Understanding microchromosomal organization and evolution in four representative woodpeckers (Picidae, Piciformes) through BAC-FISH analysis.

The genome organization of woodpeckers has several distinctive features e.g., an uncommon accumulation of repetitive sequences, enlarged Z chromosomes, and atypical diploid numbers. Despite the large diversity of species, there is a paucity of detailed cytogenomic studies for this group and we thus aimed to rectify this. Genome organization patterns and hence evolutionary change in the microchromosome formation of four species (Colaptes campestris, Veniliornis spilogaster, Melanerpes candidus, and Picumnus nebulosus) was established through fluorescence in situ hybridization using bacterial artificial chromosomes originally derived from Gallus gallus and Taeniopygia guttata. Findings suggest that P. nebulosus (2n = 110), which was described for the first time, had the most basal karyotype among species of Picidae studied here, and probably arose as a result of fissions of avian ancestral macrochromosomes. We defined a new chromosomal number for V. spilogaster (2n = 88) and demonstrated microchromosomal rearrangements involving C. campestris plus a single, unique hitherto undescribed rearrangement in V. spilogaster. This comprised an inversion after a fusion involving the ancestral microchromosome 12 (homologous to chicken microchromosome 12). We also determined that the low diploid number of M. candidus is related to microchromosome fusions. Woodpeckers thus exhibit significantly rearranged karyotypes compared to the putative ancestral karyotype.

Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

How diverse a monocentric chromosome can be? Repeatome and centromeric organization of Juncus effusus (Juncaceae).

Juncus is the largest genus of Juncaceae and was considered holocentric for a long time. Recent findings, however, indicated that 11 species from different clades of the genus have monocentric chromosomes. Thus, the Juncus centromere organization and evolution need to be reassessed. We aimed to investigate the major repetitive DNA sequences of two accessions of Juncus effusus and its centromeric structure by employing whole-genome analyses, fluorescent in situ hybridization, CENH3 immunodetection, and chromatin immunoprecipitation sequencing. We showed that the repetitive fraction of the small J. effusus genome (~270 Mbp/1C) is mainly composed of Class I and Class II transposable elements (TEs) and satellite DNAs. Three identified satellite DNA families were mainly (peri)centromeric, with two being associated with the centromeric protein CENH3, but not strictly centromeric. Two types of centromere organization were discerned in J. effusus: type 1 was characterized by a single CENH3 domain enriched with JefSAT1-155 or JefSAT2-180, whereas type 2 showed multiple CENH3 domains interrupted by other satellites, TEs or genes. Furthermore, while type 1 centromeres showed a higher degree of satellite identity along the array, type 2 centromeres had less homogenized arrays along the multiple CENH3 domains per chromosome. Although the analyses confirmed the monocentric organization of J. effusus chromosomes, our data indicate a more dynamic arrangement of J. effusus centromeres than observed for other plant species, suggesting it may constitute a transient state between mono- and holocentricity.

Satellitome Analysis in the Southern Lapwing (Vanellus chilensis) Genome: Implications for SatDNA Evolution in Charadriiform Birds.

Vanellus (Charadriidae; Charadriiformes) comprises around 20 species commonly referred to as lapwings. In this study, by integrating cytogenetic and genomic approaches, we assessed the satellite DNA (satDNA) composition of one typical species, Vanellus chilensis, with a highly conserved karyotype. We additionally underlined its role in the evolution, structure, and differentiation process of the present ZW sex chromosome system. Seven distinct satellite DNA families were identified within its genome, accumulating on the centromeres, microchromosomes, and the W chromosome. However, these identified satellite DNA families were not found in two other Charadriiformes members, namely Jacana jacana and Calidris canutus. The hybridization of microsatellite sequences revealed the presence of a few repetitive sequences in V. chilensis, with only two out of sixteen displaying positive hybridization signals. Overall, our results contribute to understanding the genomic organization and satDNA evolution in Charadriiform birds.

Differential Spreading of Microsatellites in Holocentric Chromosomes of Chagas Disease Vectors: Genomic and Evolutionary Implications.

This study focused on analyzing the distribution of microsatellites in holocentric chromosomes of the Triatominae subfamily, insect vectors of Chagas disease. We employed a non-denaturing FISH technique to determine the chromosomal distribution of sixteen microsatellites across twenty-five triatomine species, involving five genera from the two principal tribes: Triatomini and Rhodniini. Three main hybridization patterns were identified: strong signals in specific chromosomal regions, dispersed signals dependent on microsatellite abundance and the absence of signals in certain chromosomal regions or entire chromosomes. Significant variations in hybridization patterns were observed between Rhodniini and Triatomini species. Rhodniini species displayed weak and scattered hybridization signals, indicating a low abundance of microsatellites in their genomes. In contrast, Triatomini species exhibited diverse and abundant hybridization patterns, suggesting that microsatellites are a significant repetitive component in their genomes. One particularly interesting finding was the high abundance of GATA repeats, and to a lesser extent AG repeats, in the Y chromosome of all analyzed Triatomini species. In contrast, the Y chromosome of Rhodniini species did not show enrichment in GATA and AG repeats. This suggests that the richness of GATA repeats on the Y chromosome likely represents an ancestral trait specific to the Triatomini tribe. Furthermore, this information can be used to elucidate the evolutionary relationships between Triatomini and other groups of reduviids, contributing to the understanding of the subfamily's origin. Overall, this study provides a comprehensive understanding of the composition and distribution of microsatellites within Triatominae genomes, shedding light on their significance in the evolutionary processes of these species.

Supernumerary B Chromosomes of Tetragonisca fiebrigi Share Repeat Content with Standard Chromosome Set of both T. fiebrigi and Tetragonisca angustula (Apidae: Meliponini).

The stingless bees Tetragonisca angustula and Tetragonisca fiebrigi are widely distributed in Brazil, and both are commonly known as "jataí." Our goal was to investigate the possible origin of the B chromosomes in T. fiebrigi, a cytotaxonomic trait that differentiates T. fiebrigi from T. angustula. We analyzed diploid chromosome number (2n), B chromosome incidence, patterns of constitutive heterochromatin, and in situ localization of different repetitive DNA probes in T. angustula and T. fiebrigi. Both species displayed 2n = 34, with similar karyotype structures. One to three B chromosomes were observed in T. fiebrigi only. Constitutive heterochromatin was distributed on one arm of all chromosomes in both species, and T. fiebrigi B chromosomes were mainly heterochromatic with one euchromatic extremity. The (GA)15 and (CAA)10 microsatellite probes marked the euchromatic arms of all chromosomes in both species without marking the B chromosomes. The 18S ribosomal DNA (rDNA) probe marked 10 chromosomes in T. angustula and 6 A chromosomes in T. fiebrigi with an additional marking on 1B in individuals with 3B. The Tan-Bsp68I repetitive DNA probe marked the heterochromatic portion of all T. fiebrigi A and B chromosomes. This probe also marked the heterochromatic portion of all T. angustula chromosomes; therefore, both alternative hypotheses to the B chromosome origin are possible: (i) from the A chromosome complement of T. fiebrigi (intraspecific origin); or (ii) a by-product of genome reshuffling following the hybridization between T. fiebrigi and T. angustula (interspecific origin).

Cytogenetic characterization of solitary wasp Ancistrocerus flavomarginatus (Brèthes, 1906) (Hymenoptera, Vespidae) with insights into the chromosomal evolution in the genus.

Cytogenetic studies have enabled the characterization of the chromosomal macrostructure and microstructure and have contributed to the understanding of the evolution of wasp karyotypes. However, studies on Eumeninae solitary wasps are scarce. In this study, we characterized the karyotype of Ancistrocerus flavomarginatus (Brèthes, 1906) and compared it with previous data from other Ancistrocerus (Wesmael, 1836) species to shed light on the chromosomal diversity of the genus. A chromosome number of 2n = 24 in females and n = 12 in males was observed. Comparing the A. flavomarginatus karyotype with that of another Ancistrocerus species showed variations in the morphology of some chromosomal pairs. The presence of two larger chromosome pairs, almost entirely heterochromatic, and the predominance of subtelocentric chromosomes with heterochromatic short arms in A. flavomarginatus support the occurrence of fissions in Ancistrocerus. A single site of ribosomal genes was observed in A. flavomarginatus, in addition to a size polymorphism of these rDNA clusters between the homologues of some analyzed females. This polymorphism may originate from duplications/deletions due to unequal crossing-over or amplification via transposable elements. The (GA)15 microsatellite is located exclusively in euchromatic regions. Our data show that different rearrangements seem to shape chromosomal evolution in Ancistrocerus species.

Relationship between fruit phenotypes and domestication in hexaploid populations of biribá (Annona mucosa) in Brazilian Amazonia.

Background: Biribá (Annona mucosa Jacq.) is a fruit tree domesticated in Amazonia and has polyploid populations. The species presents ample phenotypic variation in fruit characteristics, including weight (100-4,000 g) and differences in carpel protrusions. Two cytotypes are recorded in the literature (2n = 28, 42) and genome size records are divergent (2C = 4.77, 5.42 and 6.00 pg). To decipher the role of polyploidy in the domestication of A. mucosa, we examined the relationships among phenotypic variation, chromosome number and genome size, and which came first, polyploidization or domestication. Methodology: We performed chromosome counts of A. mucosa from central and western Brazilian Amazonia, and estimated genome size by flow cytometry. We performed phylogenetic reconstruction with publicly available data using a Bayesian framework, time divergence analysis and reconstructed the ancestral chromosome number for the genus Annona and for A. mucosa. Results: We observed that variation in fruit phenotypes is not associated with variation in chromosome number and genome size. The most recent common ancestor of A. mucosa is inferred to be polyploid and diverged before domestication. Conclusions: We conclude that, when domesticated, A. mucosa was already polyploid and we suggest that human selection is the main evolutionary force behind fruit size and fruit morphological variation in Annona mucosa.

Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2 gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Chromosomal rearrangements and the first indication of an ♀X1 X1 X2 X2 /♂X1 X2 Y sex chromosome system in Rineloricaria fishes (Teleostei: Siluriformes).

Rineloricaria is the most diverse genus within the freshwater fish subfamily Loricariinae, and it is widely distributed in the Neotropical region. Despite limited cytogenetic data, records from southern and south-eastern Brazil suggest a high rate of chromosomal rearrangements in this genus, mirrored in remarkable inter- and intraspecific karyotype variability. In the present work, we investigated the karyotype features of Rineloricaria teffeana, an endemic representative from northern Brazil, using both conventional and molecular cytogenetic techniques. We revealed different diploid chromosome numbers (2n) between sexes (33♂/34♀), which suggests the presence of an ♀X1 X1 X2 X2 /♂X1 X2 Y multiple sex chromosome system. The male-limited Y chromosome was the largest and the only biarmed element in the karyotype, implying Y-autosome fusion as the most probable mechanism behind its origination. C-banding revealed low amounts of constitutive heterochromatin, mostly confined to the (peri)centromeric regions of most chromosomes (including the X2 and the Y) but also occupying the distal regions of a few chromosomal pairs. The chromosomal localization of the 18S ribosomal DNA (rDNA) clusters revealed a single site on chromosome pair 4, which was adjacent to the 5S rDNA cluster. Additional 5S rDNA loci were present on the autosome pair 8, X1 chromosome, and in the presumed fusion point on the Y chromosome. The probe for telomeric repeat motif (TTAGGG)n revealed signals of variable intensities at the ends of all chromosomes except for the Y chromosome, where no detectable signals were evidenced. Male-to-female comparative genomic hybridization revealed no sex-specific or sex-biased repetitive DNA accumulations, suggesting a presumably low level of neo-Y chromosome differentiation. We provide evidence that rDNA sites might have played a role in the formation of this putative multiple sex chromosome system and that chromosome fusions originate through different mechanisms among different Rineloricaria species.

On the Origin of Neo-Sex Chromosomes in the Neotropical Dragonflies Rhionaeschna bonariensis and R. planaltica (Aeshnidae, Odonata).

Odonata have holokinetic chromosomes. About 95% of species have an XX/X0 sex chromosome system, with heterogametic males. There are species with neo-XX/neo-XY sex chromosomes resulting from an X chromosome/autosome fusion. The genus Rhionaeschna includes 42 species found in the Americas. We analyzed the distribution of the nucleolar organizer region (NOR) using FISH with rDNA probes in Rhionaeschna bonariensis (n = 12 + neo-XY), R. planaltica (n = 7 + neo-XY), and Aeshna cyanea (n = 13 + X0). In R. bonariensis and A. cyanea, the NOR is located on a large pair of autosomes, which have a secondary constriction in the latter species. In R. planaltica, the NOR is located on the ancestral part of the neo-X chromosome. Meiotic analysis and FISH results in R. planaltica led to the conclusion that the neo-XY system arose by insertion of the ancestral X chromosome into an autosome. Genomic in situ hybridization, performed for the first time in Odonata, highlighted the entire neo-Y chromosome in meiosis of R. bonariensis, suggesting that it consists mainly of repetitive DNA. This feature and the terminal chiasma localization suggest an ancient origin of the neo-XY system. Our study provides new information on the origin and evolution of neo-sex chromosomes in Odonata, including new types of chromosomal rearrangements, NOR transposition, and heterochromatin accumulation.

¿Qué nos dicen los primates neotropicales bajo la mirada de la citogenética? / What do neotropical primates tell us under the look of cytogenetics?

RESUMEN Los estudios de citogenética en Primates Neotropicales (Primates: Platyrrhini) han demostrado que estos mamíferos comprenden un grupo heterogéneo a nivel cromosómico. La notable variedad de cariotipos descriptos provee evidencia significativa sobre el posible papel de los reordenamientos cromosómicos en su evolución. En el Grupo de Investigación en Biología Evolutiva (GIBE), la línea de investigación sobre el proceso de divergencia evolutiva en Platyrrhini considerando distintos aspectos de la organización del genoma se ha establecido y desarrollado de manera ininterrumpida desde hace más de 30 años. Entre los avances realizados en los últimos años se encuentra la cuantificación del tamaño del genoma en seis especies de monos caí (Cebus sp.) y dos especies de monos aulladores (Alouatta sp.) y la descripción de la composición de pares de bases en las regiones de heterocromatina constitutiva en los géneros Cebus y Ateles. Se concretaron las primeras descripciones del cariotipo y comportamiento meiótico en profase I temprana de dos especies de monos aulladores, Alouatta caraya y A. guariba clamitans. En esta última especie se identificó el primer sistema sexual de tipo pentavalente X1X2X3Y1Y2 en una especie de primate. Se caracterizó la organización de la eucromatina en términos del contenido y distribución de bases nucleotídicas AT y GC en tres especies de aulladores y en dos especies de monos caí. Estas investigaciones, entre otras, permitieron contribuir de forma original al conocimiento sobre la especiación en distintos niveles, así como sobre la arquitectura y dinámica del genoma de estos primates.

ABSTRACT Cytogenetics studies in Neotropical Primates (Primates: Platyrrhini) have shown that these mammals comprise a heterogeneous group at the chromosomal level. The remarkable variety of karyotypes described provides significant evidence on the possible role of chromosomal rearrangements in their evolution. In the Grupo de Investigación en Biología Evolutiva (GIBE), the line of research on the evolutionary divergence process in Platyrrhini considering different aspects of the organization of the genome has been established and developed uninterruptedly for more than 30 years. Among the advances made in recent years is the quantification of the genome size in six species of caí monkeys (Cebus sp.) and two species of howler monkeys (Alouatta sp.) and the description of the composition of base pairs in the constitutive heterochromatin regions in the genera Cebus and Ateles. The first descriptions were made of the karyotype and meiotic behavior in early prophase I of two species of howler monkeys, Alouatta caraya and A. guariba clamitans. In this last species, the first pentavalent-type sexual system X1X2X3Y1Y2 was identified in a primate species. The organization of euchromatin was characterized in terms of the content and distribution of AT and GC nucleotide bases in three species of howlers and in two species of caí monkeys. These, among other investigations, allowed contributing in an original way to the knowledge about speciation at different levels, as well as about the architecture and dynamics of the genome of these primates.

Using telomeric length measurements and methylation to understand the karyotype diversification of Ctenomys minutus (a small fossorial mammal).

The genus Ctenomys has been widely used in karyotype evolution studies due to the variation in their diploid numbers. Ctenomys minutus is characterized by intraspecific variation in diploid number (2n = 42, 46, 48, and 50), which makes it an interesting model to investigate genomic rearrangements mechanisms that could lead to different cytotypes in this species. Thereupon, it has been already shown that DNA methylation may participate in chromosome structure. Therefore, we aimed to investigate whether telomeres and global DNA methylation had a role in the genome rearrangements that led to this variation in C. minutus. We also realized an analysis for the presence of intrachromosomal telomeric repeats (ITRs) by fluorescence in situ hybridization. Our study demonstrated that neither telomere length nor DNA methylation had significant differences among the cytotypes. However, if only females were considered, there were significant differences for telomere length and methylation. Young individuals, regardless of their cytotypes, had the most methylated DNA. Regarding the ITRs, we found a signal on chromosome 1 in 2n = 50b. No evidence was found that telomere length or methylation could have influenced chromosomal rearrangements, although new cytotypes seem to have emerged within the distribution of parental cytotypes by the accumulation of different chromosomal rearrangements.

Rapid karyotypic evolution with high diploid number variation in a rare genus of bromeligenous frogs.

Bromeligenous Crossodactylodes is a leptodactylid genus closely related to Paratelmatobius and Scythrophrys. The diploid number in all karyotyped species of these two latter genera is 24, which diverges from the modal diploid number (2n = 22) in the family. Here, we analyzed three species of Crossodactylodes and found karyotypes with 2n = 30, 2n = 32, and 2n = 36, diploid numbers that have not been reported in any other diploid leptodactylid species to date. Reconstruction of the ancestral chromosome number indicated that the diploid number changed from 22 to 24 in the common ancestor of Crossodactylodes, Paratelmatobius, and Scythrophrys, and that progressive increases in diploid number have occurred in Crossodactylodes. The large number of telocentric/subtelocentric chromosomes in karyotypes with higher diploid numbers raises the possibility that centric fissions may have occurred during the evolution of Paratelmatobiinae. Three metacentric chromosomes, probably involved in fission events, were inferred to be present in the common ancestor of all species of Crossodactylodes, but in C. bokermanni. Chromosome mapping of the satellite DNA PcP190 suggests homology between one arm of metacentric chromosome 1 of Crossodactylodes sp. 3 and telocentric chromosome 2 of C. itambe, supporting one of the presumed centric fission events.

Multiple heterochromatin diversification events in the genome of fungus-farming ants: insights from repetitive sequences.

A substantial portion of the eukaryotic genome includes repetitive DNA, which is important for its stability, regulation, and architecture. Fungus-farming ant genomes show remarkable structural rearrangement rates that were necessary for the establishment of their agriculture-based lifestyle, highlighting the relevance of this peculiar group in understanding the repetitive portion of ant genome. Chromosomal banding studies are in accordance with genomic data because they show that repetitive heterochromatic sequences of basal and derivative Attina species are GC-rich, an uncommon trait in Formicidae. To understand the evolutionary dynamics of heterochromatin in Attina, we compared GC-rich heterochromatin patterns between the Paleoattina and Neoattina clades of this subtribe. To this end, we hybridized the Mrel-C0t probe (highly and moderately repetitive DNA) obtained from Mycetomoellerius relictus, Neoattina with GC-rich heterochromatin, in karyotypes of Paleoattina and Neoattina species. Additionally, we mapped the repetitive sequences (GA)15 and (TTAGG)6 in species of the two clades to investigate their organization and evolutionary patterns in the genome of Attina. The Mrel-C0t probe marked the heterochromatin in M. relictus, in other Mycetomoellerius spp., and in species of Mycetarotes, Cyphomyrmex, and Sericomyrmex (Neoattina). In Mycetomoellerius urichii, only pericentromeric heterochromatin was marked with Mrel-C0t. No marking was observed in Paleoattina species or in Atta and Acromyrmex (Neoattina). These results indicated that different evolutionary events led to heterochromatin differentiation in Attina. The most likely hypothesis is that GC-rich heterochromatin arose in the common ancestor of the two clades and accumulated various changes throughout evolution. The sequences (GA)15 and (TTAGG)6 located in euchromatin and telomeres, respectively, showed more homogeneous results among the species.

Satellitome analysis illuminates the evolution of ZW sex chromosomes of Triportheidae fishes (Teleostei: Characiformes).

Satellites are an abundant source of repetitive DNAs that play an essential role in the chromosomal organization and are tightly linked with the evolution of sex chromosomes. Among fishes, Triportheidae stands out as the only family where almost all species have a homeologous ZZ/ZW sex chromosomes system. While the Z chromosome is typically conserved, the W is always smaller, with variations in size and morphology between species. Here, we report an analysis of the satellitome of Triportheus auritus (TauSat) by integrating genomic and chromosomal data, with a special focus on the highly abundant and female-biased satDNAs. In addition, we investigated the evolutionary trajectories of the ZW sex chromosomes in the Triportheidae family by mapping satDNAs in selected representative species of this family. The satellitome of T. auritus comprised 53 satDNA families of which 24 were also hybridized by FISH. Most satDNAs differed significantly between sexes, with 19 out of 24 being enriched on the W chromosome of T. auritus. The number of satDNAs hybridized into the W chromosomes of T. signatus and T. albus decreased to six and four, respectively, in accordance with the size of their W chromosomes. No TauSat probes produced FISH signals on the chromosomes of Agoniates halecinus. Despite its apparent conservation, our results indicate that each species differs in the satDNA accumulation on the Z chromosome. Minimum spanning trees (MSTs), generated for three satDNA families with different patterns of FISH mapping data, revealed different homogenization rates between the Z and W chromosomes. These results were linked to different levels of recombination between them. The most abundant satDNA family (TauSat01) was exclusively hybridized in the centromeres of all 52 chromosomes of T. auritus, and its putative role in the centromere evolution was also highlighted. Our results identified a high differentiation of both ZW chromosomes regarding satellites composition, highlighting their dynamic role in the sex chromosomes evolution.

Karyotype Diversity, Mode, and Tempo of the Chromosomal Evolution of Attina (Formicidae: Myrmicinae: Attini): Is There an Upper Limit to Chromosome Number?

Ants are an important insect group that exhibits considerable diversity in chromosome numbers. Some species show only one chromosome, as in the males of the Australian bulldog ant Myrmecia croslandi, while some have as many as 60 chromosomes, as in the males of the giant Neotropical ant Dinoponera lucida. Fungus-growing ants are a diverse group in the Neotropical ant fauna, engaged in a symbiotic relationship with a basidiomycete fungus, and are widely distributed from Nearctic to Neotropical regions. Despite their importance, new chromosome counts are scarcely reported, and the marked variation in chromosome number across species has been poorly studied under phylogenetic and genome evolutionary contexts. Here, we present the results of the cytogenetic examination of fungus-farming ants and compile the cytogenetic characteristics and genome size of the species studied to date to draw insights regarding the evolutionary paths of karyotype changes and diversity. These data are coupled with a fossil-calibrated phylogenetic tree to discuss the mode and tempo of chromosomal shifting, considering whether there is an upper limit for chromosome number and genome size in ants, using fungus-farming ants as a model study. We recognize that karyotypes are generally quite variable across fungus-farming ant phylogeny, mostly between genera, and are more numerically conservative within genera. A low chromosome number, between 10 and 12 chromosomes, seems to present a notable long-term evolutionary stasis (intermediate evolutionary stasis) in fungus-farming ants. All the genome size values were inside a limited spectrum below 1 pg. Eventual departures in genome size occurred with regard to the mean of 0.38 pg, indicating that there is a genome, and likely a chromosome, number upper limit.

Against the mainstream: exceptional evolutionary stability of ZW sex chromosomes across the fish families Triportheidae and Gasteropelecidae (Teleostei: Characiformes).

Teleost fishes exhibit a breath-taking diversity of sex determination and differentiation mechanisms. They encompass at least nine sex chromosome systems with often low degree of differentiation, high rate of inter- and intra-specific variability, and frequent turnovers. Nevertheless, several mainly female heterogametic systems at an advanced stage of genetic differentiation and high evolutionary stability have been also found across teleosts, especially among Neotropical characiforms. In this study, we aim to characterize the ZZ/ZW sex chromosome system in representatives of the Triportheidae family (Triportheus auritus, Agoniates halecinus, and the basal-most species Lignobrycon myersi) and its sister clade Gasteropelecidae (Carnegiella strigata, Gasteropelecus levis, and Thoracocharax stellatus). We applied both conventional and molecular cytogenetic approaches including chromosomal mapping of 5S and 18S ribosomal DNA clusters, cross-species chromosome painting (Zoo-FISH) with sex chromosome-derived probes and comparative genomic hybridization (CGH). We identified the ZW sex chromosome system for the first time in A. halecinus and G. levis and also in C. strigata formerly reported to lack sex chromosomes. We also brought evidence for possible mechanisms underlying the sex chromosome differentiation, including inversions, repetitive DNA accumulation, and exchange of genetic material. Our Zoo-FISH experiments further strongly indicated that the ZW sex chromosomes of Triportheidae and Gasteropelecidae are homeologous, suggesting their origin before the split of these lineages (approx. 40-70 million years ago). Such extent of sex chromosome stability is almost exceptional in teleosts, and hence, these lineages afford a special opportunity to scrutinize unique evolutionary forces and pressures shaping sex chromosome evolution in fishes and vertebrates in general.

Comparative mapping of a new repetitive DNA sequence and chromosome region-specific probes unveiling rearrangements in an Amazonian frog complex.

The frog species Physalaemus ephippifer exists in the Amazonian region and harbors heteromorphic Z and W chromosomes. A genetic lineage closely related to this species was recognized based on its mitochondrial DNA and RADseq-style markers, but its taxonomic status is still unclear and has been referred to as Lineage 1 of "P. cuvieri". The heteromorphic sex chromosomes found in P. ephippifer are not present in this lineage and which of its chromosome pairs is homologous to the sex chromosomes of P. ephippifer remain to be elucidated as well as the role of such a karyotypic divergence in the evolution of these frogs. Here, we described a new family of repetitive DNA and used its chromosomal sites along with the markers detected by a probe constructed from the microdissected segment of the Z chromosome of P. ephippifer to infer chromosomal homology. We also analyzed an unnamed species that is considered to be the sister group of the clade composed of Lineage 1 of "P. cuvieri" and P. ephippifer. Our results suggest that complex rearrangements involving the chromosomes that were inferred to be homeologous to the sex chromosomes of P. ephippifer have occurred during the divergence of this group of frogs.

Chromosome Mapping of U2 snDNA in Species of Leptodactylus (Anura, Leptodactylidae).

Small nuclear RNA (snRNA) is a class of molecules involved in the processing of pre-mRNA and in regulatory cell processes. snRNAs are always associated with a set of specific proteins. The complexes are referred to as small nuclear ribonucleoproteins, and spliceosome U RNAs are their most common snRNA components. The repetitive sequences of U snDNAs have been cytogenetically mapped in several species of Arthropoda, fishes, and mammals; however, their distribution remains unknown in amphibians. Here, we show results of FISH mapping of U2 snDNA repetitive sequences in species of the amphibian genus Leptodactylus to reveal the distribution patterns of this sequence in their karyotypes. The probe hybridized in the metacentric chromosome pair 6 in Leptodactylus fuscus, L. gracilis, L. latrans, L. chaquensis, L. petersii, L. podicipinus, and L. brevipes. A different pattern was observed in L. labyrinthicus with hybridization signals in 4 chromosome pairs. The same localization of U2 gene sequences in most of the species analyzed suggests a relatively conserved pattern and a similarity of the chromosome 6 among these species of Leptodactylus.