotypic variation. Some phylogenetic related species (clade A sensu Rodri- gues et al. 2014) are proposed as bearers of the ancestral karyotype of Iguania. On the other hand, related species present variation in chromosome number and size and supernumerary elements (clade B sensu Rodrigues et al. 2014). However, two characters are highly conserved in Enyalus: the nucleolar organizing region is located on the chromosome pair number 2 and heterochromatic regions occur in the centromeric position, on M and on almost all mc (Bertolotto et al. 2002). Phylogenetic relationships within this family are still unresolved, and studies employing banding techniques associated to molecular cytogenetics should reveal unde- tected synapomorphies. Enyalius is a widely distributed genus and a potential model for biogeographical analy- ses and chromosome evolution in Squamata. The study aims to characterize the karyotype of Enyalius bouleng- eri, with classical and molecular techniques and improve knowledge about the karyotype evolution of this genus. MATERIAL AND METHODS Specimens collection Seven specimens of Enyalius boulengeri were ana- lyzed: four specimens (two females - MZUFV 1358- 1359- and two males – MZUFV 1353, 1362) from the APA Bom Jesus, Divino (20°35’52.85”S; 42°14’25.89”W) and three specimens (one female – MZUFV 1356 – and two males – MZUFV 1354-1355) from the Estação de Pesquisa, Treinamento e Educação Ambiental (EPTEA), Mata do Paraíso, Viçosa (20°48’0.40”S; 42°51’47.80”W), both in Minas Gerais State, Brazil. Proceedings were carried out according to the Animal Welfare Commis- sion of the Universidade Federal de Viçosa and the cur- rent Brazilian laws (CONCEA 1153/95). All vouchers were deposited in the herpetological collection of the Museu de Zoologia João Moojen, at the Universidade Federal de Viçosa (MZUFV), Viçosa municipality, in Minas Gerais State, Brazil. Conventional staining and molecular cytogenetic analyses The specimens were fed 24 hours before being sac- rificed. Each specimen was injected intraperitoneally with 0.1% solution of colchicine (0.1 ml per 10 g of body weight) 6 hours before euthanasia (carried out intraperi- toneally with Hypnol solution 0.01 mL. mg-1) to induce local anesthesia and pentobarbital (60 mg.kg-1 – lethal dose). Mitotic chromosomes were obtained from gut epithelial cells, according to Schmid (1978) and stained using conventional protocols (5 % Giemsa diluted in Sorensen buffer). The best metaphases were photo- graphed in digital Olympus BX53 light microscope with a DP73 Olympus camera, using the CellSens Dimen- sions® software system. Chromosome pairing and meas- urements were performed using Image Pro Plus® (IPP Version 4.5) to determine the modal value (2n) and the fundamental number (FN) for the species. Homologs were paired and grouped according to the centromere position, in decreasing size order, and classified in meta- 171Further insights into chromosomal evolution of the genus Enyalius with karyotype description of Enyalius boulengeri centrics (m), submetacentrics (sm), subtelocentrics (st) and telocentrics (t) (Green & Sessions 1991). Active NORs in the preceding interphase were identified using silver nitrate precipitation (Ag-NORs) (Howell & Black 1980), whereas the heterochromatin- rich regions were detected using C-banding protocol (Sumner 1972). The FISH technique was performed according to Pinkel et al. (1986). The 18S rDNA probe was obtained from E. boulengeri, via polymerase chain reaction (PCR) with the following primers: 18Sf (5’-CCG CTT TGG TGA CTC TTG AT-3’) and 18Sr (5’-CCG AGG ACC TCA CTA AAC CA-3’) (Gross et al. 2010). The 18S probe was labeled by nick-translation with digoxigenina 11–dUTP, and the signal detection and amplification were performed using anti-digoxigenin- rhodamine (Roche Applied Science). The DNA repetitive probes were thynilated with cy3 at the 5’ position (Sig- ma-Aldrich), using the following repetitive DNA probes: (A)30, (C)30, (CA)15, (GA)15, (GC)15, (TA)15, (CAT)10, (CAA)10, (CAG)10, (GAG)10, (CGG)10 , and the protocols followed Cioffi et al. (2011). FISH images were captured in a BX53 Olympus microscope with a XM10 camera. All procedures were carried out in the Laboratório de Sistemática Molecular BEAGLE, at the Universidade Federal de Viçosa, Viçosa municipality, Minas Gerais State, Brazil. Cytogenetic tree In order to comprehend the relationship between the phylogenetic hypothesis and cytogenetic data of the genus Enyalius, we overlapped our results on Rodri- gues et al. (2014) hypothesis. The cytogenetic data were derived from the available data on literature (Gorman et al. 1967; Pellegrino et al. 1999; Bertolotto et al. 2002; Bertolotto 2006; Rodrigues et al. 2006; Rodrigues et al. 2014). The species tree was reconstructed in TNT 1.6 (Goloboff et al. 2008), and some rearrangements were made on Figtree software v1.3.1 (Rambaut 2009) and Illustrator v. CS3. RESULTS A karyotype with diploid number equal to 2n = 36 chromosomes comprised of 12 macrochromosomes (M) and 24 microchromosomes (mc) (12M + 24mc) char- acterized the E. boulengeri populations (Figure 1). The M pairs 1, 3, 4, and 5 are metacentrics, and the pairs 2 and 6 are submetacentrics in all metaphases. The karyo- type formula was 8m + 4sm + 24mc, with fundamental number (FN) equal to 24. A secondary constriction was observed in the distal end of the long arm of chromo- some pair 2 (Figure 1). Heteromorphic sex chromosomes or supernumerary elements were not detected in any of these specimens. The NORs were detected at the subtelomeric region of the long arm from both homologues on chromosome pair 2. NORs location corresponded to the conspicuous secondary constriction observed with Giemsa stain, and to the location of the 18S rDNA probe (Figure 1B). The C-banding results did not highlight heterochromatin regions from any chromosome pair. This result is prob- able related to the technique used in this study, once the presence of heterochromatin are reported to the other species of the genus (Bertolotto, 2006). Repetitive DNA probes (GA)15 showed conspicuous accumulation on the pericentromeric region of chromosome pairs 1 and 6 and several mc (Figure 2A), whereas (GC)15 probe marked the pericentromeric region of the first chromo- some pair and a few mc (Figure 2B). Repetitive FISH patterns obtained with (CAT)10 accumulated on the centromeric and terminal regions of some macrochro- mosomes and several microchromosomes (Figure 2C). The repetitive DNA probes (A)30, (C)30, (CA)15, (TA)15, (CAA)10, (CAG)10, (CGG)10, and (GAG)10 did not label any chromosome pair. The two clades of Enyalius (Rodrigues et al. 2014) diverged on their cytogenetic patterns (Figure 3). The clade A (composed of five species with cytogenetic data available for three of them) presents the same chromo- some formula (8m + 4sm + 24mc), and one species with cytogenetically differentiated sex chromosomes (Enya- lius perditus 2). On the other hand, clade B (composed of seven species, six of them with cytogenetic data avail- able), comprises species with high levels of caryological instability. The species possess different formulae (i. e. E. pictus: 8m + 4sm + 24mc and E. bibronii: 8m + 2sm + 2t + 24mc), as well as B chromosomes (i. e. E. bilin- eatus: 8m + 4sm+ 24mc + 1B/2B), sex chromosomes (E. bilinetus and E. leechii), besides some unusual telocen- tric chromosomes (i. e. E. catenatus 1: 6m + 2sm + 6t + 24mc and E. erythroceneus: 24t + 24mc). DISCUSSION Enyalius boulengeri showed a 2n = 36 (12M + 24mc) karyotype that is ubiquitous among species of the genus and pleurodont Iguania (Gorman et al. 1967; Pellegrino et al. 1999; Bertolotto et al. 2002; Bertolotto 2006; Rodri- gues et al. 2006; Rodrigues et al. 2013). Furthermore, it was observed a similarity between the nucleolar organizing region (NOR) and the 18S rDNA labeling at the distal end of the long arm of both 172 Cynthia Aparecida Valiati Barreto et al. homologues on chromosome pair 2. This same pattern (similarity between NOR and 18S rDNA probes and labeling in chromosome pair 2) is reported for all oth- er species of the genus Enyalius and from Leiosauridae family (Bertolotto et al. 2002; Bertolotto 2006; Rodri- gues et al. 2006). Here, the first Enyalius species was labelled with repetitive DNA probes, providing addi- tional karyological data. This result will contributes to disentangling the phylogenetic relationships within the genus when similar studies are available to other spe- cies of the genus. The fundamental number of 24 is also shared with the other species from clade A of Enyalius (E. perditus and E. iheringii). The invariable number of mc (24 mc) in all species of the genus seems to be the rule in Squamata. Although clades A and B (Rodrigues et al. 2014) differ on macrochromosome constitution, mc are the same on all species of Enyalius. Thus, mc seem to be constituted by DNA sequences that represent a con- served part of the karyotypes of Enyalius. Patterns of repetitive DNA probes within this genus will be inform- ative to test this hypothesis. Two techniques corroborate that NORs are located on the subterminal region of the long arm of the second chromosome pair in E. boulengeri, with eight species of the genus presenting this same pattern (Bertolotto et al. 2002; Bertolotto 2006; Rodrigues et al. 2006). The con- servation of chromosomal position of NORs also sug- gests the stability of this chromosome segment in Leio- sauridae family (Bertolotto et al. 2002). The pattern of NOR banding should representing a phylogenetic sign for close related species. In addition, another pattern grabbing attention was the location of Ag-NOR and FISH 18S rDNA probe in the same chromosome region, which has been reported for several species from differ- ent families of clade Iguania (i. e. Agamidae (Patawang et al. 2015), Leiosauridae (Bertolotto et al. 2002), Liola- emidae (Bertolotto et al. 1996), Polychrotidae (Bertolotto et al. 2001), and Tropiduridae (Kasahara et al. 1987). Figure 1. Giemsa-stained karyotypes of Enyalius boulengeri. Insets present chromosome pairs with Ag-NOR (above) and 18S rDNA (below) identified on chromosome pair number 2. Scale bars indicate 5 μm. Figure 2. Mitotic chromosomes of Enyalius boulengeri labeled with the repetitive DNA probes: A: (GA)15; B: (GC)15; C: (CAT)10. Scale bar indicated 10 μm. 173Further insights into chromosomal evolution of the genus Enyalius with karyotype description of Enyalius boulengeri E. boulengeri showed distribution of microsatel- lite repeats on pericentromeric and centromeric regions of the macrochromosome and many microchromo- ssomes. No uneven accumulation of repetitive DNA probes was observed in the chromosome pairs, which corroborates the hypothesis this species have no system of sexual chromosomes. Studies using FISH to evalu- ate the genome distribution of microsatellite repeats on sex chromosomes was realized by Porkorna, 2011, this study showed certain microsatellite sequences are exten- sively accumulated over the whole length or parts of the W chromosome in Eremias velox (Pokorná et al. 2011). Giovannotti et al, (2018) isolated the repetitive element IMO-TaqI satDNA in several species of Lacertidae and found this element to be very abundant in the constitu- tive heterochromatin of the W-sex chromosome of the four Lacerta species investigated. On the other hand, repetitive probes also have evidenced chromosome sta- bility among some species and populations of the Sci- nax perpusillus group and Ololygon tripui (Peixoto et al. 2015; Peixoto et al. 2016). Our results highlighted that the chromosomal for- mula reported in E. boulengeri (8m + 4sm + 24mc) is shared with all species with described data in clade A. This pattern are possible result of the phylogenet- ic relation among the species that occurs in warmer climates in Sout heastern a nd nor t heastern Brazi l and once they belonged to the same clade inside the Enyalius genus. This character has been considered an ancestral karyotype for pleurodont Iguania, which includes the Leiosauridae family (Paull et al. 1976; Bertolotto et al. 2002) and might also be present in the two remaining species with unknown karyotypes (E. brasiliensis and E. perditus 1). Clade B members inhab- it the cooler climates in Southeastern and Southern Brazil (Rodrigues et al. 2014), and presents a second distinct trend on chromosome evolution in Iguania, showing considerable karyotype variability. This trend is exemplified by Anolis, Norops, Ctenonotus also with relation to sex chromosomes (Castiglia et al. 2013; Giovannotti et al. 2017; Lisachov et al. 2019; Kichigin et al. 2019). For instance, some species present super- numerary chromosomes, different chromosomal for- mulae, besides some unusual telocentric chromosomes. In E. erythroceneus (24t + 24mc), the number of telo- centric chromosomes seems to derive from f ission events of chromosomes, assuming that the ancestral karyotype is 8m + 4sm. The karyotypes of two species in Clade B may also indicate fission events: Enyalius catenatus and E. bibroni are evidenced by the presence of telomeric elements (6m + 2sm + 6t + 24mc and 8m + 2sm + 2t + 24mc, respectively). Figure 3. Compiled information about cytogenetic data in the genus Enyalius and some related species on a condensed phylogenetic hypothesis resulting from the analyses of Rodrigues et al. (2014). 174 Cynthia Aparecida Valiati Barreto et al. CONCLUSION Our results support classical cytogenetics (diploid number, FN and NOR number and location) as an effi- cient tool to characterize lizard species in a family level, which corroborates the position of E. boulengeri within its genus. Repetitive DNA probes complement this con- servative pattern and, if apply to other species of the genus, may allow to detect synapomorphies so as to improve knowledge about chromosome evolution and phylogenetic relationship within this genus. ACKNOWLEDGEMENTS The authors are grateful to the anonymous reviewers that read the first draft of this manuscript. We also thank Priscilla Hote for the collection of some individuals. FUNDING This work is a contribution to the project “Biogeo- grafia e Conservação da anurofauna no Complexo Ser- rano da Mantiqueira, Sudeste do Brasil” supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Project #068437-2014/06). The authors acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their financial support. REFERENCES Bertolotto CEV, Pellegrino KCM, Rodrigues MT, Yonena- ga-Yassuda Y. 2002. 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