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 Cyphomyrmex transversus Emery, 1894, Sericomyrmex maravalhas Ješovnik et Schultz, 2017, and Mycetomoellerius relictus (Borgmeier, 1934), to provide insights into the chromosomal evolution in these genera and to investigate the presence the GC-rich heterochromatin in these species. Cyphomyrmex transversus (2n = 18, 10m + 2sm + 6a) and S. maravalhas (2n = 48, 28m + 20sm) showed karyotypes distinct from other species from their genera. Mycetomoellerius relictus (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.

Physical localization of 45S rDNA in Cymbopogon and the analysis of differential distribution of rDNA in homologous chromosomes of Cymbopogon winterianus

Cymbopogon, commonly known as lemon grass, is one of the most important aromatic grasses having therapeutic and medicinal values. FISH signals on somatic chromosome spreads off Cymbopogon species indicated the localization of 45S rDNA on the terminal region of short arms of a chromosome pair. A considerable interspecific variation in the intensity of 45S rDNA hybridization signals was observed in the cultivars of Cymbopogon winterianus and Cymbopogon flexuosus. Furthermore, in all the varieties of C. winterianus namely Bio-13, Manjari and Medini, a differential distribution of 45S rDNA was observed in a heterologous pair of chromosomes 1. The development of C. winterianus var. Manjari through gamma radiation may be responsible for breakage of fragile rDNA site from one of the chromosomes of this heterologous chromosome pair. While, in other two varieties of C. winterianus (Bio-13 and Medini), this variability may be because of evolutionary speciation due to natural cross among two species of Cymbopogon which was fixed through clonal propagation. However, in both the situations these changes were fixed by vegetative method of propagation which is general mode of reproduction in the case of C. winterianus.

Variation in Ribosomal DNA in the Genus Trifolium (Fabaceae)

The genus Trifolium L. is characterized by basic chromosome numbers 8, 7, 6, and 5. We conducted a genus-wide study of ribosomal DNA (rDNA) structure variability in diploids and polyploids to gain insight into evolutionary history. We used fluorescent in situ hybridization to newly investigate rDNA variation by number and position in 30 Trifolium species. Evolutionary history among species was examined using 85 available sequences of internal transcribed spacer 1 (ITS1) of 35S rDNA. In diploid species with ancestral basic chromosome number (x = 8), one pair of 5S and 26S rDNA in separate or adjacent positions on a pair of chromosomes was prevalent. Genomes of species with reduced basic chromosome numbers were characterized by increased number of signals determined on one pair of chromosomes or all chromosomes. Increased number of signals was observed also in diploids Trifolium alpestre and Trifolium microcephalum and in polyploids. Sequence alignment revealed ITS1 sequences with mostly single nucleotide polymorphisms, and ITS1 diversity was greater in diploids with reduced basic chromosome numbers compared to diploids with ancestral basic chromosome number (x = 8) and polyploids. Our results suggest the presence of one 5S rDNA site and one 26S rDNA site as an ancestral state.

Physical localization of 45S rDNA in Cymbopogon and the analysis of differential distribution of rDNA in homologous chromosomes of Cymbopogon winterianus

Cymbopogon, commonly known as lemon grass, is one of the most important aromatic grasses having therapeutic and medicinal values. FISH signals on somatic chromosome spreads off Cymbopogon species indicated the localization of 45S rDNA on the terminal region of short arms of a chromosome pair. A considerable interspecific variation in the intensity of 45S rDNA hybridization signals was observed in the cultivars of Cymbopogon winterianus and Cymbopogon flexuosus. Furthermore, in all the varieties of Cymbopogon winterianus namely Bio-13, Manjari and Medini, a differential distribution of 45S rDNA was observed in a heterologous pair of chromosome 1. The development of Cymbopogon winterianus var. Manjari through gamma radiation may be responsible for breakage of fragile rDNA site from one of the chromosomes of this heterologous chromosome pair. While, in other two varieties of Cymbopogon winterianus (Bio-13 and Medini), this variability may be because of evolutionary speciation due to natural cross among two species of Cymbopogon which was fixed through clonal propagation. However, in both the situations these changes were fixed by vegetative method of propagation which is general mode of reproduction in the case of Cymbopogon winterianus.

Comparative Cytogenetic Analysis of Three Eumeninae Species (Hymenoptera, Vespidae)

Eumeninae represents the largest subfamily within Vespidae, with 3,600 species described. Of these, only 18 have been cytogenetically analysed. In the present study, we used both classical and molecular techniques to characterise and compare the karyotypes of 3 Eumeninae species, namely, <i>Ancistrocerus</i> sp., <i>Pachodynerus grandis,</i> and <i>Pachodynerus nasidens</i>. <i>Ancistrocerus</i> sp. presented a haploid chromosome number of n = 12, with the first 2 chromosomes of the karyotype being almost entirely heterochromatic and much larger than the remaining chromosomes. The 2 <i>Pachodynerus</i> species presented the same chromosome number (n = 11 and 2n = 22) but displayed different karyotypic formulae<i>.</i> Additionally, chromosomal polymorphisms were observed in the analysed <i>P. nasidens</i> female. In the 3 species, heterochromatin was located in one of the chromosome arms. Fluorochrome staining revealed a balanced composition of AT and GC bases within the chromatin for each of the 3 species, except for few regions that were visibly GC-rich. All species had a single 18S rDNA site that co-localised with GC-rich regions; however, this localisation varied from species to species and not all GC-rich regions corresponded to ribosomal genes. Based on the cytogenetic data obtained here, we discuss the possible numerical/structural rearrangements that may be involved in the karyotypic evolution of the 3 studied species. In addition to the first description of the molecular cytogenetic characteristics of the Eumeninae subfamily and the genus <i>Pachodynerus</i>, this study also provides a relevant contribution towards the discussion of chromosomal evolution in Eumeninae wasps.

Analysis of small and large subunit rDNA introns from several ectomycorrhizal fungi species

The small (18S) and large (28S) nuclear ribosomal DNA (rDNA) introns have been researched and sequenced in a variety of ectomycorrhizal fungal taxa in this study, it is found that both 18S and 28S rDNA would contain introns and display some degree variation in size, nucleotide sequences and insertion positions within the same fungi species (Meliniomyces). Under investigations among the tested isolates, 18S rDNA has four sites for intron insertions, 28S rDNA has two sites for intron insertions. Both 18S and 28S rDNA introns among the tested isolates belong to group I introns with a set of secondary structure elements designated P1-P10 helics and loops. We found a 12 nt nucleotide sequences TACCACAGGGAT at site 2 in the 3’-end of 28S rDNA, site 2 introns just insert the upstream or the downstream of the12 nt nucleotide sequences. Afters sequence analysis of all 18S and 28S rDNA introns from tested isolates, three high conserved regions around 30 nt nucleotides (conserved 1, conserved 2, conserved 3) and identical nucleotides can be found. Conserved 1, conserved 2 and conserved 3 regions have high GC content, GC percentage is almost more than 60%. From our results, it seems that the more convenient host sites, intron sequences and secondary structures, or isolates for 18S and 28S rDNA intron insertion and deletion, the more popular they are. No matter 18S rDNA introns or 18S rDNA introns among tested isolates, complementary base pairing at the splicing sites in P1-IGS-P10 tertiary helix around 5’-end introns and exons were weak.

Paracentric Inversions Differentiate the Conservative Karyotypes in Two Centropomus Species (Teleostei: Centropomidae)

Centropomus is the sole genus of the Centropomidae family (Teleostei), comprising 12 species widely distributed throughout the Western Atlantic and Eastern Pacific, with 6 of them occurring in the Western Atlantic in extensive sympatry. Their life history and phylogenetic relationships are well characterized; however, aspects of chromosomal evolution are still unknown. Here, cytogenetic analyses of 2 Centropomus species of great economic value (C. undecimalis and C. mexicanus) were performed using conventional (Giemsa, Ag-NOR, and fluorochrome staining, C- and replication banding) and molecular (chromosomal mapping of 18S and 5S rDNA, H2A-H2B and H3 hisDNA, and (TTAGGG)n repeats) approaches. The karyotypes of both species were composed of 48 solely acrocentric chromosomes (2n = 48; FN = 48), but the single ribosomal site was located in varying positions in the long arms of the second largest chromosome pair. Replication bands were generally similar, although conspicuous differences were observed in some chromosome regions. In both species, the histone H3 genes were located on 3 apparently homeologous chromosome pairs, but the exact position of these clusters differed slightly. Interspecific hisDNA and rDNA site displacements can indicate the occurrence of multiple paracentric inversions during the evolutionary diversification of the Centropomus genomes. Although the karyotypes remained similar in both species, our data demonstrate an unsuspected microstructural reorganization between them, driven most likely by a series of paracentric inversions.

Genome differentiation, natural hybridisation and taxonomic relationships among Eleocharis viridans, E. niederleinii and E. ramboana (Cyperaceae)

The role of natural hybridisation and genome changes in the differentiation and speciation of Eleocharis (Cyperaceae) was addressed through the study of the following three closely related species of the polyphyletic series Tenuissimae: Eleocharis viridans Kük. ex Osten, E. ramboana R.Trevis & Boldrini and E. niederleinii Boech., which often reproduce asexually. Molecular and cytogenetic data were used to understand the genomic and karyotypic relationships in the group. Genomes were compared using internal transcribed spacer–cleaved amplified polymorphic sequence (ITS-CAPS) marker and confirmed with random amplified polymorphic DNA, which allowed identification of different genetic groups, with clear evidence of natural hybrids. Karyotype analysis showed numerical variation from 2n = 20–42, with occurrence of chromosome heteromorphisms and polymorphisms, including variability in 35S rDNA site numbers. Meiotic studies demonstrated irregular pairing in some samples, which is associated with hybridisation and asexual reproduction. Genomic in situ hybridisation (GISH) reactions were conducted using two well defined genetic groups as probes, with 2n = 20 and normal meiosis. Probes were tested against each one of the genetic groups and showed positive, partial and negative GISH results, which supported the molecular analysis data. The results indicated that the three studied species are undergoing an intense process of genomic and karyotypic re-arrangement, which results in overlapping of morphological and genomic characteristics. The present study has exemplified the value of an integrative taxonomic approach to solve conflicts in species delimitation in groups undergoing hybridisation.