Simple SummaryAnts are an important insect group that includes a considerable number of species. Along with this diversity in species, they also exhibit a wide variation in chromosome numbers, from 1 up to 60 chromosomes. DNA molecules can be counted in a specific stage of the cell life cycle and quantified. These DNA molecules are very tightly packed together with several proteins and are called chromosomes. Each species shows a specific number of chromosomes with different shapes and sizes, as well as different quantities of DNA. We can use such information (the number of chromosomes, shape of the chromosomes, and quantity of DNA) as morphological attributes to study evolution at the species level. In this study, we describe new karyotypes of several ant species. In addition, from previous studies, we have compiled all the available information regarding the chromosome number and DNA quantity in fungus-farming ant cells. Different processes, called rearrangements, can change chromosomes over time, producing new character states. Such states can be tracked, along with the species and groups of similar species, using their relationships to identify patterns. We use DNA sequences to reconstruct the relationships of fungus-farming ant species (molecular phylogeny). By comparing such phylogeny with the chromosome number and DNA quantity, we discuss the evolution of chromosomes and DNA quantity (or genome size), and the potential limits to these features across fungus-farming ants. 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.
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