Karyotype conservation and genomic organization of repetitive sequences in the leaf-cutting ant Atta cephalotes (Linnaeus, 1758) (Formicidae: Myrmicinae).

Leaf-cutting ants are among the New World's most conspicuous and studied ant species due to their notable ecological and economic roles. Cytogenetic studies carried out in Atta show remarkable karyotype conservation among the species. We performed classical cytogenetics and physical mapping of repetitive sequences in the leaf-cutting ant Atta cephalotes (Linnaeus, 1758), the type species of the genus. Our goal was to test the karyotype conservation in Atta and to understand the genomic organization and diversity regarding repetitive sequences in leaf-cutting ants. Atta cephalotes showed 2n = 22 (18m + 2sm + 2st) chromosomes. The heterochromatin followed a centromeric pattern, and the GC-rich regions and 18S rDNA clusters were co-located interstitially in the 4th metacentric pair. These cytogenetic characteristics were observed in other Atta species that had previously been studied, confirming the karyotype conservation in Atta. Evolutionary implications regarding the conservation of the chromosome number in leaf-cutting ants are discussed. Telomeric motif (TTAGG)n was detected in A. cephalotes as observed in other ants. Five out of the 11 microsatellites showed a scattered distribution exclusively on euchromatic areas of the chromosomes. Repetitive sequences mapped on the chromosomes of A. cephalotes are the first insights into genomic organization and diversity in leaf-cutting ants, useful in further comparative studies.

Developing Oligo Probes for Chromosomes Identification in Hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L., 2n = 20) is a valuable crop that is successfully used as a food, technical and medicinal crop. It is a dioecious plant with an XX\XY sex determination system. Some chromosomes of C. sativa have almost the same lengths and centromeric indexes. Cytogenetic markers help to distinguish similar plant chromosomes, including sex chromosomes, which is important for the breeding process. Two repeats (CS-1 and CS-237) were used to develop labeled oligo-probes for rapid and low-cost oligo-FISH. These oligos can be recommended for use as cytological markers to distinguish sex chromosomes (X and Y) and somatic chromosome pairs 3, 6, and 8 by rapid oligo-FISH in a short time.

The Nuclear 35S rDNA World in Plant Systematics and Evolution: A Primer of Cautions and Common Misconceptions in Cytogenetic Studies.

The ubiquitous presence of rRNA genes in nuclear, plastid, and mitochondrial genomes has provided an opportunity to use genomic markers to infer patterns of molecular and organismic evolution as well as to assess systematic issues throughout the tree of life. The number, size, location, and activity of the 35S rDNA cistrons in plant karyotypes have been used as conventional cytogenetic landmarks. Their scrutiny has been useful to infer patterns of chromosomal evolution and the data have been used as a proxy for assessing species discrimination, population differentiation and evolutionary relationships. The correct interpretation of rDNA markers in plant taxonomy and evolution is not free of drawbacks given the complexities derived from the lability of the genetic architecture, the diverse patterns of molecular change, and the fate and evolutionary dynamics of the rDNA units in hybrids and polyploid species. In addition, the terminology used by independent authors is somewhat vague, which often complicates comparisons. To date, no efforts have been reported addressing the potential problems and limitations involved in generating, utilizing, and interpreting the data from the 35S rDNA in cytogenetics. This review discusses the main technical and conceptual limitations of these rDNA markers obtained by cytological and karyological experimental work, in order to clarify biological and evolutionary inferences postulated in a systematic and phylogenetic context. Also, we provide clarification for some ambiguity and misconceptions in terminology usually found in published work that may help to improve the usage of the 35S ribosomal world in plant evolution.

Chromosome Evolution in the Genus Partamona (Apidae: Meliponini), with Comments on B Chromosome Origin.

The genus Partamona includes 33 species of stingless bees, of which 11 were studied cytogenetically. The main goal of this study was to propose a hypothesis about chromosomal evolution in Partamona by combining molecular and cytogenetic data. Cytogenetic analyses were performed on 3 Partamona species. In addition, the molecular phylogeny included mitochondrial sequences of 11 species. Although the diploid number was constant within the genus, 2n = 34, B chromosomes were reported in 7 species. Cytogenetic data showed karyotypic variations related to chromosome morphology and the amount and distribution of heterochromatin and repetitive DNA. The molecular phylogenetic reconstruction corroborated the monophyly of the genus and separated the 2 clades (A and B). This separation was also observed in the cytogenetic data, in which species within each clade shared most of the cytogenetic characteristics. Furthermore, our data suggested that the B chromosome in the genus Partamona likely originated from a common ancestor of the species that have it in clade B and, through interspecific hybridization, it appeared only in Partamona rustica from clade A. Based on the above, Partamona is an interesting genus for further investigations using molecular mapping of B chromosomes as well as for broadening phylogenetic data.

Cytogenetic markers and their spatial distribution in a population living in proximity to areas sprayed with pesticides.

Human populations are in contact with potentially toxic substances in varying amounts, if the exposure is work-related or direct, generally the amount of toxin is usually greater than if the exposure is environmental through the drifts that occur. It was proposed to determine the existence of genotoxic damage evaluated through Chromosomal Aberrations and Micronuclei assays and their spatial distribution pattern, as well as the possible relationship between that damage and the values found in biochemical biomarkers, in groups of individuals environmental exposure (respiratory exposure) to mixtures of pesticides, in the province of Córdoba-Argentina. Biochemical and hematological determinations were made in each samples. The results reveal that the monitoring of human populations through the analysis of cytogenetic markers enabled the detection of direct damage in man caused by polluting substances and the results were obtained rapidly. The disadvantage of this type of study is the inability to estimate the degree of exposure.

Karyotype structure and cytogenetic markers of Amoimyrmex bruchi and Amoimyrmex silvestrii: contribution to understanding leaf-cutting ant relationships.

Leaf-cutting ants are considered the most important herbivores in terrestrial environments throughout the Neotropics. Amoimyrmex Cristiano, Cardoso, & Sandoval, 2020 is the sister clade of the remaining leaf-cutting ants from the genera Atta and Acromyrmex. Amoimyrmex striatus was the only species cytogenetically studied within the genus and shares the same chromosomal number as Atta, bearing 22 chromosomes, whereas Acromyrmex bears 38 chromosomes, with the exception of the social parasite Acromyrmex ameliae (2n = 36). Our objective here was to cytogenetically analyze the species of Amoimyrmex bruchi and Amoimyrmex silvestrii, as well as to describe the karyotype of these sister species, using an integrative approach using classical and molecular cytogenetics. We aimed to characterize the cytogenetic markers that contribute to the systematics and taxonomy of the genus. Our results showed that the karyotypes of these two species are very similar, with an identical chromosome number (2n = 22), chromosome morphology (2K = 20m + 2sm), and location of 18S rDNA and telomeric repeat TTAGG on the chromosomes. However, the microsatellite probe GA(15) showed variation across the species and populations studied. We suggest that both species diverged relatively recently and are unmistakably sisters because of the many shared characteristics, including the highly conserved karyotypes.

T-Cell Acute Lymphoblastic Leukemia: Biomarkers and Their Clinical Usefulness.

T-cell acute lymphoblastic leukemias (T-ALL) are immature lymphoid tumors localizing in the bone marrow, mediastinum, central nervous system, and lymphoid organs. They account for 10-15% of pediatric and about 25% of adult acute lymphoblastic leukemia (ALL) cases. It is a widely heterogeneous disease that is caused by the co-occurrence of multiple genetic abnormalities, which are acquired over time, and once accumulated, lead to full-blown leukemia. Recurrently affected genes deregulate pivotal cell processes, such as cycling (CDKN1B, RB1, TP53), signaling transduction (RAS pathway, IL7R/JAK/STAT, PI3K/AKT), epigenetics (PRC2 members, PHF6), and protein translation (RPL10, CNOT3). A remarkable role is played by NOTCH1 and CDKN2A, as they are altered in more than half of the cases. The activation of the NOTCH1 signaling affects thymocyte specification and development, while CDKN2A haploinsufficiency/inactivation, promotes cell cycle progression. Among recurrently involved oncogenes, a major role is exerted by T-cell-specific transcription factors, whose deregulated expression interferes with normal thymocyte development and causes a stage-specific differentiation arrest. Hence, TAL and/or LMO deregulation is typical of T-ALL with a mature phenotype (sCD3 positive) that of TLX1, NKX2-1, or TLX3, of cortical T-ALL (CD1a positive); HOXA and MEF2C are instead over-expressed in subsets of Early T-cell Precursor (ETP; immature phenotype) and early T-ALL. Among immature T-ALL, genomic alterations, that cause BCL11B transcriptional deregulation, identify a specific genetic subgroup. Although comprehensive cytogenetic and molecular studies have shed light on the genetic background of T-ALL, biomarkers are not currently adopted in the diagnostic workup of T-ALL, and only a limited number of studies have assessed their clinical implications. In this review, we will focus on recurrent T-ALL abnormalities that define specific leukemogenic pathways and on oncogenes/oncosuppressors that can serve as diagnostic biomarkers. Moreover, we will discuss how the complex genomic profile of T-ALL can be used to address and test innovative/targeted therapeutic options.

Determination of cytogenetic markers for biological monitoring in coypu (Myocastor coypu).

Cytogenetic tests are used to assess the influence of physical and chemical factors with potential mutagenic and genotoxic properties on the animal organism. The test results make it possible to eliminate mutagens, as well as helping predict possible genetic consequences in animal cells and assess animal resistance. The aim of this study was to examine, using cytogenetic tests, the spontaneous chromosome and DNA damage in coypu lymphocytes. Four tests: fragile site (FS), bleomycin (BLM), micronucleus, (MN) and comet were used for the first time in coypu cells. The averages with standard deviations obtained in the research were as follows: 3.30 ± 0.80 fragile sites/cell; 0.63 ± 0.80 BLM damage/cell; 6.10 ± 0.53% binucleated cells with MN; and 3.24 ± 0.63% DNA in tail. The present analysis showed high interindividual variation in spontaneous chromosomal and DNA damage levels. In the case of micronucleus, fragile sites, and comet assays, the differences between animals were statistically significant. The data suggest that these assays are sensitive enough to detect some effects on an individual animal and can be proposed as tools for coypu biomonitoring.

Integrated Karyotypes of Diploid and Tetraploid Carrizo Citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf.) as Determined by Sequential Multicolor Fluorescence in situ Hybridization With Tandemly Repeated DNA Sequences.

Carrizo citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf., CC] is one of the most widely used rootstocks in citriculture worldwide, but its cytogenetic study has been hampered by its inherent small size, morphological similarity to mitotic chromosomes, and lack of accessible cytological landmarks. In our previous study, a spontaneously occurring tetraploid CC seedling was discovered. The main goals of this study were to elucidate the chromosome constitution and construct the karyotypes of diploid CC rootstock and its corresponding spontaneously occurring tetraploid. To accomplish these, the chromosomal characteristics were investigated by sequential multicolor fluorescence in situ hybridization (FISH) with eight properly labeled repetitive DNA sequences, including a centromere-like repeat, four satellite repeats, two rDNAs, and an oligonucleotide of telomeric (TTTAGGG) n repeat. The results nicely demonstrated that these repetitive DNAs are reliable cytogenetic markers that collectively facilitate simultaneous and unequivocal identification of homologous chromosome pairs. Based on chromosome size and morphology together with FISH patterns of repetitive DNAs, an integrated karyotype of CC rootstock was constructed, consisting of 2n = 2x = 12m (1sat) + 6sm with karyotype asymmetry degree being divided into 2B category. Cytogenetically speaking, the variable and asymmetric distribution patterns of these repetitive DNAs were fully confirmed the hybrid nature of CC rootstock. In addition, comparative distribution patterns and chromosomal localizations of these repetitive DNAs convincingly showed that this tetraploid CC material arose from somatic chromosome doubling of diploid CC rootstock. This study revealed, for the first time, the integrated karyotype and chromosomal characteristics of this important citrus rootstock as well as its spontaneously occurring tetraploid plant. Furthermore, this study is a good prospective model for study species with morphologically indistinguishable small chromosomes.

Cytogenetic characterization of Ameivula ocellifera (Spix, 1825) (Squamata, Teiidae) from the brazilian northeast / Caracterização citogenética de Ameivula ocellifera (Spix, 1825) (Squamata, Teiidae) do nordeste brasileiro

Ameivula is as a new genus of Teiidae family that emerged after extensive revision of species that comprised the former complex of species called Cnemidophorus group. Its species has a wide distribution from the northeast of Brazil to northern Argentina. Cytogenetic studies in the Teiidae family have shown that karyotypical data are important tools in phylogenetic and systematic studies within this group allowing to determine the position of species in the family. Thus, this study aimed to describe the karyotype of Ameivulaocellifera (Spix, 1825) from Picos, Piauí state in the Brazilian Northeast. Specimens were collected from August 2014 to October 2015 using interception traps and pitfalls, mounted randomly along the Caatinga area. The animals were collected and transported to Federal Institute of Piauí, campus Picos, where was carried out all laboratory procedures. Individuals analyzed showed a diploid number of 2n = 50 for both sexes, with karyotype composed by 30 macrochromosomes and 20 microchromosomes of telocentric and subtelocentric types. There were no heteromorphic sex chromosomes in the studied specimens. C-band technique evidenced the heterochromatic blocks in pericentromeric and telomeric regions of chromosomes. The nucleolar organizing regions appeared as a simple unit located at the terminal portion of the long arm of chromosomal pair number 5. The chromosomal characteristics of A. ocellifera analyzed do not show divergences regarding individuals from other regions. However, the nucleolar organizing regions seems to be a good chromosomal marker that permits to distinguish the species already studied.

Ameivula é um novo gênero da família Teiidae que surgiu após extensa revisão de espécies que compuseram o antigo complexo de espécies chamado grupo Cnemidophorus. Suas espécies têm uma ampla distribuição do nordeste do Brasil ao norte da Argentina. Estudos citogenéticos na família Teiidae mostraram que os dados cariotípicos são ferramentas importantes em estudos filogenéticos e sistemáticos dentro deste grupo, permitindo determinar a posição das espécies na família. Assim, este estudo tem como objetivo descrever o cariótipo de Ameivula ocellifera (Spix, 1825) de Picos, no nordeste brasileiro. Os espécimes foram coletados de agosto de 2014 a outubro de 2015 utilizando armadilhas de interceptação e armadilhas, montadas aleatoriamente ao longo da área da Caatinga. Os animais foram coletados e transportados para o Instituto Federal do Piauí, campus Picos, onde foram realizados todos os procedimentos laboratoriais. Os indivíduos analisados apresentaram um número diploide de 2n = 50 para ambos os sexos, com cariótipo composto por 30 macrocromossomos e 20 microcromossomos dos tipos telocêntrico e subtelocêntrico. Não houve cromossomos sexuais heteromórficos nos espécimes estudados. A técnica da banda C evidenciou os blocos heterocromáticos nas regiões pericentroméricas e teloméricas dos cromossomos. As regiões organizadoras de nucléolos apareceram como uma unidade simples localizada na porção terminal do braço longo do par cromossômico número 5. As características cromossômicas de A. ocellifera analisadas não mostram divergências em relação a indivíduos de outras regiões. No entanto, as regiões organizadoras de nucléolos parecem ser um bom marcador cromossômico que permite distinguir as espécies já estudadas.

Development of new cytogenetic markers for Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang.

Thinopyrum ponticum (Podpera, 1902) Z.-W. Liu & R.-C.Wang, 1993 is an important polyploid wild perennial Triticeae species that is widely used as a source of valuable genes for wheat but its genomic constitution has long been debated. For its chromosome identification, only a limited set of FISH probes has been used. The development of new cytogenetic markers for Th. ponticum chromosomes is of great importance both for cytogenetic characterization of wheat-wheatgrass hybrids and for fundamental comparative studies of phylogenetic relationships between species. Here, we report on the development of five cytogenetic markers for Th. ponticum based on repetitive satellite DNA of which sequences were selected from the whole genome sequence of Aegilops tauschii Cosson, 1849. Using real-time quantitative PCR we estimated the abundance of the found repeats: P720 and P427 had the highest abundance and P132, P332 and P170 had lower quantity in Th. ponticum genome. Using fluorescence in situ hybridization (FISH) we localized five repeats to different regions of the chromosomes of Th. ponticum. Using reprobing multicolor FISH we colocalized the probes between each other. The distribution of these found repeats in the Triticeae genomes and its usability as cytogenetic markers for chromosomes of Th. ponticum are discussed.

Pipeline for the Rapid Development of Cytogenetic Markers Using Genomic Data of Related Species.

Repetitive DNA including tandem repeats (TRs) is a significant part of most eukaryotic genomes. TRs include rapidly evolving satellite DNA (satDNA) that can be shared by closely related species, their abundance may be associated with evolutionary divergence, and they have been widely used for chromosome karyotyping using fluorescence in situ hybridization (FISH). The recent progress in the development of whole-genome sequencing and bioinformatics tools enables rapid and cost-effective searches for TRs including satDNA that can be converted into molecular cytogenetic markers. In the case of closely related taxa, the genome sequence of one species (donor) can be used as a base for the development of chromosome markers for related species or genomes (target). Here, we present a pipeline for rapid and high-throughput screening for new satDNA TRs in whole-genome sequencing of the donor genome and the development of chromosome markers based on them that can be applied in the target genome. One of the main peculiarities of the developed pipeline is that preliminary estimation of TR abundance using qPCR and ranking found TRs according to their copy number in the target genome; it facilitates the selection of the most prospective (most abundant) TRs that can be converted into cytogenetic markers. Another feature of our pipeline is the probe preparation for FISH using PCR with primers designed on the aligned TR unit sequences and the genomic DNA of a target species as a template that enables amplification of a whole pool of monomers inherent in the chromosomes of the target species. We demonstrate the efficiency of the developed pipeline by the example of FISH probes developed for A, B, and R subgenome chromosomes of hexaploid triticale (BBAARR) based on a bioinformatics analysis of the D genome of Aegilops tauschii (DD) whole-genome sequence. Our pipeline can be used to develop chromosome markers in closely related species for comparative cytogenetics in evolutionary and breeding studies.

Chromosomal Evolution and Cytotaxonomy in Wrasses (Perciformes; Labridae).

The wrasses (family Labridae) represent a suitable model to understand chromosomal evolution and to test the efficacy of cytotaxonomy since they display a remarkable karyotypic variation, rarely reported in marine Perciformes, as well as a high number of species and complex systematics. Therefore, we provided new chromosomal data in 5 labrids from South Atlantic (Doratonotus megalepis, Halichoeres dimidiatus, Halichoeres penrosei, Thalassoma noronhanum, and Xyrichtys novacula) and carried out a detailed comparative analysis of karyotypic data in Labridae using multivariate approaches. Basal diploid values (2n = 48) were observed in most of species studied in the present work but D. megalepis (2n = 46), along with distinct karyotype formulae. Single 18S rDNA sites interspersed with GC-rich heterochromatin were also commonly reported except for both Halichoeres species (2 18S rDNA-bearing pairs), following a species-specific pattern. These data show the high rates of chromosomal evolution in wrasses, ranging from microstructural rearrangements to centric fusions. A revision of chromosomal data in Labridae based on multivariate analysis of 74 taxa allowed inferring karyoevolutionary trends within tribes and genera of wrasses. The dendrogram obtained was in agreement with recent systematic hypotheses. In spite of the independent occurrence of some chromosomal rearrangements, karyoevolutionary trends could be identified within tribes of Labridae. Moreover, the karyotypic features are also suitable as cytotaxonomic markers of wrasses.

Towards a FISH-based karyotype of Rosa L. (Rosaceae).

The genus Rosa Linnaeus, 1753 has important economic value in ornamental sector and many breeding activities are going on supported by molecular studies. However, the cytogenetic studies of rose species are scarce and mainly focused on chromosome counting and chromosome morphology-based karyotyping. Due to the small size of the chromosomes and a high frequency of polyploidy in the genus, karyotyping is very challenging for rose species and requires FISH-based cytogenetic markers to be applied. Therefore, in this work the aim is to establish a FISH-based karyotype for Rosa wichurana (Crépin, 1888), a rose species with several benefits for advanced molecular cytogenetic studies of genus Rosa (Kirov et al. 2015a). It is shown that FISH signals from 5S, 45S and an Arabidopsis-type telomeric repeat are distributed on five (1, 2, 4, 5 and 7) of seven chromosome pairs. In addition, it is demonstrated that the interstitial telomeric repeat sequences (ITR) are located in the centromeric regions of four chromosome pairs. Using low hybridization stringency for ITR visualization, we showed that the number of ITR signals increases four times (1-4 signals). This study is the first to propose a FISH-based Rosa wichurana karyotype for the reliable identification of chromosomes. The possible origin of Rosa wichurana ITR loci is discussed.

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae (Siluriformes): a report case in Corydoras carlae Nijssen & Isbrücker, 1983.

Corydoras Lacepède, 1803 is the most specious genus of Corydoradinae subfamily and many of its species are still unknown in relation to molecular cytogenetic markers. However, the diploid number and karyotypic formula were recorded for many species of this group. In current study, we provided the first cytogenetic information of Corydoras carlae Nijssen & Isbrücker, 1983, an endemic fish species from Iguassu River basin, Paraná State, Brazil. The individuals were collected in Florido River, a tributary of Iguassu River and analysed with respect to diploid number, heterochromatin distribution pattern, Ag-NORs and mapping of 5S and 18S ribosomal genes. The karyotype of this species comprises 46 chromosomes arranged in 22m+22sm+2st. The heterochromatin is distributed in centromeric and pericentromeric positions in most of the chromosomes, and also associated with NORs. The Ag-NORs were detected in the terminal position on the long arm of the metacentric pair 6. The double-FISH technique showed that 5S rDNA and 18S rDNA were co-localized in the terminal portion on the long arm of the metacentric pair 6. This condition of co-localization of ribosomal genes in Corydoras carlae seems to represent a marker for this species.

The role of chromosomal fusion in the karyotypic evolution of the genus Ageneiosus (Siluriformes: Auchenipteridae)

Ageneiosus is the most widely distributed genus of the family Auchenipteridae among South American river basins. Although chromosome studies in the family are scarce, this genus has the largest number of analyzed species, with 2n = 54 to 56 chromosomes, differing from the rest of the family (2n = 58). This study aimed to analyze Ageneiosus inermis from the Araguaia River basin. The diploid number found was of 56 chromosomes. Heterochromatin was allocated in terminal region of most chromosomes, plus a pericentromeric heterochromatic block in pair 1, a pair distinguished by size in relation to other chromosomes pairs. AgNORs were detected in only one submetacentric chromosome pair, which was confirmed by FISH. 5S rDNA was present in only one metacentric chromosome pair. Hybridization with [TTAGGG]n sequence marked the telomeres of all chromosomes, in addition to an ITS in the proximal region of the short arm of pair 1. The repetitive [GATA]n sequence was dispersed, with preferential location in terminal region of the chromosomes. Ageneiosus has a genomic organization somewhat different when compared to other Auchenipteridae species. Evidences indicate that a chromosomal fusion originated the first metacentric chromosome pair in A. inermis, rearrangement which may be a basal event for the genus.

Ageneiosus é o gênero da família Auchenipteridae mais amplamente distribuído em bacias da América do Sul. Apesar dos estudos cromossômicos nesta família serem escassos, este gênero tem o maior número de espécies analisadas, com número diploide variando de 54 a 56 cromossomos, o que difere do restante da família (2n = 58). Este estudo objetivou analisar Ageneiosus inermis da bacia do rio Araguaia. O número diploide encontrado foi de 56 cromossomos. A heterocromatina se mostrou localizada na região terminal da maioria dos cromossomos, além de um bloco heterocromático pericentromérico no par 1, um par facilmente distinguível no cariótipo pelo seu maior tamanho quando comparado aos outros pares do complemento. AgRONs foram detectadas em somente um par de cromossomos submetacêntricos, que foi confirmado pela FISH. 5S rDNA se mostrou presente em somente um par de cromossomos metacêntricos. A hibridização com a sequência [TTAGGG]n marcou os telômeros de todos os cromossomos, além de um ITS (sequência telomérica intersticial) na região proximal do braço curto do par 1. A sequência repetitiva [GATA]n se mostrou dispersa, com localização preferencial na região terminal dos cromossomos. Ageneiosus apresenta uma organização genômica um pouco diferente quando comparada a outras espécies de Auchenipteridae. As evidências indicam que uma fusão cromossômica originou o primeiro par de cromossomos metacêntricos de A. inermis, rearranjo que parece ser um evento basal para o gênero.