Chromosomal organization of different repetitive sequences in four wasp species of the genus Trypoxylon Latreille (Hymenoptera: Crabronidae) and insights into the composition of wasp telomeres.

This study characterizes the chromosomal organization of DNA repetitive sequences and the karyotypic evolution in four representatives of the solitary wasp genus Trypoxylon using conventional and molecular cytogenetic techniques. Our findings present the first cytogenetic data for Trypoxylon rogenhoferi (2n = 30) and Trypoxylon albonigrum (2n = 32), while the karyotypes of Trypoxylon nitidum (2n = 30) and Trypoxylon lactitarse (2n = 30) were similar to those previously described. Fluorochrome staining and microsatellite distribution data revealed differences in the constitutive heterochromatin composition among species. Trypoxylon nitidum and T. albonigrum exhibited one major rDNA cluster, potentially representing an ancestral pattern for aculeate Hymenoptera, while T. rogenhoferi and T. lactitarse showed two pericentromeric rRNA gene sites, suggesting amplification events in their ancestral clade. The (TCAGG)n motif hybridized in the terminal regions of the chromosomes in all four Trypoxylon species, which may suggest that this sequence represents DNA telomeric repeat. Notably, the presence of this repetitive sequence in the centromeric regions of certain chromosome pairs in two species supports the hypothesis of chromosomal fusions or inversions in the ancestral karyotype of Trypoxylon. The study expands the chromosomal mapping data of repetitive sequences in wasps and offers insights into the dynamic evolutionary landscape of karyotypes in these insects.

Conventional cytogenetics and microsatellite chromosomal distribution in social wasp Mischocyttarus cassununga (Ihering, 1903) (Vespidae, Polistinae, Mischocyttarini).

Cytogenetics has allowed the investigation of chromosomal diversity and repetitive genomic content in wasps. In this study, we characterized the karyotype of the social wasp Mischocyttarus cassununga using conventional cytogenetics and chromosomal mapping of repetitive sequences. This study was undertaken to extend our understanding of the genomic organization of repetitive DNA in social wasps and is the first molecular cytogenetic insight into the genus Mischocyttarus. The karyotype of M. cassununga had a chromosome number of 2n = 64 for females and n = 32 for males. Constitutive heterochromatin exhibited three distribution patterns: centromeric and pericentromeric regions along the smaller arms and extending almost the entire chromosome. The major ribosomal DNA sites were located on chromosome pair in females and one chromosome in males. Positive signals for the microsatellite probes (GA)n and (GAG)n were observed in the euchromatic regions of all chromosomes. The microsatellites, (CGG)n, (TAT)n, (TTAGG)n, and (TCAGG)n were not observed in any region of the chromosomes. Our results contrast with those previously obtained for Polybia fastidiosuscula, which showed that the microsatellites (GAG)n, (CGG)n, (TAT)n, (TTAGG)n, and (TCAGG)n are located predominantly in constitutive heterochromatin. This suggests variations in the diversity and chromosomal organization of repetitive sequences in the genomes of social wasps.

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.

First Report of the Tramp ant Technomyrmex vitiensis Mann, 1921 (Formicidae: Dolichoderinae) in Brazil with Cytogenetic and Sperm Structure Data and an Updated Key to Brazilian Dolichoderinae Genera.

Invasive ants are usually harmful taxa and are considered a potential problem to biodiversity due to their negative ecological impacts, as they can outcompete native ant species. Ten such species are reported in Brazil. In this study, we report for the first time the Asian tramp ant Technomyrmex vitiensis Mann, 1921 at the municipality of Oiapoque, in the Brazilian Amazon. The colony studied contained workers, intercastes, males and larvae, which provided sperm structure and cytogenetic data. Considering the unprecedented report of the genus Technomyrmex as well as the recent finding of the primarily Australian genus Leptomyrmex in Brazil, we present a revised key for the workers of Brazilian Dolichoderinae genera. Technomyrmex vitiensis presented 2n = 16 chromosomes; all metacentrics and comparative cytogenetics on the genus is provided. A single rDNA 18S site located in intrachromosomal region was observed in this species, which is a common trait in ants. The spermatozoa of T. vitiensis had a filiform shape, with 78.13 (± 1.96) µm of total length and 11.43 (± 0.51) µm of nucleus length. Total and nucleus sperm size length fit with the known variation observed in other ant species. The occurrence of T. vitiensis in Brazil is probably a result of traffic between French Guiana and the Amapá state. Cytogenetics and sperm structures of T. vitiensis enhance the biological knowledge of this tramp species. We highlight the scarce knowledge of ant diversity in the state of Amapá and the consequences that the presence of this species may have in this region.

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.

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.

Decay of homologous chromosome pairs and discovery of males in the thelytokous fungus-growing ant Mycocepurus smithii.

The prevalent mode of reproduction among ants is arrhenotokous parthenogenesis where unfertilized eggs give rise to haploid males and fertilized eggs develop into diploid females. Some ant species are capable of thelytokous parthenogenesis, a type of asexual reproduction where females develop from unfertilized diploid eggs. Thelytoky is well-documented in more than 20 ant species. Cytogenetic data are available for six species demonstrating that some thelytokous ant species are capable of producing males occasionally as well as maintaining their chromosome numbers and proper chromosome pairings. Mycocepurus smithii is a thelytokous fungus-growing ant species that inhabits large parts of Central and South America. Cytogenetic data are unavailable for M. smithii and male individuals were never documented for this species, although the presence of males is expected because genetic recombination was observed in a few sexually reproducing populations in Brazil and haploid sperm was documented from the spermathecae of M. smithii queens. This study aims at comparatively studying asexual and sexual populations of M. smithii using classical and molecular cytogenetic methods to test whether karyotype configuration is modified according to the mode of reproduction in M. smithii. Moreover, we report the discovery of M. smithii males from a sexually reproducing population in the Brazilian state Pará, diagnose the male of M. smithii, and morphologically characterize their spermatozoa. Karyotypic variation was observed within the asexual population (2n = 9, 10, or 11), whereas the chromosome number was fixed in the sexual population (2n = 14, n = 7). Identical karyotypes were maintained within individual M. smithii colonies and karyotype variation was only observed between colonies. In asexual individuals, the karyomorphs showed a decay of homologous chromosome pairs, especially in individuals with the karyomorph 2n = 11, which is potentially caused by relaxed natural selection on proper chromosome pairing. In contrast, females in the sexual population showed proper homologous chromosome pairings. In individuals of both asexual and sexual populations, we find that heterochromatin was localized in centromeric regions and on the short arms of the chromosomes, GC-rich regions were associated with heterochromatic regions, and 18S rDNA genes were located on the largest chromosome pair. This comparative cytogenetic analysis contributes to our understanding about the cytological mechanisms associated with thelytokous parthenogenesis in ants and suggests the decay of chromosome structure in the absence of meiosis and genetic recombination.

Cytogenetic Analysis of the Fungus-Farming Ant Cyphomyrmex rimosus (Spinola, 1851) (Formicidae: Myrmicinae: Attini) Highlights Karyotypic Variation.

The fungus-farming ant genus Cyphomyrmex (subtribe Attina, clade Neoattina) comprises 23 described species that are widely distributed throughout the Neotropics. Species within Cyphomyrmex have taxonomic issues such as Cyphomyrmex rimosus (Spinola, 1851) which is likely a species complex. Cytogenetics is a useful tool for evolutionary studies and understanding species with dubious taxonomy. In this study, we characterized the karyotype of C. rimosus from Viçosa, Minas Gerais State, southeastern Brazil using classical and molecular cytogenetic techniques to enrich the chromosomal information about Cyphomyrmex. The karyotype of C. rimosus from the rainforest of southeastern Brazil (2n = 22, 18m + 4sm) notably contrasts with that previously described for this species in Panama (2n = 32). This intraspecific chromosomal variation suggests the existence of a species complex within this taxon according to the previous hypothesis derived from morphological analysis. We detected GC-rich heterochromatic regions in C. rimosus and, using repetitive DNA probes, showed that this heterochromatin shares repetitive sequences with other Neoattina species already studied, enhancing the importance of this specific genome region in the understanding of Attina evolution. Mapping of microsatellite (GA)15 on C. rimosus was restricted to the euchromatic regions of all chromosomes. The single intrachromosomal rDNA sites observed in C. rimosus follow the general genomic organization trend of ribosomal genes in Formicidae. Our study extends the data of chromosome mapping on Cyphomyrmex and reinforces the importance of cytogenetic studies in different localities to better understand taxonomic issues in widely distributed taxa such as C. rimosus.

Distribution of GC-rich heterochromatin and ribosomal genes in three fungus-farming ants (Myrmicinae, Attini, Attina): insights on chromosomal evolution.

Cytogenetic studies on fungus-farming ants have shown remarkable karyotype diversity, suggesting different chromosomal rearrangements involved in karyotype evolution in some genera. A notable cytogenetic characteristic in this ant group is the presence of GC-rich heterochromatin in the karyotypes of some ancient and derivative species. It was hypothesized that this GC-rich heterochromatin may have a common origin in fungus-farming ants, and the increase in species studied is important for understanding this question. In addition, many genera within the subtribe Attina have few or no cytogenetically studied species; therefore, the processes that shaped their chromosomal evolution remain obscure. Thus, in this study, we karyotyped, through classical and molecular cytogenetic techniques, the fungus-farming ants Cyphomyrmextransversus Emery, 1894, Sericomyrmexmaravalhas Jesovnik et Schultz, 2017, and Mycetomoelleriusrelictus (Borgmeier, 1934), to provide insights into the chromosomal evolution in these genera and to investigate the presence the GC-rich heterochromatin in these species. Cyphomyrmextransversus (2n = 18, 10m + 2sm + 6a) and S.maravalhas (2n = 48, 28m + 20sm) showed karyotypes distinct from other species from their genera. Mycetomoelleriusrelictus (2n = 20, 20m) presented the same karyotype as the colonies previously studied. Notably, C.transversus presented the lowest chromosomal number for the genus and a distinct karyotype from the other two previously observed for this species, showing the existence of a possible species complex and the need for its taxonomic revision. Chromosomal banding data revealed GC-rich heterochromatin in all three species, which increased the number of genera with this characteristic, supporting the hypothesis of a common origin of GC-rich heterochromatin in Attina. Although a single chromosomal pair carries rDNA genes in all studied species, the positions of these rDNA clusters varied. The rDNA genes were located in the intrachromosomal region in C.transversus and M.relictus, and in the terminal region of S.maravalhas. The combination of our molecular cytogenetic data and observations from previous studies corroborates that a single rDNA site located in the intrachromosomal region is a plesiomorphic condition in Attina. In addition, cytogenetic data obtained suggest centric fission events in Sericomyrmex Mayr, 1865, and the occurrence of inversions as the origin of the location of the ribosomal genes in M.relictus and S.maravalhas. This study provides new insights into the chromosomal evolution of fungus-farming ants.

Cytogenetic variability in four species of Gnamptogenys Roger, 1863 (Formicidae: Ectatomminae) showing chromosomal polymorphisms, species complex, and cryptic species.

Gnamptogenys includes 138 described species that are widely distributed, with high diversity, in the Neotropics. Some Neotropical species have taxonomic issues, as is the case with Gnamptogenys striatula, for which morphological variations have been observed between different populations. For the ant species with taxonomic issues, classical and molecular cytogenetic studies have assisted in the resolution of these issues. Cytogenetic studies of Gnamptogenys are scarce and have only been reported for 14 taxa. These reports have rarely presented chromosomal morphology. Considering the importance of the taxonomic revision of some species, such as G. striatula, the present study cytogenetically characterized four species of Gnamptogenys: G. striatula, G. moelleri, G. regularis, and G. triangularis, discussing their phylogenetic and biogeographic characteristics. The number of chromosomes ranged from 2n = 26 to 2n = 44, with distinct karyotypes at both species and population levels. All four species presented a pair of 18S rDNA gene markers that coincided with GC-rich regions. In the case of G. striatula from the Atlantic rainforest, a chromosomal polymorphism was observed, with chromosomal translocations being the likely origin of this polymorphism. Two populations of G. striatula showed karyotype differences, thus corroborating previous morphological data indicating the existence of a species complex in this taxon. In addition, G. regularis showed a polymorphism involving a chromosome pair bearing ribosomal genes, possibly caused by unequal crossing-over. Although G. moelleri has a well-defined taxonomy, a population from the eastern Amazon rainforest presented a divergent karyotype from the Atlantic rainforest populations, suggesting the existence of a cryptic species in this taxon.

Comparative physical mapping of 18S rDNA in the karyotypes of six leafcutter ant species of the genera Atta and Acromyrmex (Formicidae: Myrmicinae).

Leafcutter ants of the Atta and Acromyrmex genera are important plagues in different cultures. Cytogenetic data on chromosome number, morphology, and chromosomal banding pattern are only available for 17 species of leafcutter ants. Molecular cytogenetic data for the detection of ribosomal genes by the FISH technique are scarce, and only 15 Neotropical ant species have been studied. This study aimed to physically map the 18S ribosomal RNA genes (rDNA) of six leafcutter ants belonging to the genera Atta and Acromyrmex using FISH. The results were compared with data on the fluorochrome CMA3 currently available for these species. All analyzed species presented the 18S rDNA on one pair of chromosomes. In Acromyrmex subterraneus molestans and Ac. aspersus, FISH signals were observed in the terminal region of the short arm of the largest subtelocentric pair, while in Atta bisphaerica, A. laevigata, and A. sexdens, FISH signals were observed in the interstitial region of the long arm of the fourth metacentric pair. In Acromyrmex striatus, 18S rDNA was located in the interstitial region of the second metacentric pair. The karyotypic formula for Ac. aspersus was 2n = 38 (8m + 10sm + 16st + 4a), representing the first report in this species. The observed 18S rDNA regions in A. laevigata, A. sexdens, A. bisphaerica, Ac. aspersus, and Ac. subterraneus molestans corresponded to the CMA3+ bands, while in Ac. striatus, several GC-rich bands and one pair of 18S rDNA bands were observed. No differential bands were visible using the DAPI fluorochrome. Karyotype uniformity with previously studied Atta spp. was also observed at the level of molecular cytogenetics using 18S rDNA FISH. A difference in the size of the chromosomal pair carrying the 18S rDNA gene was observed in Ac. striatus (2n = 22) and Atta spp. (2n = 22) highlighting the dissimilarity between these species. The results from the present study contribute to the description of 18S rDNA clusters in Neotropical ants.

Cytogenetic data on the threatened leafcutter ant Atta robusta Borgmeier, 1939 (Formicidae: Myrmicinae: Attini).

The karyotype of the threatened ant species Atta robusta is described so as to establish the evolutionary relationships of this taxon with other leafcutter ants. Standard Giemsa staining, C-banding, NOR banding, fluorochromes CMA3/DAPI, Hsc-FA technique and Fluorescence in situ Hybridization (FISH) using 18S rDNA probe were conducted on a population from Aracruz, state of Espírito Santo, Brazil, allowing for comparisons with data available on Atta and other fungus-growing ant species. The diploid chromosome number observed for A. robusta was 2n=22, and the karyotypic formula was 18m+2sm+2st. Heterochromatic blocks were observed in the centromeric region of most chromosomes, where one pair of metacentric chromosomes is characterized by a GC-rich heterochromatic band in the interstitial region of its long arm. The detection of 18S rDNA using FISH confirmed the presence of single NOR for A. robusta. This is the first report of rDNA 18S detection using FISH for leafcutter ants. The cytogenetic results of this study confirm the information available for Atta and allow us to confirm the conserved chromosome number, morphology and banding pattern within the genus for the taxa studied to date, which included species from three out of the four groups of Atta indicated by molecular data. The accumulation of cytogenetic data on fungus-growing ants enhances the understanding of the genomic evolutionary patterns of Atta, since it belongs to a group of recent origin between the most well studied ants. Cytogenetic data does not indicate restrictions in relocation or reintroduction in areas where populations were extinct due to the conserved karyotype. This study allows for cytogenetic comparison of A. robusta with other ants of Atta, emphasizing the importance of chromosomal information for species conservation.