Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 29-38, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1106 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Luísa Antônia Campos Bar- ros, Gisele Amaro Teixeira, Paulo Castro Ferreira, Rodrigo Batista Lod, Linda Inês Silveira, Frédéric Petit- clerc, Jérôme Orivel, Hilton Jeferson Alves Cardoso de Aguiar (2021) Cytoge- netic survey of eight ant species from the Amazon rainforest. Caryologia 74(4): 29-38. doi: 10.36253/caryologia-1106 Received: October 02, 2020 Accepted: September 24, 2021 Published: March 08, 2022 Copyright: © 2021 Luísa Antônia Cam- pos Barros, Gisele Amaro Teixeira, Paulo Castro Ferreira, Rodrigo Batista Lod, Linda Inês Silveira, Frédéric Petit- clerc, Jérôme Orivel, Hilton Jeferson Alves Cardoso de Aguiar. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID HJACA: 0000-0001-7738-1460 GAT: 0000-0002-7106-5798 LACB: 0000-0002-1501-4734 LIS: 0000-0003-4172-4489 FP: 0000-0002-2015-1258 JO: 0000-0002-5636-3228 Cytogenetic survey of eight ant species from the Amazon rainforest Luísa Antônia Campos Barros1, Gisele Amaro Teixeira2, Paulo Castro Ferreira1, Rodrigo Batista Lod1, Linda Inês Silveira3, Frédéric Petit- clerc4, Jérôme Orivel4, Hilton Jeferson Alves Cardoso de Aguiar1,5,* 1 Universidade Federal do Amapá, Campus Binacional, Oiapoque, 68980-000, Brazil 2 Programa de Pós-graduação em Biologia Celular e Estrutural, Universidade Federal de Viçosa, Viçosa, Brazil 3 Programa de Pós-graduação em Biologia Animal, Universidade Federal de Viçosa, Viço- sa, Brazil 4 CNRS, UMR EcoFoG, AgroParisTech, CIRAD, INRA, Université de Guyane, Université des Antilles, Campus Agronomique, Kourou, France 5 Programa de Pós-graduação em Biodiversidade Tropical, Universidade Federal do Amapá, Macapá, Brazil *Corresponding author. E-mail: hilton@unifap.br Abstract. The scarce information regarding ant diversity in the state of Amapá and lack of cytogenetic data of species from the Amazon region can hide ant biodiversi- ty information that may be detectable with affordable cytogenetic techniques. In this study, we describe the karyotypes of eight ant taxa collected from Amazonian localities in French Guiana and Brazil. Chromosome numbers ranged from 2n = 18 to 2n = 68. The following chromosome numbers were observed for each species: Azteca sp. group chartifex 2n = 28; Dolichoderus bidens (Linnaeus, 1758) 2n = 18; Gnamptogenys tortu- olosa (Smith, 1858) 2n = 44; Camponotus renggeri Emery, 1894 n = 20; Pseudomyrmex unicolor (Smith, 1855) 2n = 68 and n = 34; Apterostigma sp. pilosum complex 2n = 46; Odontomachus bauri Emery, 1892 2n = 44, and Wasmannia auropunctata (Roger, 1863) 2n = 32. The karyotypes of P. unicolor, G. tortuolosa, and O. bauri are reported here for the first time. Our data enabled comparisons between chromosomal data of some spe- cies from Amazon and Atlantic rainforests. We also highlight the methods used for the ant chromosome classification. Keywords: karyotype, chromosome evolution, biodiversity, Formicidae, Neotropics, taxonomy. INTRODUCTION The classical cytogenetic approach utilizes a single dye, orcein or Giemsa (Liehr 2017), without previous trypsin-treatment, for the study of chromo- somes and has also been denoted as beta karyology by White (reviewed by Petitpierre 2009). The low cost of classical cytogenetics allows more extensive sampling and plays a vital role in the discovery and understanding of diver- 30 Luísa Antônia Campos Barros et al. sity in different organisms (Zacharopoulou et al. 2017; Di-Nizo et al. 2017; Cioffi et al. 2018). In hymenopteran cytogenetics, chromosomes can be obtained from live larvae using a stereomicroscope and chemicals, even from distant localities such as those in Amazonia. The technique provided by Imai et al. (1988) enables the use of artisanal procedures with rus- tic material such as empty pill packs to keep the ganglia in hypotonic solution and syringes for their dissocia- tion on the slides. Important taxonomic insights may be achieved from karyotype information and, according to Schubert (2011), efforts must be made to avoid los- ing such data. The resolution of sampling issues is par- ticularly important in population-level approaches for understanding taxonomic problems (Petitpierre 2011; Cioffi et al. 2018; Chèvre et al. 2018). To date, classical cytogenetic studies are routinely performed for many organisms (Petitpierre 2009; Liehr 2017), thus support- ing the accuracy and validity of their results. Karyotype configuration can be useful for spe- cies delimitation, as karyotypes with structural and/ or numerical differences may not pair properly during meiosis (King 1993). This kind of chromosomal variation can affect fertility in heterozygotes and, in extreme cases, lead to sterility caused by gamete aneuploidy. Remarkable examples of chromosome number distinctness in close- ly related species or within the same species have been reported. For instance, in the Cervidae species Muntia- cus muntjak (Zimmermann, 1780), females possess 2n = 6 chromosomes and males possess 2n = 7, and Muntiacus reevesi (Ogilby, 1839) has a distinct chromosomal organi- zation of 2n = 46 (Wurster and Benirschke 1970). Recent examples of intraspecific chromosomal variations in ants have been observed from different populations within the species. For instance, different cytotypes have been found in Holcoponera striatula (Mayr, 1884) (as Gnamptogenys striatula) (2n = 32, 34), Holcoponera moelleri Forel, 1912 (as Gnamptogenys moelleri) (2n = 34, 44) (Teixeira et al. 2020), and Mycetophylax morschi (Emery, 1888) (2n = 26, 28, 30) (Micolino et al. 2019). Karyological information is currently available for approximately 800 species of ants distributed across the world (reviewed by Lorite and Palomeque 2010; Car- doso et al. 2018; Mariano et al. 2019). Neotropical ant species have been targeted for cytogenetic studies since the first surveys conducted by Crozier (1970) in South America, including Brazil, and by Goñi et al. (1983) in Uruguay. Pioneering studies in ant cytogenetics in Bra- zil were performed by Fadini and Pompolo (1996) and Mariano et al. (2000) and, since then, there has been a steady increase in the number of cytogenetic researches in ants using different approaches. Thus far, more than 180 ant taxa have been cytogenetically studied in the Neotropics, most of them from the Atlantic rainforest in Brazil (reviewed by Mariano et al. 2019). In the Amazo- nian region, karyological information is limited to that obtained from species restricted to French Guiana and Brazil (reviewed by Aguiar et al. 2020). In this study, we describe the karyotypes of eight ant species from the Amazon rainforest using a comparative approach with available population data, as our contri- bution toward understanding the evolutionary pattern of ant diversity in the Neotropics. MATERIALS AND METHODS Ant colonies were collected by active search in French Guiana at Kourou and Sinnamary, and in Brazil at Oiapoque, state of Amapá and Açailândia, state of Mara- nhão (Table 1). Adult voucher specimens were deposited into the ant collection at the Laboratório de Mirmecologia do Centro de Pesquisas do Cacau (CPDC/Brazil) in Bahia, Brazil, under records #5802, #5803, and #5816. Mitotic chromosomes were obtained from the cer- ebral ganglia of the larvae after meconium elimination, as described by Imai et al. (1988). The chromosome number and morphology of metaphases were analyzed using conventional 4% Giemsa staining. Chromosomes were arranged in order of decreasing size and based on the ratio of the chromosome arm lengths (r = long arm/ short arm), i.e., on the centromeric position, accord- ing to the classification proposed by Levan et al. (1964). The chromosomes were measured and classified as m = metacentric (r = 1–1.7), sm = submetacentric (r = 1.7–3), st = subtelocentric (r = 3–7), and a = acrocentric (r > 7). Chromosomes were organized using Corel Photopaint X3 and measured using Image Pro Plus. Reflexions on the nomenclature used to classify ant chro- mosomes Imai (1991) proposed a detailed chromosomal nomenclature based on heterochromatin location; how- ever, a classification based on this type of chromatin is impractical because large (detectable) heterochromatic blocks are not present in many ant groups. Addition- ally, the use of chromosome measurements diminishes subjectivity and enables karyotype comparisons between populations or species. Analysis of the karyotypes of Acromyrmex spp. (reviewed by Barros et al. 2021) using the nomencla- ture of Levan et al. (1964) allowed for the detection of dissimilarities in the karyotypic formula caused by the 31Cytogenetic survey of eight ant species from the Amazon rainforest variations in short arm size due to differential hetero- chromatin growth. Among the Atta spp., differences were not detected even with chromosome classification using chromosomal measurements (Barros et al. 2014), but variations could be identified by karyomorphomet- ric comparison with the leaf-cutting ant Amoimyrmex striatus (Roger, 1863) (Cristiano et al. 2013). Amoimyr- mex striatus, in addition to two other species, currently belongs to the new genus Amoimyrmex (Cristiano et al. 2020). The nomenclature of Levan et al. (1964) is typically used for chromosomal classification of different organ- isms such as plants (Winterfeld et al. 2018; Sadeghian et al. 2019), spiders (Araújo et al. 2020), beetles (Şendoğan and Alpagut-Keskin 2016), bees (Lopes et al. 2021), wasps (Tavares and Teixeira 2021), velvet worms (reviewed by Duarte et al. 2020), and fishes (Brandão et al. 2018). Recent ant cytogenetic studies have focused on measure- ments of chromosomes (Barros et al. 2010, 2014, 2016; Cristiano et al. 2013, 2017, Santos et al. 2016, Micolino et al. 2019, 2020; Teixeira et al. 2020). We suggest the use of the standardized chromosomal nomenclature employing measurements described by Levan et al. (1964) in For- micidae as well as in Hymenoptera, thereby allowing for comparisons between the species and populations. We also suggest the use of less condensed chromosomes and care with centromeric location (primary constriction) to diminish subjectivity in chromosome measurements. This chromosome classification based on measurements will also facilitate access to data on ant cytogenetics by researchers working on other organisms and could likely contribute to a better understanding of ant chromosomal diversity and evolution. RESULTS AND DISCUSSION We analyzed the chromosomes of eight ant species, eight genera, and six subfamilies. Our analysis presents the first karyological records for Pseudomyrmex unicolor (Smith, 1855), Gnamptogenys tortuolosa (Smith, 1858), and Odontomachus bauri Emery, 1892. Three species have already been described for the Atlantic rainforest, and showed karyotypic similarities. Unique karyotypes were detected in two different species complexes, sug- gesting genera revision. Subfamily Dolichoderinae Azteca sp. group chartifex presented 2n = 28, 10m + 4sm + 6st + 8a (Figure 1A). Previously, karyological data for only five taxa from the genus Azteca were available; four of these taxa were characterized as 2n = 28 and one, Azteca alfari Emery, 1893, as 2n = 26 (reviewed by Mari- ano et al. 2019). The karyotype of Azteca chartifex Emery, 1896 from French Guiana is 2n = 28, 10M + 18A (Mari- ano et al. 2019). If we group the chromosomes of Azteca Table 1. Ant species collected from the Amazon rainforest and analyzed using classical cytogenetics. Collection sites, sample sizes (numbers of colonies/individuals), diploid (2n) and haploid (n) chromosome numbers, and karyotype formula. Ant species Locality Col./Ind. 2n (n) Karyotype formula 2n / (n) Subfamily Dolichoderinae Azteca sp. group chartifex La Montagne des Singes, Kourou, FG 1/8 28 10m + 4sm + 6st + 8a Dolichoderus bidens (Linnaeus, 1758) Chácara du Rona, Oiapoque-AP, BR 2/11 18 14m + 4sm Subfamily Ectatomminae Gnamptogenys tortuolosa (Smith, 1858) * Sinnamary, FG 1/4 44 12m + 17sm + 15st Subfamily Formicinae Camponotus renggeri Emery, 1894 Campus Agronomique, Kourou, FG 1/4 (20) (2sm + 17st + 1a) Subfamily Myrmicinae Apterostigma sp. pilosum complex La Montagne des Singes, Kourou, FG 2/6 46 6m + 18sm + 16st + 6a Wasmannia auropunctata (Roger, 1863) Chácara du Rona, Oiapoque-AP, BR 1/5 32 16m + 10sm + 6st Subfamily Ponerinae Odontomachus bauri Emery, 1892 * Açailândia-MA, BR 1/7 44 6sm + 24st + 14a Subfamily Pseudomyrmecinae Pseudomyrmex unicolor (Smith, 1855) * Campus Agronomique, Kourou, FG 2/5 68 56m + 12sm (56m+12sm) (34) (28m + 6sm) Abbreviations: * first cytogenetic report; BR = Brazil, FG = French Guiana; Brazilian states: AP = Amapá, MA = Maranhão. 32 Luísa Antônia Campos Barros et al. sp. group chartifex from the present study into two cat- egories, partially in accordance with Imai et al. (1988), as with Az. chartifex, the karyotypic formula is 14M + 14A. This seems to indicate differences in chromosome mor- phology between the two taxa, which corroborates the morphological data. Data from molecular cytogenetic studies may contribute to corroborate these two taxa. Colonies of Dolichoderus bidens (Linnaeus, 1758) were found in carton nests built on the abaxial surface of leaves of the family Musaceae. The behavior of the workers was particularly aggressive. There are several records of D. bidens in French Guiana (Franco et al. 2019) and a single record in the neighboring Brazilian state of Amapá, in Serra do Navio, the center of the state (Kempf 1959). To date, there has been no report of D. bidens inhabiting areas between these regions, which are approximately 400 km apart. Dolichoderus bidens showed a karyotype of 2n = 18, 14m + 4sm (Figure 1B) in our study. Heterochromatic blocks around the centromeric/pericentromeric area of the chromosomes were identified (Figure 2A). Until now, the karyotype of D. bidens was only available for speci- mens collected in the Atlantic rainforest of Ilhéus, Bahia (Santos et al. 2016). Our results for the specimens col- lected from the Amazon rainforest showed similarities between these two rainforest populations, with subtle variations due to measurement divergences. In contrast, in a recent study, Dolichoderus imitator Emery, 1894 showed remarkable karyotypic differences between the population from the Amazon rainforest (2n = 46) and that from the Atlantic rainforest (2n = 38) (Santos et al. 2016; Aguiar et al. 2020). Subfamily Ectatomminae Gnamptogenys tortuolosa, which is included in the Neotropical sulcata group, presented 2n = 44, 12m + 17sm + 15st (Figure 1C). As observed previously by Imai (1991), using standard Giemsa staining, all chro- mosomes showed heterochromatic blocks restricted to the pericentromeric region and the short arms of subte- locentric pairs (Figure 2B). Cytogenetic data for 14 taxa of Gnamptogenys are available, including representa- tives of the mordax, striatula, and rastrata Neotropi- cal groups (reviewed by Teixeira et al. 2020). This is the first chromosomal record for the sulcata group. The high chromosome number (2n >12, according to Imai et al. 1994) and the high number of subtelocentric pairs with heterochromatin in the short arms suggest that centric fission rearrangements could have played an important role during the evolution of G. tortuolosa, as other spe- Figure 1. Karyotypes of ant species from subfamilies: Dolichoderinae - (A) Azteca sp. group chartifex (2n = 28), (B) Dolichoderus bidens (2n = 18); Ectatomminae - (C) Gnamptogenys tortuolosa (2n = 44); and Formicinae - (D) Camponotus renggeri (n = 20). Box in (C) show size heteromophism of the long arm of pair 22 in G. tortuolosa, with one homologous submetacentric and the other subtelocentric chromosome. Scale bars = 5 µm. 33Cytogenetic survey of eight ant species from the Amazon rainforest cies of Gnamptogenys do not typically have a large num- ber of chromosomes (Teixeira et al. 2020). Mariano et al. (2015) have proposed that centric fissions are important in the evolution of this genus and, although there are scarce cytogenetic data concerning the sulcata group, these fissions appear to play an important role in this group. Heteromorphism involving the long arm of chro- mosome pair 22 was observed in G. tortuolosa, which resulted in differences in the morphology of homologous chromosomes, with one chromosome being submeta- centric and the other subtelocentric (Figure 1C, box). The two chromosome variants are different in size and, therefore, processes that duplicate or delete chromatin could have been involved in the origin of this hetero- morphism. Subfamily Formicinae The nest of Camponotus renggeri Emery, 1894 col- lected during the present study was found on fallen rot- ten wood. In Oiapoque, north of the state of Amapá, Brazil, we also observed underground nests, as previ- ously reported by Ronque et al. (2016). It is important to note that it is rarer to find C. renggeri nests in rainforest areas than in savannah regions, including the Amazoni- an savannahs (Aguiar, Barros personal observation). The colony of C. renggeri from the Amazon rainfor- est showed n = 20, 2sm + 17st + 1a (Figure 1D). Colonies from other localities, such as the Amazonian savannah located at Macapá and the savannahs of Cerrado in the states of Mato Grosso (Aguiar et al. 2017) and Goiás (Vieira and Santana 2020), also showed n = 20 chromo- somes. The presence of a secondary constriction on the short arm of a subtelocentric chromosome of medium size (pair 5) suggests the presence of rDNA clusters. Two chromosome-rDNA bearer pairs, a submetacentric pair and a subtelocentric pair of medium size, have previ- ously been reported for this species (Aguiar et al. 2017). This is in contrast to that observed in the sister species Camponotus rufipes (Fabricius, 1775) and Camponotus (Myrmothrix) spp., which show a single submetacentric rDNA-bearer pair (Aguiar et al. 2017). Several chromo- somal polymorphisms are associated with Camponotus (Myrmothrix) spp., but no variation was observed among the males analyzed in this study. Subfamily Myrmicinae Wasmannia auropunctata Roger (1863) presented 2n = 32, 16m + 10sm + 6st (Figure 3A). Its karyotype showed the same chromosome number and similar mor- phology to that of the Atlantic rainforest population (Souza et al. 2011). Although Souza et al. (2011) used a different chromosome classification method (Imai 1991), without the use of chromosome measurements, the kar- ytoype is similar to that obtained in this study, being possible to recognize all chromosome pairs. A chromo- somal polymorphism was detected in ants from French Guiana (Aguiar et al. 2020, see Figure 5b, since the kar- Figure 2. Metaphases showing heterochromatic blocks (arrowheads) via 4% Giemsa staining in (A) Dolichoderus bidens (2n = 18) on cen- tromeric and pericentromeric regions and (B) Gnamptogenys tortuolosa (2n = 44) on pericentromeric regions of all chromosomes and short arms of subtelocentric pairs. Scale bar = 5 µm. 34 Luísa Antônia Campos Barros et al. yotype is incorrectly written in Table 1); however, ants collected at Oiapoque, Brazil (about 200 km away) did not show karyotype variations. The comparison between the karyotypes of specimens from these two localities provided insights into the polymorphism observed in French Guiana. A submetacentric chromosome, which corresponds to the largest chromosome of the karyotype in ants from French Guiana, is absent in specimens from Oiapoque, so we can infer that this particular chromo- some originated from a chromosomal rearrangement that need to be further investigated. The Apterostigma sp. pilosum complex was charac- terized as 2n = 46, 6m + 18sm + 16st + 6a (Figure 3B). The chromosome number among Apterostigma ranges from 2n = 20 to 2n = 46 (Mariano et al. 2019). The genus Apterostigma contains six taxa that have been cytoge- netically analyzed, but only half of the species have been taxonomically described. The Apterostigma pilosum complex is composed of nine similar species and is con- sidered to be taxonomically difficult to resolve (Lattke 1997). Some species were placed in synonymy of Apter- ostigma mayri Forel, 1893 by Weber (1958). Apterostigma mayri and Apterostigma sp. pilosum complex showed distinct chromosome numbers of 2n = 24 and 2n = 46, respectively, although both are included within the pilo- sum complex (Lattke 1997). The karyotypes with a lower chromosome number show more meta/submetacentric chromosomes when compared to species with higher chromosome numbers, including members of the Apter- ostigma sp. pilosum complex. This suggests that centric fission rearrangements seem to be a part of the chro- mosomal evolution of the genus Apterostigma. A taxon Figure 3. Karyotypes of ant species from subfamilies: Myrmicinae - (A) Wasmannia auropunctata (2n = 32), (B) Apterostigma sp. pilosum complex (2n = 46); Ponerinae - (C), (D) Odontomachus bauri (2n = 44); and Pseudomyrmecinae - (F), (G) Pseudomyrmex unicolor (2n = 68, n = 34). The boxes show polymorphism for subtelocentric chromosome pair 22 in O. bauri: (C) homozygous individual with small arms and (D) heterozygous individual with a distinctive large subtelocentric chromosome. Scale bars = 5 µm 35Cytogenetic survey of eight ant species from the Amazon rainforest from French Guiana showed a distinct and intermediate number of chromosomes (2n = 32) (Mariano et al. 2011) compared to that in the Apterostigma sp. described here. Cytogenetic data highlight the need for revision of the pilosum complex and the genus Apterostigma. Subfamily Ponerinae Odontomachus bauri showed 2n = 44, 6sm + 24st + 14a (Figure 3C-D). This species is included in the hae- matodus group, and all the studied species have the same chromosome number, 2n = 44 (reviewed in Santos et al. 2010). However, variations in chromosomal mor- phology exist among species and provide insights into the mode of karyotypic evolution in this group (Aguiar et al. 2020). Differential heterochromatin growth after centric fission events may have played a role in the chro- mosomal evolution of the haematodus group according to Imai et al. (1994). The O. bauri karyotype, according to the morphological variations due to heterochromatin growth on short arms, is derived within the haemato- dus group (see Aguiar et al. 2020) and corroborates the molecular phylogenetic position (Larabee et al. 2016). The long arm of the second subtelocentric pair of O. bauri collected from the Amazon rainforest showed a size polymorphism that was observed in individuals of the same colony. Homozygous individuals harbored two smaller subtelocentric chromosomes (Figure 3C, box). Only heterozygous individuals showed a distinctive large subtelocentric chromosome (Figure 3D, box). No indi- viduals with two large subtelocentric chromosomes were observed. This type of chromosome size polymorphism has been observed in several ant species (e.g., Barros et al. 2013; Teixeira et al. 2020) and can originate from unequal crossing-over or translocations that cause visi- ble chromosomal deletions or duplications (Schubert and Lysak 2011; Barros et al. 2013). Subfamily Pseudomyrmecinae Pseudomyrmex unicolor has been reported from Ser- ra do Navio in the state of Amapá (Kempf 1959); howev- er, it was also reported by different researchers in French Guiana (Franco et al. 2019) highlighting the scarcity of myrmecological studies in the state of Amapá. Pseudo- myrmex unicolor was characterized as having 2n = 68, 56m + 12sm and n = 34, 28m + 6sm (Figure 3E, F); a similarly high chromosome number is present in Pseu- domyrmex gracilis (Fabricius, 1804) (2n = 70) obtained from the Atlantic rainforest. Cytogenetic information is available for seven Pseudomyrmex spp. ranging from 2n = 24 to 2n = 70 (Sposito et al. 2006). Despite having high chromosome numbers, only metacentric and submeta- centric chromosomes were detected in P. unicolor. Poly- ploidy does not appear to be an important factor in the chromosomal evolution of ants (Lorite and Palomeque 2010) and, thus far, there is no evidence indicating poly- ploidization among Pseudomyrmex spp. (Tsutsui et al. 2008; Ardila-Garcia et al. 2010). The presence of heterochromatin blocks on the short arms of chromosomes of P. unicolor suggests that the “heterochromatic growth” after centric fissions (Imai et al. 1994) occurred during the chromosomal evolu- tion of this species. Although this process is not well understood (Hirai et al. 1994), it may involve distinct mechanisms that enlarge the size of heterochromatic blocks on the chromosomes, such as slippage saltatory amplification, which contributes to an increase in the amount of DNA; unequal crossing-over, which extends the heterochromatin among homologous regions; and also distribution by ectopic recombination among non- homologous chromosomes (Hirai 2020). The dispersion of rDNA on terminal regions indicates the involvement of different mechanisms (Hirai 2020). The increase in heterochromatin after chromosome fission has been previously suggested as a mechanism of chromosomal evolution in leaf-cutting ants of the genus Acromyrmex (Barros et al. 2016). FINAL REMARKS As there are few ant cytogenetic studies at the pop- ulation level, we conducted the karyotypic analysis of some ant species from the Amazon rainforest and car- ried out a comparative analysis with the populations of the Atlantic rainforest to detect karyotypic similarities and dissimilarities between them. Despite its simplicity, classical cytogenetics can reveal chromosomal variations that may affect the ability of a species to generate fertile progeny. This study highlights the need for a taxonomic revision of the Apterostigma pilosum complex and the Azteca chartifex group. Structural variations provide insights into the chromosomal evolution responsible for the polymorphisms detected in this study in W. auro- punctata and O. bauri, as well as the heteromorphism in G. tortuolosa. GEOLOCATION INFORMATION Ant colonies were collected from the following locations in French Guiana: La Montagne des Sing- 36 Luísa Antônia Campos Barros et al. es, Kourou (5.07225, -52.69407), Campus Agronom- ique, Kourou (5.17312, -52.65480), and Sinnamar y (5.28482, -52.91403). Colonies were collected in Brazil at Oiapoque, Amapá (3.84151, -51.84112), and Açailândia, Maranhão (-4.84200, -47.29667) (Table 1). ACKNOWLEDGMENTS We are grateful to Sr. Rona (in memoriam) for pro- viding us access to the field collection and Denilce Men- eses Lopes for use of the equipment at the Laboratório de Citogenética de Insetos at Universidade Federal de Viçosa. PCF and RBL thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their scholarships. GAT and LIS acknowledge the schol- arships provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Financial support for this study was provided by “Investissement d’Avenir” grants managed by the French Agence Nationale de la Recherche (DRIIHM ref. ANR-11-LABX-0010 and CEBA ref. ANR-10-LABX-25-01), by the PO-FEDER 2014-2020, Région Guyane (BiNG, ref. GY0007194), and by the Pro- grama de Auxílio ao Pesquisador – PAPESQ/UNIFAP. Sampling authorizations were provided by ICMBio/SIS- BIO in Brazil (accession numbers: 32459 and 62598) and by ABSCH-CNA-FR-240495-2 in French Guiana (acces- sion number: TREL1820249A/61). REFERENCES Aguiar HJAC, Barros LAC, Alves DR, Mariano CSF, Delabie JHC, Pompolo SG. 2017. Cytogenetic stud- ies on populations of Camponotus rufipes (Fabricius, 1775) and Camponotus renggeri Emery, 1894 (Formi- cidae: Formicinae). PLoS ONE 12(5):e0177702. Aguiar HJAC, Barros LAC, Silveira LI, Petitclerc F, Etienne S, Orivel J. 2020. Cytogenetic data for six- teen ant species from North-eastern Amazonia with phylogenetic insights into three subfamilies. 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