Genetics and Molecular Biology 43, 4, e20200149 (2020) Copyright © 2020, Sociedade Brasileira de Genética. DOI: https://doi.org/10.1590/1678-4685-GMB-2020-0149 Research Article Animal Genetics Extensive genomic reshuffling involved in the karyotype evolution of genus Cerradomys (Rodentia: Sigmodontinae: Oryzomyini) Camilla Bruno Di-Nizo1 , Malcolm Andrew Ferguson-Smith2, Maria José de J. Silva1 1Instituto Butantan, Laboratório de Ecologia e Evolução, São Paulo, SP, Brazil. 2Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. Rodents of the genus Cerradomys belong to the tribe Oryzomyini and present high chromosome variability with diploid numbers ranging from 2n=46 to 60. Classical cytogenetics and fluorescence in situ hybridization (FISH) with telomeric and whole chromosome-specific probes of another Oryzomyini, Oligoryzomys moojeni (OMO), were used to assess the karyotype evolution of the genus. Results were integrated into a molecular phylogeny to infer the hypothetical direc- tion of chromosome changes. The telomeric FISH showed signals in telomeres in species that diverged early in the phy- logeny, plus interstitial telomeric signals (ITS) in some species from the most derived clades (C. langguthi, C. vivoi, C. goytaca, and C. subflavus). Chromosome painting revealed homology from 23 segments of C. maracajuensis and C. marinhus to 32 of C. vivoi. Extensive chromosome reorganization was responsible for karyotypic differences in closely related species. Major drivers for genomic reshuffling were in tandem and centric fusion, fission, paracentric and pericentric inversions or centromere repositioning. Chromosome evolution was associated with an increase and de- crease in diploid number in different lineages and ITS indicate remnants of ancient telomeres. Cytogenetics results cor- roborates that C. goytaca is not a junior synonym of C. subflavus since the karyotypic differences found may lead to reproductive isolation. Keywords: Chromosomal evolution, GTG-banding, Oryzomyini, ZOO-FISH. Received: May 13, 2020; Accepted: September 07, 2020. Introduction vicino, 2002; Bonvicino, 2003; Percequillo et al., 2008; Cerradomys is a rodent genus of the tribe Oryzomyini, Tavares et al., 2011; Bonvicino et al., 2014). distributed in open vegetations of South America from Besides the cytotaxonomic importance, cytogenetics northeastern Brazil to southeastern Bolivia and northwestern reveals substantial chromosomal variation mainly due to Paraguay. It was previously described as a subgenus of Robertsonian rearrangements, pericentric inversion and sex Oryzomys (Oryzomys subflavus) (Weksler et al., 2006; Per- chromosome polymorphisms. This makes the genus an ex- cequillo, 2015). From 1981 to 2002, different karyotypes cellent group for studies of karyotype evolution (Maia and were described suggesting that the genus was not monotypic Hulak, 1981; Almeida and Yonenaga-Yassuda, 1985). (Maia and Hulak, 1981; Almeida and Yonenaga-Yassuda, It has been shown that classic cytogenetic studies can 1985; Svartman and Almeida, 1992; Andrades-Miranda et fail to detect interspecific chromosomal homologies in al., 2002), and this was confirmed later using molecular and groups with great chromosomal variability, such as rodents morphological studies (Bonvicino and Moreira, 2001; of the tribe Oryzomyini, and that chromosome painting stud- Langguth and Bonvicino, 2002; Bonvicino, 2003). Then, ies provide the required resolution (Ferguson-Smith et al., Oryzomys subflavus became Oryzomys gr. subflavus and, in 1998). Thus, such studies were able to detect syntenic seg- 2006, this group of species was raised to the genus category ments and shed light on the rearrangements occurring Cerradomys (Weksler et al., 2006). throughout the chromosomal evolution of this tribe (Naga- Currently, eight species are formally described and machi et al., 2013; Di-Nizo et al., 2015; Suárez et al., 2015; cytogenetic information has been an important identifying Oliveira Da Silva et al., 2017). These studies become even tool: Cerradomys akroai (2n=60, FN=74), C. goytaca more informative when linked to a phylogeny, since they al- (2n=54, FN=66), C. langguthi (2n=50, 49, 48, and 46, low the recovery of possible trajectories of chromosomal FN=56), C. maracajuensis (2n=56, FN=58), C. marinhus changes (Di-Nizo et al., 2015; Sotero-Caio et al., 2015; (2n=56, FN=54), C. scotti (2n=58, FN=70 and 72), C. sub- Suárez et al., 2015). Until now, molecular cytogenetic stud- flavus (2n=54, 55 and 56, FN=62-64) and C. vivoi (2n=50, ies of the Cerradomys genus have been scarce (Nagamachi FN=64) (Svartman and Almeida, 1992; Langguth and Bon- et al., 2013) and its karyotype evolution remains to be ex- plored. Send correspondence to Camilla Bruno Di-Nizo. Instituto Butantan, The aim of this work is to investigate chromosomal Laboratório de Ecologia e Evolução, Avenida Vital Brazil, 1500, homologies among Cerradomys species and to infer the rear- São Paulo, SP, Brazil. E-mail: [email protected] rangements that have occurred during the karyotype evolu- 2 Di-Nizo et al. tion of the genus. To achieve these goals, we have performed fibroblast cell culture (Freshney, 1986), and metaphases of classic cytogenetics, FISH with telomeric probes and chro- C. akroai, C. scotti, C. goytaca, C. langguthi, and C. sub- mosome painting using Oligoryzomys moojeni (OMO, flavus with 2n=55 and 2n=56 were obtained in vivo from 2n=70) whole chromosome probes. Oligoryzomys is another spleen and bone marrow (Ford and Hamerton, 1956). All genus of the tribe Oryzomyini that belongs to a sister clade samples were analyzed (at least 30 metaphases from each in- of Cerradomys (Weksler et al., 2006); both lineages di- dividual) using conventional Giemsa staining, CBG-ban- verged approximately 5 Mya (Leite et al., 2014). In addition, ding (Sumner, 1972) and GTG-banding (Seabright, 1971). we performed molecular phylogenetic analyses to infer the hypothetical polarity of chromosome changes. Fluorescence in situ Hybridization (FISH) Fluorescence in situ hybridization with telomeric Material and Methods probes (TTAGGG)n labeled with Fluorescein isothiocyanate (FITC) was carried out on all samples following the recom- Chromosome preparation and classical cytogenetics mended protocol (Telomere PNA FISH Kit, Code No. Samples comprise 10 individuals referred to here as K5325, DAKO). Slides were counterstained with 4’,6-Dia- Cerradomys marinhus (CMARI); C. maracajuensis midine-2’-phenylindole dihydrochloride (DAPI) with anti- (CMARA); C. akroai (CAK); C. scotti (CSC); C. langguthi fade mounting medium Vectashield. Metaphases were ana- (CLA); C. vivoi (CVI); C. goytaca (CGO) and C. subflavus lyzed with an Axiophot fluorescence microscope (Carl (CSU) (Table 1). Zeiss) using the software ISIS (Metasystem) that can overlap The animals surveyed by the authors were live trapped images of filters for DAPI and FITC. In some cases, meta- under ICMBio licences (numbers 11603-1 and 24003-4) of phases were analyzed in an Axioskop 40 epifluorescence mi- Instituto Chico Mendes de Conservação da Biodiversidade. croscope (Carl Zeiss) equipped with the AxionVision soft- Some specimens were captured by collaborators under their ware and propidium iodide (PI) was added to the respective licenses (Table S1). Animals were euthanized ac- fluorescence antifade solution (0.5 mL/mL) to visualize cording to “Guidelines for Animal use” (Sikes et al., 2011) chromosomes. under permission of the Comissão de Ética para Uso de Chromosome painting was performed on metaphases Animais do Instituto Butantan (CEUAIB 1151/13). of one representative of each species - C. marinhus, C. Skins, skulls and partial skeletons of C. maracajuen- maracajuensis, C. scotti, C. langguthi, C. vivoi, C. goytaca sis, C. marinhus and C. langguthi were deposited at the and C. subflavus with 2n=54 - (Table 1), except for C. akroai Museu Nacional da Universidade Federal do Rio de Janeiro and C. subflavus (2n=55 and 56), which lacked sufficient (MN); C. akroai, C. scotti, C. vivoi and C. subflavus were de- metaphases. posited in the Museu de Zoologia da Universidade de São Twenty specific single painting probes from Oligory- Paulo (MZUSP) and C. goytaca was deposited at the Núcleo zomys moojeni with 2n=70, FN=72 obtained by fluorescen- de Pesquisa em Ecologia e Desenvolvimento Sócio-Am- ce-activated chromosome sorting (FACS) were used (OMO biental de Macaé (NPM). Cell suspensions or fibroblast cells Xa, OMO 1–8, 11, 16, 17, 25–30, 33, and 34; see Di-Nizo et are deposited in the Laboratório de Ecologia e Evolução do al., 2015). The probes were made by degenerate oligonu- Instituto Butantan. cleotide-primed polymerase chain reaction (DOP-PCR) and Metaphases of C. marinhus, C. maracajuensis, C. vi- labeled with biotin-16-dUTP (Telenius et al., 1992; Yang et voi, and C. subflavus with 2n=54 were obtained in vitro from al., 1995). FISH was performed according to previous stud- Table 1 - Specimens analyzed in this work. Species Voucher Locality 2n FN Sex FISH OMO Phylogeny C. marinhus (CMARI) CRB1835 Cocos, BA 56 54 M X X C. maracajuensis (CMARA) MN71687 Parque Nacional Emas, GO 56 58 M X X C. akroai (CAK) MZUSP30347 Uruçuí-Una, PI 60 76 M - X C. scotti (CSC) MJJS189 Serra das Galés, GO 58 72 F X X C. langguthi (CLA) JFV474 Piracuruca, PI 46 56 M X X C. vivoi (CVI) BIO555 Mucugê, BA 50 64 M X - C. goytaca (CGO) NPM933 Restinga de Jurubatiba, RJ 54 66 F X X C. subflavus (CSU) CIT2053 Itirapina, SP 56 64 M - X