Cytogenetic studies in Trachymyrmex holmgreni Wheeler , 1925 ( Formicidae : Myrmicinae ) by conventional and molecular methods

Over the past several decades, ant cytogenetic studies have focused on chromosome number and morphology; however, recently, additional information concerning heterochromatin composition and 45S rDNA location has become accessible. The fungus-growing ants are a peculiar ant group that cultivates fungus for food, and Trachymyrmex is suspected to be the sister group of leafcutter ants. Cytogenetic data are so far available for sixn Trachymyrmex species. The present study aimed to increase the knowledge about Trachymyrmex cytogenetics by the chromosomal characterization of Trachymyrmex holmgreni including the karyotyping, fluorochromes staining, 18S rDNA, and microsatellite (GA)15 fluorescence in situ hybridization (FISH). Karyotyped samples from four ant colonies showed 2n = 20 metacentric chromosomes. Centromeric heterochromatin rich in GC base pairs was detected in all chromosomes. FISH revealed the presence of rDNA clusters on the fourth chromosome pair, and an intense spreading of the microsatellite (GA)15 including exclusively euchromatic areas of the chromosomes. The GC-rich heterochromatin observed in different ant species may have a common origin and, thus, phylogenetic implication that needs to be further investigated. To the best of our knowledge, this study is the first report of the use of chromosomal physical location of repetitive DNA sequences by means of microsatellite probes in Formicidae.


Introduction
Fungus-growing ants are found exclusively in the New World, primarily in the Neotropical region (Mayhé-Nunes & Jaffé, 1998;Schultz & Meier, 1995), and are suggested to have originated around 50 Mya (Chapela et al., 1994;Schultz considered by Schultz and Brady (2008) to be closely related to the leafcutter ants.Trachymyrmex is possibly a paraphyletic group (Schultz & Brady, 2008;Mehdiabadi & Schultz, 2010) and taxonomic uncertainties remain.
Ant cytogenetics has drawn the attention of myrmecologists (Delabie et al., 2012) and is useful in phylogenetic, taxonomic, evolutionary and conservation applications (e.g., Cristiano et al., 2013;Barros et al., 2015;Santos et al., 2016;Aguiar et al., 2017).Cytogenetic studies in fungus-growing ants are scarce, especially for the so called "lower attine" group (reviewed in Barros et al., 2011).Cytogenetic data in Trachymyrmex are available for six species (Table 1), and the chromosome numbers range from 2n = 12 to 2n = 22 chromosomes.Trachymyrmex septentrionalis (McCook 1881), included in the T. septentrionalis group, is closely related to leafcutter ants (Schultz & Brady 2008), and presents 2n=20 chromosomes (Murakami et al., 1998).This chromosome number is similar to that observed in Atta spp.already studied, and also Ac. striatus, with 2n = 22, although minor chromosome morphology differences are observed between these two group species.The other leafcutter clade, with the other Acromyrmex, present 2n = 38, meaning, a derived karyotype was originated by centric fissions, according to Barros et al. (2016).
Recently, the detection of specific microsatellites as landmarks has been used in different organisms including insects such as orthopterans (Milani & Cabral-de-Mello, 2014, Palacios-Gimenez et al. 2015, Palacios-Gimenez & Cabral-de-Mello, 2015).These microsatellites can be useful markers in evolutionary studies.Microsatellites distribution is highly variable in the species genomes, and can be found in specific regions or dispersed throughout the chromosomes (Sumner, 2003).Many of the studied species present a scattered distribution pattern of microsatellites along the chromosomes.
Regarding the phylogenetic position of Trachymyrmex within the "higher attine" group, and the absence of previous physical mapping of rDNA genes data in this genus, this study aimed to describe the fungus-growing ant T. holmgreni, Wheeler 1925, included in the Iheringi group (reviewed in Mayhé-Nunes & Brandão, 2005) by means of classical and molecular cytogenetics.

Material and Methods
Four colonies of T. holmgreni were collected in Itutinga, State of Minas Gerais, Brazil (21º 17' S; 44º 39' W), on July 29 th , 2012.Sample collection was done under the authorization of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for the collection of biological material issued to Luísa Antônia Campos Barros (SISBIO accession number 32459).Ant vouchers (workers) were identified by Jacques Hubert Charles Delabie and deposited in the reference collection at the Laboratório de Mirmecologia, Centro de Pesquisas do Cacau (CPDC/Brazil) under the record #5725.
The metaphases were obtained from cerebral ganglia of the larvae after meconium elimination, according to Imai et al. (1988).To determine the morphology of the chromosomes, a total of 10 metaphases were measured and chromosomes were classified according to Levan et al. (1964).Corel Photopaint X3® and Image Pro Plus® were the softwares used for mounting the chromosomal karyotype and measurements, respectively.For subsequent techniques, at least 15 metaphases were analyzed.At least five individuals per colony were analyzed.
Microsatellite (GA) 15 was used as probe in the physical location of repetitive DNA.The sequence of this probe was directly labeled with Cyanine-3 (Cy3) in the 5' terminal during synthesis by Sigma (St. Louis, MO, USA).The microsatellite hybridization procedures were performed according to Kubat et al. (2008), with the modifications of Cioffi et al. (2010).
The metaphases were observed and documented using a fluorescence microscope Olympus BX 60, coupled with capture system Q-Color3 Olympus ® images, using the software Q capture ® with the filters WB (450-480 nm), WU (330-385 nm) and WG (510-550 nm) for analyzing CMA 3 , DAPI and rhodamine, respectively.The metaphases labeled with the microsatellite (GA) 15 probe were observed using a microscope Olympus BX 53F coupled with an Olympus MX10 camera and the image software CellSens ® with the filter WG (510-550 nm) for the probe rich in Cy3 and WU forthe chromosomes (330-385 nm).

Discussion
The karyotype presented by T. holmgreni is similar in number and morphology to that of T. septentrionalis (Murakami et al., 1998), a species closely related to leafcutter ants (Schultz & Brady 2008), and T. relictus (Barros et al., 2013).The predominance of metacentric chromosomes is a karyotypic characteristic of the species of Trachymyrmex studied so far (Murakami et al., 1988;Barros et al., 2013Barros et al., , 2014a)).The heterochromatin pattern observed in T. holmgreni is similar to that previously described in T. fuscus (2n = 18) (Barros et al., 2014a), with centromeric and pericentromeric bands that coincided with GC-rich regions (CMA 3 + ).Chromosomal fusion hypothesis was suggested for the taxa with 2n = 12 owing to the low chromosome number and the presence of interstitial heterochromatic blocks (Murakami et al., 1998).Considering the chromosome number available for Trachymyrmex spp.(Table 1), associated with the location and composition of heterochromatin for the studied species (Murakami et al., 1998;Barros et al., 2013Barros et al., , 2014, present study), present study), centric fusion rearrangements seems to have occurred during the chromosomal evolution of this genus.Further cytogenetic studies will enable more robust inferences.
Although T. holmgreni had presented multiple GCrich bands, only a single pair of NOR-bearing chromosomes was observed, demonstrating that most of the GC-rich heterochromatin bands in this species do not correspond to the 18S ribosomal genes.This was also observed in other fungusgrowing ants such as M. goeldii (Barros et al., 2010(Barros et al., , 2012)), A. niger (Barros et al., 2016) and Ac.striatus (Cristiano et al., 2013, Teixeira et al. 2017).
Cytogenetic data of T. holmgreni in the present study showed predominance of metacentric chromosomes, multiple GC-rich heterochromatic bands and a single 18S rDNA pair: similar chromosomal traits that are observed in Ac. striatus (Cristiano et al., 2013).However, most Acromyrmex (2n = 38) and all Atta species already studied have a single pair of 18S rDNA rich in GC (Barros et al., 2014b(Barros et al., , 2015(Barros et al., , 2016;;Teixeira et al., 2017).It is suggested that Ac. striatus is the sister group of the remaining leafcutter ants (Cristiano et al., 2013), and the GC-rich patterns observed in Ac. striatus which are also found in T. holmgreni and T. fuscus may have a common origin and, thus, a phylogenetic implication.This must be further investigated in other Trachymyrmex and other fungus-growing ant groups.
Usually NORs are rich in GC base pairs (Sumner, 2003).Another peculiar observation was described in D. voraginosus in which GC-rich regions did not correspond with rDNA 45S clusters (Santos et al. 2016), indicating the importance of the extension of rDNA mapping in Formicidae.Regions with differential staining with DAPI, indicative of regions rich in AT base pairs, were not observed in T. holmgreni, a similar pattern to that observed in other ants such as T. fuscus Emery, 1934 (Barros et al. 2014a), Atta spp.(Barros et al. 2014b(Barros et al. , 2015)), Dolichoderus (Santos et al., 2016), and Pseudoponera (Correia et al., 2016).
The repetitive probe (GA) 15 presented dispersed distribution in the euchromatic regions of the chromosomes.The dispersed pattern of the (GA) 15 microsatellite differs from that observed in the orthopteran Abracris flavolineata (De Geer, 1773),which presented specific euchromatic and heterochromatic bands (Milani & Cabral de Mello, 2014).In Formicidae, there are no data of chromosomal physical location using repetitive DNA sequences for comparisons.However, initial descriptive data can generate new insights into the comprehension of the ant genome.These data open new possibilities for population and evolutionary studies and the use of additional probes.
Fig 1d) rich in GC-base pairs (Fig 1b) was observed in all chromosomes.Differentially, AT base pairs rich regions were not found (Fig 1c), but only negative regions complementing the fluorochrome CMA 3 (Fig 1d).The detection of ribosomal 18S genes by FISH analysis showed bands in the centromeric region of the fourth pair of metacentric chromosomes (Fig 1e).The results indicated an intense spreading of the dinucleotide microsatellite (GA) 15 in the T. holmgreni genome including only euchromatic areas of the chromosomes (Fig 1f).

Fig 1 .
Fig 1. Conventional and molecular cytogenetics of mitotic cells of Trachymyrmex holmgreni.a) Diploid karyotype arranged in descending order of size (2n = 20), b) CMA 3 and c) DAPI staining for the detection of GC and AT rich blocks, respectively, d) C-banding for heterochromatin detection, e) FISH analysis for 18S rDNA, and f) FISH analysis for dinucleotide microsatellites repeats (GA)15.