# An Integrative Approach With Cytogenetic and Molecular Data of the Leaf-Cutting Ant Acromyrmex hystrix (Latreille, 1802) Highlights a Notably Derived Karyotype in the Genus

Luísa Antônia Campos Barros, Gisele Amaro Teixeira, Hugo de Azevedo Werneck, Sandrine Etienne, Jérôme Orivel, Hilton Jeferson Alves Cardoso de Aguiar

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Luísa Antônia Campos Barros, Gisele Amaro Teixeira, Hugo de Azevedo Werneck, Sandrine Etienne, Jérôme Orivel, et al.. An Integrative Approach With Cytogenetic and Molecular Data of the Leaf-Cutting Ant Acromyrmex hystrix (Latreille, 1802) Highlights a Notably Derived Karyotype in the Genus. Zoologica Scripta, 2026, 55 (1), pp.145-152. 10.1111/zsc.70007. hal-05468439

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An integrative approach with cytogenetic and molecular data of the leaf-cutting ant *Acromyrmex hystrix* (Latreille, 1802) highlights a notably derived karyotype in the genus

"A Notably Derived Karyotype in the Leaf-Cutting Ant *Acromyrmex hystrix*: An Integrative Cytogenetic and Molecular Approach"

Luísa Antônia Campos Barros<sup>a*</sup>, Gisele Amaro Teixeira<sup>a,b</sup>, Hugo de Azevedo Werneck<sup>b</sup>, Sandrine Etienne<sup>c</sup>, Jérôme Orivel<sup>d</sup>, Hilton Jeferson Alves Cardoso de Aguiar<sup>a,e</sup>

<sup>a</sup> Universidade Federal do Amapá, Campus Binacional – Oiapoque, n°3051, Bairro Universidade, Oiapoque, Amapá, 68980-000, Brazil;

<sup>b</sup> Departamento de Biologia Geral, Universidade Federal de Viçosa, Campus Universitário, Viçosa, MG 36570-900, Brazil;

<sup>c</sup> INRAE Experimental Domain - Godet - 97131 Petit-Canal, Guadeloupe;

<sup>d</sup> CNRS, UMR EcoFoG, AgroParisTech, CIRAD, INRAE, Université de Guyane, Université des Antilles, Campus Agronomique, BP 316, Kourou 97379, CEDEX, France;

<sup>e</sup> Programa de Pós-graduação em Biodiversidade Tropical, Universidade Federal do Amapá, Campus Marco Zero do Equador, Macapá, Amapá, 68.903-419, Brazil.

* Corresponding author:

Luísa Antônia Campos Barros; E-mail: luufv@yahoo.com.br

Running tittle: Cytogenetic and molecular phylogeny of the ant *Acromyrmex hystrix*
30 **Abstract**

31 Fungus-growing ants are a peculiar ant group that cultivates fungi for food. Within 32 this group, the so-called leaf-cutting ants (Atta Fabricius, 1804 and Acromyrmex 33 Mayr, 1865) hold significant ecological and economic importance. Previous 34 studies have highlighted conserved chromosome numbers with 2n=22 in Atta and 35 2n=38 in Acromyrmex, with the single exception of the social parasite 36 Acromyrmex ameliae De Souza et al. 2007, with 2n=36, as a result of 37 Robertsonian fusion. Although Acromyrmex hystrix (Latreille, 1802) is the type 38 species of Acromyrmex it is one of the least-known species of this genus. In this 39 study, we described its karyotype and inferred the phylogenetic relationships 40 within Acromyrmex. A distinct chromosome number was detected for the species, 41 with 2n=28 chromosomes and a karyotypic formula of 10m+16sm+2st. Our 42 phylogenetic reconstruction revealed Ac. hystrix constituted a separate clade 43 within Acromyrmex. This karyotype is derived within Acromyrmex with a 44 chromosome number reduction from 2n=38 to 28. Cytogenetic and molecular 45 data revealed the evolutionary divergence of Ac. hystrix within Acromyrmex. 46 Although leaf-cutting ants are among the most studied ants, many gaps remain 47 in our understanding of these insects. The inclusion of additional species in the 48 molecular phylogeny, coupled with chromosomal information, could provide 49 insights into the evolution of Acromyrmex. 50

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## 52 Introduction

53 Leaf-cutting ants are dominant herbivores and ecosystem engineers (Hölldobler & Wilson, 1990, Swanson et al., 2019). These ants are found exclusively on the 54 American continent, predominantly in tropical areas (Delabie et al., 2011), and 55 are considered the most derived representatives within the Attina subtribe 56 (Mehdiabadi & Schultz, 2010). Currently, 34 valid species of *Acromyrmex* Mayr, 57 1865 and 15 species of *Atta* Fabricius, 1804 (including a fossil species) (Bolton, 58 2024) have been described, and some species are considered major pests in 59 agriculture (Della Lucia et al., 2014; Britto et al., 2016). Leaf-cutting ants form a 60 monophyletic group (Mehdiabadi & Schultz, 2010) and new insights into the 61 relationship between *Atta* and *Acromyrmex* arose after the investigation of 62 *Amoimyrmex striatus* (Roger, 1863) (cited as *Acromyrmex striatus*) using 63 molecular and cytogenetic tools (Cristiano et al., 2013). The genus *Amoimyrmex* 64 Cristiano et al. 2020 is considered a sister group of the remaining leaf-cutting 65 ants *Acromyrmex* and *Atta* and is a distinct monophyletic group within Attina 66 (Cristiano et al., 2020). To date, there is no molecular phylogeny that includes all 67 *Acromyrmex* species. Based on morphological traits, the genus *Acromyrmex* has 68 been subdivided into two subgenera, *Acromyrmex* and *Moellerius*, with the latter 69 considered the most derived (Mayhé-Nunes, 1991). However, all the molecular 70 phylogeny data published on *Acromyrmex* suggests that the two *Acromyrmex* 71 subgenera do not represent natural groups (Sumner et al., 2004; Cristiano et al., 72 2013, 2020; Barrera et al., 2022; Mera-Rodríguez et al., 2024). Thus, the addition 73 of more species could improve our understanding of the evolutionary 74 relationships within the genus. In *Atta*, the available molecular phylogeny shows 75 that the genus is monophyletic and divided into four groups (Archeatta, Epiatta, 76 Neoatta, and *Atta s. stricto*) (Bacci et al., 2009; Barrera et al., 2022). 77

78 Cytogenetic studies on leaf-cutting ants are available for six of the 15 existing 79 *Atta* species (reviewed by Mariano et al., 2019 and Teixeira et al., 2022) included 80 in three of the four monophyletic groups established by molecular phylogenies 81 (Bacci et al., 2009; Barrera et al., 2022). To date, there is no chromosomal 82 information on species belonging to the Archeatta clade, which is composed of 83 *Atta texana* (Buckley, 1860), *Atta insularis* Guérin-Méneville, 1844, and *Atta 84 mexicana* (Smith, 1858). The karyotype of *Atta* species is conserved, considering
all cytogenetic traits analyzed. All *Atta* species studied thus far have 2n=22 chromosomes (18m + 2sm + 2st) (Barros et al., 2014, 2015; revised by Teixeira et al., 2022).

In *Acromyrmex*, 18 of 34 species have been karyotyped, and the chromosome number 2n=38 is observed in almost all species, including both subgenera (revised by Barros et al., 2021; Barros et al., 2024). The only exception is the social parasite *Ac. ameliae* De Souza et al. 2007, with 2n=36 chromosomes. It has been suggested that its karyotype is derived in comparison to other *Acromyrmex* species owing to chromosomal fusion during its evolution as a social parasite (Barros et al., 2021), highlighting the importance of studying species with peculiar biological traits.

The lack of cytogenetic information for approximately half of the known *Acromyrmex* species, together with large variations in their ecology and distribution, precludes any general considerations. *Acromyrmex hystrix* (Latreille, 1802), the type species of this genus, is among the least studied. To contribute to a better understanding of the mechanisms involved in the chromosomal evolution of *Acromyrmex*, our objectives were to: i) describe the karyotype of *Ac. hystrix* using classical and molecular cytogenetics, and ii) report the phylogenetic position of *Ac. hystrix* within *Acromyrmex*.

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## 105 Materials and methods

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### 107 Field sampling and material deposition

Part of a colony of *Ac. hystrix* was collected in Petit Saut, French Guiana (5.068256, -53.048379) on August 23rd, 2018. The nest was located at the base of a tube-like root system and covered by a mass of vegetal debris. It resembled a 1-meter-long rug that stretched from the ground to the trunk of a living tree. The fungus was located inside this “rug.” There was no indication of chamber organization within the nest. The detection of these nests is rare and appears to be associated only with highly undisturbed environments. Interestingly, the specimens behaved far more aggressively compared to other species in the genus.
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## Chromosome preparation and karyotype analysis

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Mitotic metaphases were obtained from cerebral ganglia of worker larvae after meconium elimination, according to Imai et al. (1988). A total of 23 individuals were cytogenetically analyzed including classical and molecular cytogenetics.

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Chromosome number and morphology of metaphases were analyzed using conventional 4% Giemsa staining. To determine the morphology of the chromosomes, a total of 10 metaphases were measured and chromosomes were classified according to Levan et al. (1964). This classification considers the ratio between the long arm (l) and the short arm (p) to obtain the following chromosome morphology: 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 in karyotypes using Adobe Photoshop 2021 (version 22.2) and measured using Image Pro Plus.

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## Banding techniques and molecular cytogenetics

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At least 30 metaphases from three individuals were analyzed for each cytogenetic technique. C-banding was performed according to Sumner (1972).

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Sequential staining with the fluorochrome chromomycin A₃ (CMA₃) and 4'-diamidino-2-phenylindole (DAPI) was performed to detect specific GC- and AT-rich regions following Schweizer (1980), except for the distamycin removal.

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Ribosomal 18S genes were detected by FISH, following Pinkel et al. (1986). The 18S rDNA probe was obtained via PCR (polymerase chain reaction) amplification employing the primers rDNA 18SF1 (5'-GTC ATA GCT TTG TCT CAA AGA-3') and 18SR1.1 (5'-CGC AAA TGA AAC TTT TTT AAT CT-3') designed for the bee *Melipona quinquefasciata* Lepeletier, 1836 (Pereira, 2006). Total DNA from the ant *Camponotus rufipes* (Fabricius, 1775) was used as template in PCR reactions. 18S rDNA probes were labeled maintaining the conditions for PCR amplification (Pereira, 2006) by an indirect method using digoxigenin-11-dUTP (Roche, Mannheim, Germany), and the FISH signals were detected with anti-digoxigenin-rhodamine (Roche Applied Science), following the manufacturer's protocol. The metaphases were photographed using an Olympus BX60

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microscope, linked to a Q Color 3 Olympus image capture system with the filters WU (330-385 nm), and WG (510-550 nm) used for analyzing DAPI, and rhodamine, respectively.

The microsatellite (GA)₁₅ and telomeric motif (TTAGG)₆ were used as probes in FISH experiments. The probe sequence 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 Pinkel et al. (1986) with the following modifications: metaphase chromosomes were denatured in 70% formamide/2x SSC at 75°C for 5 min; the probes were hybridized with chromosomes in 20 µL of hybridization mix (200 ng of labeled probe, 2x SSC, 50% formamide, and 10% dextrane sulfate). This hybridization mix was heated for 10 min at 85°C, and the slides were kept in a moist chamber at 37°C overnight. Then, the slides were washed in 4x SSC/Tween and dehydrated in an alcohol series. The antibody incubation step was omitted since the microsatellite probe was directly marked. Finally, counterstaining with DAPI (DAPI Fluoroshield, Sigma Aldrich) was performed.

## DNA extraction, PCR amplification, and sequencing

Workers of the same colony were collected and stored in absolute ethanol, before DNA extraction and phylogenetic analysis. The DNA extraction was performed from a single leg of a specimen of *Ac. hystrix* using the "HotShot" protocol (Truett et al., 2000). The DNA purification was performed on column resin Sephadex® G50 superfine (GE Healthcare) in MultiScreen MILLIPORE® (ref. MSHVN4550) plates. DNA quality was quantified through electrophoresis on 0.8% (w/v) agarose gel dyed with GelRed 10,000x. Extracted DNA was amplified by PCR and DNA nuclear sequences for wingless (wg), long-wave rhodopsin (LW Rh), elongation factor 1-alpha F1 (EF1αF1) genes were obtained using previously published primers (Ward & Downie 2005; Brady et al., 2006). Amplicon sequencing was performed using Sanger sequencing by Fasteris (Plan-les-Ouates, Switzerland).

## Sequence alignment and molecular phylogeny
182
183 To recover the phylogenetic relationship, we downloaded from GenBank 136
184 sequences to ingroup composed by *Acromyrmex*, *Atta* and *Amoimyrmex* genera
185 and 12 sequences to outgroup using the genera *Sericomyrmex*,
186 *Paratrachymyrmex*, and *Mycetomoellerius*. The gene fragments used were the
187 exons of wg, LW Rh, and EF1αF1 (see Supporting Information 1).

188 The alignments were performed in the MAFFT v.7 (Katoh and Standley, 2013).
189 All genic fragments were aligned using the iterative refinement methods with
190 FFT-NS-i strategy (Katoh et al., 2005). The aligned sequences were visually
191 checked in the MEGA v.11 (Tamura et al., 2021). Each fragment was considered
192 as one partition and the data were concatenated in one matrix in the software
193 Sequence Matrix (Vaidya et al., 2011).

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195 The Bayesian inference analysis was performed in MrBayes v. 3.2.7 (Ronquist et
196 al., 2012) using the CIPRES Science gateway online server (Miller et al., 2010).
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198 We performed two independent runs with four chains for each one. The mixed
199 evolutionary models (Ronquist & Huelsenbeck 2003) with invariant proportion
200 and Gamma correction were implemented to all partitions. The MCMC (Markov
201 Chain Monte Carlo) was run with 20,000,000 generations and the trees were
202 verified using the Tracer v.1.5 program (Rambaut & Drummond, 2009), observing
203 the final PSRF (Potential Scale Reduction Factor = 1.00), which compares the
204 variance between the chains with the variance within each chain, in addition to
205 the ESS values (Estimated Sample Size ≥ 200). The 25% of the initial MCMC
206 generations were excluded, the remainder was used to calculate a consensus
207 tree. The frequency of each branch in the majority consensus represents the
208 value of the posterior probability (PP) of the Bayesian formula. All trees were
209 edited in the FigTree v program. 1.3.1 (Rambaut, 2009).
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## Results

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### *Classical and molecular cytogenetics*
212 *Acromyrmex hystrix* showed 2n=28 chromosomes in all analyzed individuals, 213 with a karyotypic formula of 2n = 10m + 16sm + 2st (Fig. 1a). The chromosomes 214 were arranged in pairs and measured.

215 Heterochromatic blocks were identified in the centromeric regions of the 216 chromosomes; however, additional bands on the two highest pairs were detected 217 after C-banding (Fig. 1b). GC-rich bands ($CMA_3^+$) were detected in the interstitial 218 region of the second highest submetacentric chromosome pair (pair 7th) (Fig. 1c). 219 The exact location can be better observed in less condensed metaphases (Fig. 2a). $CMA_3^+$ additional bands are also detected on the small arms of two 220 chromosome pairs, 5th and 11th (Fig. 1c). AT-rich bands (DAPI+) are visible in the 221 interstitial region of the highest submetacentric pair (pair 6th) (Fig. 1d, Fig. 2b). 222

223 *Acromyrmex hystrix* showed a single 18S rDNA site, detected on the second- 224 highest submetacentric pair (Fig. 1e). The dinucleotide microsatellite ($GA_n$) 225 displayed an intense spreading pattern throughout the genome of *Ac. hystrix*, 226 restricted to the euchromatic regions of the chromosomes, except for the 227 interstitial region of the large arm of the 6th, 7th, and 12th chromosome pairs (Fig. 228 1f). The telomeric motif (TTAGG)$_n$ was detected at both ends of all chromosomes 229 in *Ac. hystrix* (Fig. 1g).

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231 **Phylogenetic analysis**

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233 The alignment with the four concatenated gene fragments had a total length of 234 1,220 base pairs. Our topology recovered the leaf-cutting ants as a monophyletic 235 group, as well as the genera *Atta*, *Amoimyrmex*, and *Acromyrmex*. Within 236 *Acromyrmex*, the species *Ac. hystrix* belonged to a separate clade (Fig. 3).

237

238 **Discussion**

239 According to the Bayesian consensus phylogeny based on the concatenated 240 sequences, our topology of the phylogenetic tree of the leaf-cutting ants was 241 similar to that observed by Cristiano et al. (2020) and Barrera et al. (2022), who 242 used nuclear genes and ultraconserved elements (UCEs), respectively. Although
243 *Acromyrmex* presented some branches of polytomy, several relationships were 244 consistent with those reported in previous studies (Cristiano et al., 2020; Barrera 245 et al., 2022). The positioning of the *hystrix* clade, as evidenced by our phylogeny 246 analysis, was further supported by chromosomal and biological data.

247 With 2n=28, *Ac. hystrix* shows a unique karyotype among leaf-cutting ants, which 248 appears intermediate, on one hand, between the genera *Amoimyrmex* and *Atta*, 249 both with 2n=22, and, on the other hand, *Acromyrmex* with 2n=38. The karyotype 250 of *Ac. hystrix* revealed a second karyotypic variation in *Acromyrmex*, a genus 251 previously considered to be conserved with 2n=38 in 17 out of 18 species 252 cytogenetically analyzed to date, except for *A. ameliae* with 2n=36 (reviewed by 253 Barros et al., 2016; Barros et al., 2021; Barros et al., 2024). Considering that *Ac.* 254 *ameliae* presents a derived karyotype in *Acromyrmex* and is associated with 255 social parasitism, the unique karyotype of *Ac. hystrix* represents an independent 256 variation that is not associated with social parasitism.

257 Molecular phylogeny revealed that *Ac. hystrix* is located in a distinct position 258 within *Acromyrmex*. Considering that *Ac. balzani* (Emery, 1890), *Ac. echinatior* 259 (Forel, 1899), and other species of the sister clade to *Ac. hystrix*, such as *Ac.* 260 *aspersus* (Smith, 1858), *Ac. rugosus* (Smith, 1858), *Ac. subterraneus* (Forel, 261 1893), and *Ac. disciger* (Mayr, 1887), all possess 2n=38 chromosomes (reviewed 262 by Barros et al., 2021), we recognize *Ac. hystrix* as having a derived karyotype 263 within *Acromyrmex*, characterized by a chromosome number reduction from 264 2n=38 to 28. Five chromosome fusions, including NOR-bearing chromosome pair 265 relocation, are likely involved because intrachromosomal rDNA clusters in *Ac.* 266 *hystrix*, a derived pattern in the genus, seem to be a consequence of fusions and 267 other rearrangements during the evolution of this species (Barros et al., 2021).

268 Considering the chromosomal rearrangements of fusion type, we expected most 269 chromosomes to be metacentric and large. However, due to the location of the 270 species in molecular phylogeny, additional complex chromosome 271 rearrangements should be involved in the karyotype evolution of the species. 272 Interestingly, both *Ac. balzani* and *Ac. echinatior*, which are located out of the 273 internal *Acromyrmex* clade, possess derived karyological features, including a 274 distinct pattern for the st1 pair and a higher number of acrocentric chromosomes, 275 respectively (Barros et al., 2016).
276 Chromosome reduction likely shaped the distinct karyotype of the social parasite
277 *Ac. ameliae* (Barros et al., 2021), with Robertsonian fusion being the most
278 plausible rearrangement in this species. *Acromyrmex hystrix* is not a social
279 parasite, and the chromosomal reduction involved in shaping this karyotype
280 should be considered exceptional in the genus. The karyotype configuration
281 suggested that this *Acromyrmex* species belongs to an isolated lineage, as
282 demonstrated by the long branch of the recovered molecular phylogeny. The
283 presence of the canonical telomere sequence (TTAGG)n in the terminal region of
284 both ends of the chromosomes of *Ac. hystrix* seems to corroborate the
285 occurrence of Robertsonian fusion as the origin of the species karyotype, instead
286 of tandem fusion (see discussion in Barros et al. 2021).

287 *Acromymrex hystrix* exhibits a unique pattern of heterochromatin, which is
288 exclusively found in centromeric blocks. In contrast, it was more complex in all
289 other studied *Acromyrmex* spp., being restricted to the short arms of
290 submetacentric and subtelocentric chromosomes, while only metacentric
291 chromosomes bear such centromeric heterochromatic blocks. The centromeric
292 heterochromatin pattern observed in *Ac. hystrix* seems to support the
293 Robertsonian fusion hypothesis. This is because the two subtelocentric
294 chromosomes would retain the heterochromatic blocks on the centromere after
295 fusion, thereby reducing the number of chromosomes in the karyotype.

296 Multiple GC-rich bands were detected on the chromosomes of *Ac. hystrix* in
297 addition to ribosomal clusters. This configuration was also observed in two other
298 *Acromyrmex* species, *Ac. niger* (Smith, F. 1858) and *Ac. rugosus* (Barros et al.,
299 2016), and in *Am. striatus* (Cristiano et al., 2013). However, in *Atta* spp. and most
300 *Acromyrmex* spp., only the ribosomal DNA sites (a single pair) are GC-rich. In
301 other Attina species and ants in general, rDNA clusters are GC-rich and flanked
302 by heterochromatic segments (reviewed by Teixeira et al., 2021). The GC band
303 pattern observed in *Ac. hystrix* and other leaf-cutting ants, where the GC bands
304 are located on the short arms of two chromosome pairs, suggests the genomic
305 growth of GC-rich heterochromatin. This discovery greatly enhances our
306 understanding of karyotype evolution patterns in Attina.

307 Single rDNA clusters restricted to intrachromosomal regions are common among
308 Neotropical ant species (Teixeira et al., 2021, Damasceno et al., 2024). In *Ac*.
hystrix, the rDNA clusters were restricted to the interstitial region of the large arm of the 2nd sm pair. This pattern is similar to that observed in *Atta* spp. and *Amoimyrmex* spp. This intrachromosomal pattern differs from that of the majority of known *Acromyrmex* spp., in which these clusters are located in the terminal region of the largest st, and is considered a derived trait in the genus *Acromyrmex* (see discussion in Barros et al., 2021).

AT-rich bands were detected on the long arm of the 6th chromosome colocalized with heterochromatic blocks. This trait is not observed in any *Atta* spp., *Acromyrmex* spp., or *Am. striatus* (reviewed by Teixeira et al., 2021), suggesting that this is a derived character state in leaf-cutting ants restricted to *Ac. hystrix*. AT-rich specific regions are uncommon among ants. To date, they have been found only in other fungus-farming ants of the genus *Mycetophylax* (Cardoso et al., 2014), the little fire ant *Wasmannia auropunctata* (Roger 1863) (Souza et al., 2011), and *Crematogaster tenuicula* (Silveira et al., 2024). In all of these ant groups, this trait apparently originated independently.

Euchromatic regions of the genome are usually enriched by microsatellites (Toth et al., 2000; Li et al., 2004) and all chromosomes of *Ac. hystrix* contained (GA)n in the euchromatin of both chromosome arms, except for the heterochromatic centromeric regions. In addition, these microsatellites were not observed in the pericentromeric region of the long arm of the 6th and 7th chromosomes due to AT-rich heterochromatin and 18S rDNA clusters, respectively. This spread pattern restricted to euchromatic regions was observed in different genera of fungus-growing ants (Teixeira et al., 2022) and other hymenopteran insects (Marchioro et al., 2020; Cunha et al., 2023). This indicated a non-random distribution of microsatellites in the ant genome. The presence of (GA)n in both arms of all chromosomes of *Ac. hystrix* (2n=28) contrasts with that previously observed in *Acromyrmex* spp. (2n=38), in which this microsatellite is restricted to the long arms of some submetacentric and subtelocentric chromosomes, and to both arms of metacentric chromosomes (Teixeira et al., 2022). These data suggest the substantial role played by Robertsonian fusions in the origin of the derived karyotype of *Ac. hystrix*. In Neotropical stingless bees, similar microsatellite patterns were observed among different species; specifically, bees with a higher chromosome number (n=17) had microsatellites restricted to a single
chromosome arm. Nevertheless, chromosomes of species with low chromosome numbers (n=8, 9, and 15) carry these repetitive sequences in both arms, suggesting the involvement of fusion events (Cunha et al., 2023).

Although leaf-cutting ants are among the most studied members of their family, there are still many gaps in the understanding of these insects. Even though Ac. *hystrix* is the type species of the genus *Acromyrmex* (Mayr, 1865), biological information about this species remains scarce, especially in terms of its bioecology (Weber, 1946; 1947; Gonçalves, 1961). This species is found throughout South America, particularly in the Amazonian regions of Venezuela, Peru, and Brazil. Ac. *hystrix* also occurs in the northeastern and central regions of Brazil (Gonçalves, 1961; Weber, 1972; Dáttilo et al., 2010; Delabie et al., 2011). It is a rainforest species with a recognizable nest, covered with a thick layer of straw, and is frequently found with tube-like roots (Weber, 1946; Gonçalves, 1961).

This integrative study sheds light on the lesser-known species, *Acromyrmex hystrix*, and its role in the evolution and diversity of this genus. It also underscores the need to describe the karyotype of many *Acromyrmex* species, calling into question the presumed conserved chromosomal configuration (2n=38) of this genus. The initial molecular phylogeny revealed a diverse range of lineages within *Acromyrmex*, prompting further investigation to include all species in a comprehensive analysis of karyotype evolution in these leaf-cutting ants.

363

## 364 Acknowledgements

We are grateful to Dr. Jacques H. C. Delabie for the species identification, and Dr. Céline Leroy for field assistance. This study was supported by Fundação de Amparo à Pesquisa do Amapá (FAPEAP, #286), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 420512/2023-3). GAT thanks CNPq for the scholarship granted by PDJ/CNPq (150041/2023-3).

370

## 371 Disclosure

The authors declare no conflict of interest.

372
373 References

374

375 Aguiar, H. J. A. C., Barros, L. A. C., Silveira, L. I., Petitclerc, F., Etienne, S., & 376 Orivel, J. (2020) Cytogenetic data for sixteen ant species from North-eastern 377 Amazonia with phylogenetic insights into three subfamilies. *Comparative 378 Cytogenetics*, 14(1), 43-60. https://doi.org/10.3897/compcytogen.v14i1.46692

379 Bacci, M., Solomon, S. E., Mueller, U. G., Martins, V. G., Carvalho, A. O., Vieira, 380 L. G., & Silva-Pinhati, A. C. O. (2009) Phylogeny of leafcutter ants in the genus 381 Atta Fabricius (Formicidae: Attini) based on mitochondrial and nuclear DNA 382 sequences. *Molecular Phylogenetics and Evolution*, 51(3), 427-437. 383 https://doi.org/10.1016/j.ympev.2008.11.005

384 Barros, L.A.C., Teixeira, G.A., Aguiar, H.J.A.C., Mariano, C.S.F., Delabie, J.H.C., 385 & Pompolo, S.G. (2014) Banding patterns of three leafcutter ant species of the 386 genus *Atta* (Formicidae: Myrmicinae) and chromosomal inferences. *Florida 387 Entomologist*, 97, 1694-1701. https://doi.org/10.1653/024.097.0444

388 Barros, L. A. C., Aguiar, H. J. A. C., Teixeira, G. A., Mariano, C. S. F., Teixeira, 389 M. C., Delabie, J. H. C., & Pompolo, S. G. (2015) Cytogenetic data on the 390 threatened leafcutter ant *Atta robusta* Borgmeier, 1939 (Formicidae: Myrmicinae: 391 Attini). *Comptes Rendus Biologies*, 38 (10), 660-665. 392 https://doi.org/10.1016/j.crvi.2015.07.006

393 Barros, L. A. C., Aguiar, H. J. A. C., Mariano, C. S. F., Andrade-Souza, V., Costa, 394 M. A., Delabie, J. H.C., & Pompolo, S. G. (2016) Cytogenetic data on six leafcutter 395 ants of the genus *Acromyrmex* Mayr, 1865 (Hymenoptera, Formicidae, 396 Myrmicinae): insights into chromosome evolution and taxonomic implications. 397 *Comparative Cytogenetics*, 10 (2), 229-243. 398 https://doi.org/10.3897/CompCytogen.v10i2.7612

399 Barros, L. A. C., Teixeira, G. A., Aguiar, H. J. A. C., Lopes, D. M., & Pompolo, 400 S.G. (2018) Cytogenetic studies in *Trachymyrmex holmgreni* Wheeler, 1925 401 (Formicidae: Myrmicinae) by conventional and molecular methods. *Sociobiology 402* 65(2), 185-190. https://doi.org/10.13102/sociobiology.v65i2.2214

403 Barros, L. A. C., Teixeira, G. A., Lod, R. B., Arnhold, A., & de Aguiar, H. J. A. C. 404 (2024). Chromosome Mapping of Ribosomal DNA Clusters in Four Leaf-cutting 405 Ant Species of the Genus *Acromyrmex*, with Description of a Triploid Individual 406 in *A. laticeps*. *Sociobiology*, 71(3), e10403. 407 https://doi.org/10.13102/sociobiology.v71i3.10403

408 Bolton, B. (2024) An online catalog of the ants of the world. Available from 409 https://antcat.org. (accessed February 19, 2024).

410 Britto, J. S., Forti, L. C., de Oliveira, M. A., Zanetti, R., Wilcken, C. F., Zanuncio, 411 J. C., ... & Silva-Camargo, R. (2016) Use of alternatives to PFOS, its salts and 412 PFOSF for the control of leaf-cutting ants *Atta* and *Acromyrmex*. *International 413 Journal of Environmental Research*, 3, 11-92.

414 Cardoso, D.C., Pompolo, S.G., Cristiano, M. P., &Tavares, M.G. (2014) The role 415 of fusion in ant chromosome evolution: insights from cytogenetic analysis using 416 a molecular phylogenetic approach in the genus *Mycetophylax*. *PLoS ONE*, 9(1), 417 e87473. https://doi.org/10.1371/journal.pone.0087473
418 Cioffi M. B., Kejnovsky E., & Bertollo L. A. C. (2010) The Chromosomal Distribution of Microsatellite Repeats in the Genome of the Wolf Fish *Hoplias malabaricus*, Focusing on the Sex Chromosomes. *Cytogenetic and Genome Research*, 132, 289-296. https://doi.org/10.1159/000322058

422 Cristiano, M. P., Cardoso, D. C., & Fernandes-Salomão, T. M. (2013) Cytogenetic and molecular analyses reveal a divergence between *Acromyrmex striatus* (Roger, 1863) and other congeneric species: taxonomic implications. *PLoS ONE*, 8(3), e59784. https://doi.org/10.1371/journal.pone.0059784

426 Cristiano, M. P., Cardoso, D. C., Sandoval-Gómez, V. E., & Simões-Gomes, F. C. (2020) *Amoimyrmex* Cristiano, Cardoso & Sandoval, gen. nov. (Hymenoptera: Formicidae): a new genus of leaf-cutting ants revealed by multilocus molecular phylogenetic and morphological analyses. *Austral Entomology*, 59(4), 643-676. https://doi.org/10.1111/aen.12493

431 Dátillo, W., Vicente R. E., Nunes R. V., & Carvalho M. S. G. (2010) Primeiro registro da quenquém cisco-da-Amazônia *Acromyrmex hystrix* (Latreille) (Formicidae: Myrmicinae) para o estado do Maranhão, Brasil. *EntomoBrasilis*, 3(3), 92-93. https://doi.org/10.12741/ebrasilis.v3i3.109

435 Delabie, J. H. C., Alves, H. S. R., Strenzel, G. M. R., Carmo, A. F. R., & Nascimento, I. C. (2011) Distribuição das formigas cortadeiras dos gêneros *Acromyrmex* e *Atta* no Novo Mundo. In: Della-Lucia TMC (Ed.) Formigas cortadeiras: da bioecologia ao manejo. 1ª. Editora da Universidade Federal de Viçosa, Viçosa, 80-101.

440 Della Lucia, T. M. C., Gandra, L. C., & Guedes, R. N. (2014) Managing leaf-cutting ants: peculiarities, trends and challenges. *Pest Management Science*, 70(1), 14-23. https://doi.org/10.1002/ps.3660

443 Fernández, F. V., Castro-Huertas, & Serna, F. (2015) Hormigas cortadoras de hojas de Colombia: *Acromyrmex y Atta* (Hymenoptera: Formicidae). Fauna de Colombia, Monografía No.5. Bogotá D.C., Colombia: Instituto de Ciencias Naturales, Universidad Nacional de Colombia, 350 pp.

447 Forti, L. C., De Andrade, M. L., Andrade, A. P. P., Lopes, J. F., & Ramos, V. M. (2006) Bionomics and identification of *Acromyrmex* (Hymenoptera: Formicidae) through an illustrated key. *Sociobiology*, 48(1), 135-153.

450 Gonçalves, C.R. (1961) O gênero *Acromyrmex* no Brasil (Hym. Formicidae). *Studia Entomologica*, 4(1-4), 113-180.

452 Hölldobler, B. and Wilson, E.O. (1990) The ants. Harvard University Press.

453 Imai, H., Taylor, R. W., Crosland, M. W., & Crozier, R. H. (1988) Modes of spontaneous chromosomal mutation and karyotype evolution in ants with reference to the minimum interaction hypothesis. *Japanese Journal of Genetics*, 63, 159-185.

457 Mayhé-Nunes, A. J. (1991) Estudo de *Acromyrmex* (Hymenoptera, Formicidae) com ocorrência constatada no Brasil: subsídios para uma análise filogenética [Dissertation]. Programa de Pós-graduação em Entomologia. Viçosa: Universidade Federal de Viçosa.
461 Mehdiabadi, N. J., & Schultz, T. R. (2010) Natural history and phylogeny of the fungus-farming ants (Hymenoptera: Formicidae: Myrmicinae: Attini). *Myrmecological News*, 13, 37-55.

462
463 Mera-Rodríguez, D., Fernández-Marín, H., & Rabeling, C. (2024). Phylogenomic approach to integrative taxonomy resolves a century-old taxonomic puzzle and the evolutionary history of the *Acromyrmex octospinosus* species complex. *Systematic Entomology*, 2025, 1-26. https://doi.org/10.1111/syen.1266526

468 Silveira L. I., Teixeira G. A., Barros L. A. C., Dergam J. A., & Aguiar H. J. A. C. 469 Chromosomal diversity in *Crematogaster* Lund, 1831 (Formicidae: Myrmicinae) 470 from the Amazon rainforest. *Genome*, 68, 1-12. doi: 10.1139/gen-2023-0130.

471 Sumner, A. T. 1972. A simple technique for demonstrating centromeric heterochromatin. *Experimental Cell Research*, 83, 304-306. https://doi.org/10.1016/0014-4827(72)90558-7.

474 Sumner, S., Aanen, D. K., Delabie, J., & Boomsma, J. J. (2004) The evolution of social parasitism in *Acromyrmex* leaf-cutting ants: a test of Emery's rule. *Insectes Sociaux*, 51(1), 37-42.

477 Swanson, A. C., Schwendenmann, L., Allen, M. F., Aronson, E. L., Artavia-León, A., Dierick, D., ... & Zelikova, T. J. (2019) Welcome to the *Atta* world: A framework for understanding the effects of leaf-cutter ants on ecosystem functions. *Functional Ecology*, 33(8), 1386-1399. https://doi.org/10.1111/1365-2435.13319

481 Teixeira, G. A., Barros, L. A. C., Aguiar, H. J. A. C., & Pompolo, S. G. (2017) 482 Comparative physical mapping of 18S rDNA in the karyotypes of six leafcutter 483 ant species of the genera *Atta* and *Acromyrmex* (Formicidae: Myrmicinae). 484 *Genetica*, 145, 351-357. https://doi.org/0.1007/s10709-017-9970-1

485 Teixeira, G. A., Aguiar, H. J. A. C., Petitclerc, F., Orivel J., Lopes, D. M., & Barros, L. A. C. (2021) Evolutionary insights into the genomic organization of major 486 ribosomal DNA in ant chromosomes. *Insect Molecular Biology*, 30(3), 340-354. 487
488 https://doi.org/10.1111/imb.12699

489 Teixeira, G. A., Barros, L. A. C., de Aguiar, H. J. A. C., & Lopes, D. M. (2022) 490 Multiple heterochromatin diversification events in the genome of fungus-farming 491 ants: insights from repetitive sequences. *Chromosoma*, 131(1), 59-75. 492 https://doi.org/10.1007/s00412-022-00770-7

493 Ward, P. S., Brady, S. G., Fisher, B. L., & Schultz, T. R. (2015) The evolution of 494 Myrmicine ants: phylogeny and biogeography of a hyperdiverse ant clade 495 (Hymenoptera: Formicidae). *Systematic Entomology*, 40(1), 61-81. 496 https://doi.org/10.1111/syen.12090

497 Weber, N. A. (1946) The biology of the fungus-growing ants. Part 9. The British 498 Guiana species. Part IX. The British Guiana species. *Revista de Entomologia*, 499 17,114-172.

500 Weber, N. A. (1947) Lower Orinoco River fungus-growing ants (Hymenoptera: Formicidae, Attini). Boletín de Entomología Venezolana, 6, 143-161.
501
**Figure Legends**

**Figure 1.** Diploid karyotype of *Acromyrmex hystrix* (2n=28): (a) conventional staining with Giemsa; (b) detection of heterochromatin using C-banding, indicated by dark blocks; (c) detection of GC-rich regions with chromomycin A₃ fluorochrome (arrows); (d) detection of AT rich regions with DAPI fluorochrome (arrowheads); fluorescence *in situ* hybridization with different repetitive DNA probes (red regions); (e) 18S rDNA, (f) (GA)₁₅, and (g) (TTAGG)₆. Bars = 5 µm.

**Figure 2.** Details of two chromosome pairs of *Acromyrmex hystrix* showing conventional staining, fluorochrome CMA₃ and DAPI. a) 7th chromosome pair highlighting GC-rich bands (CMA₃⁺) in the interstitial region and complementary AT negative bands (DAPI⁺); b) 6th chromosome pair highlighting the presence of AT-rich bands (DAPI⁺) detected in the interstitial region and complementary GC-negative bands (CMA₃⁻). Bars = 5 µm.

**Figure 3.** Phylogenetic tree based on Bayesian inference of some leaf-cutting ants, including *Acromyrmex hystrix*, estimated with concatenated data from three gene fragments (wg, LW Rh, and EF1αF1). The values above the branches correspond to the Bayesian posterior probabilities (PP). Additional non-leaf-cutting "higher attine" ants are included as out-groups.
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