Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(4): 29-39, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-172 Citation: M.N. Moura, D.C. Cardoso, B.C.L. Baldez, M.P. Cristiano (2019) Genome size in ants: retrospect and prospect. Caryologia 72(4): 29-39. doi: 10.13128/cayologia-172 Published: December 23, 2019 Copyright: © 2019 M.N. Moura, D.C. Cardoso, B.C.L. Baldez, M.P. Cristiano. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, 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. Genome size in ants: retrospect and prospect Mariana Neves Moura1,2, Danon Clemes Cardoso2,*, Brenda Carla Lima Baldez3, Maykon Passos Cristiano2,* 1 Programa de Pós- Graduação em Ecologia, , Universidade Federal de Viçosa, CEP: 36.570–000, Viçosa, Minas Gerais, Brazil 2 Departamento de Biodiversidade, Evolução e Meio Ambiente, Universidade Federal Ouro Preiato – UFOP, CEP: 35.400–000, Ouro Preto, Minas Gerais, Brazil 3 Programa de Pós- Graduação em Ecologia de Biomas Tropicais, Universidade Federal de Ouro Preto, CEP: 35.400–000, Ouro Preto, Minas Gerais, Brazil *Corresponding authors: danon@ufop.edu.br; maykon@ufop.edu.br Abstract. Genome size is very useful in studies regarding taxonomy, evolution, and reproductive biology in many animal groups, including insects. Herein, we assembled the information about genome size in ants, compiling the DNA content estimated so far, in order to evaluate the methods, the tissues and the internal standard applied to estimate the genomes size. All values were placed in a phylogenetic tree to put it in an evolutionary context and the means of the subfamilies were further compared statisti- cally to investigate changes and trends in the variation across taxa. The compiled data resulted in 86 specimens of ants, comprising 69 different species. This number repre- sents 0.52% of the total number of 13,369 ant species described, covering only 40 from 333 valid extant genera. The average Formicidae genome size was 0.36 pg (± 0.13). Most of the estimates were obtained through flow cytometry (83.5%), commonly using brain tissues, with Drosophila melanogaster as internal standard (76%). Differences in DNA content of ant species may be related to differences in the amount of heterochro- matin and is not related with chromosome number. The evaluation of the genome size estimations currently available for ants has highlighted their scarcity. Such information would be valuable as independent data for the study of ant diversity and evolution- ary biology. Further, we conclude that the standardization of the techniques used and a large–scale study on ant genome size are urgently required, given the importance of this insect group and the needs for the improvement in our knowledge on ant genome. Keywords. C-value, DNA content, Genetic diversity, Genome, Evolution, Phylogeny. INTRODUCTION Ants comprise a monophyletic group with approximately 13,369 val- id species distributed throughout the planet, with exception of extreme northern and southern latitudes (Bolton, 2018). They are one of the largest groups among insects in species diversity and biomass and together with some wasps and bees, are known as eusocial insects and comprise the order Hymenoptera (Hölldobler and Wilson, 1990; Ardila–Garcia et al., 2010). 30 Mariana Neves Moura et al. They represent an important insect group to investigate the relationship between the genealogical lineages and the distribution patterns of species, due to their occur- rence in different habitats of the most diverse ecosystems (Goodisman et al., 2008). Currently, the family Formici- dae is divided into 17 extant and 3 extinct subfamilies, spanning 333 valid extant genera and 154 extinct genera (Bolton, 2018). The subfamily Myrmicinae is the largest and most diverse subfamily worldwide, covering about 47% of all ant species (Françoso and Brandão, 1993; Brandão, 1999). Genome size, also named DNA content, DNA amount, or DNA C–value, has been described as a trait that ‘uniquely lies at the intersection of phenotype and genotype’, and the genome size of eukaryotes varies over five orders of magnitude, with a distribution skewed toward small values, around 2 picograms (pg) (Oliver et al., 2007). This variation does not seem to be corre- lated with the complexity of the organism or with the number of genes in eukaryotes, leading to what is called the “C–value paradox” (Moore, 1984; Gregory, 2001, 2005a; Eddy, 2012). It has been questioned, for example, why similar organisms with similar amounts of coding sequence have different amounts of DNA. While changes in gene sequences are often slow and gradual, changes in genome size can be rapid and abrupt as a consequence of chromosomal rearrangements or duplications (Alberts et al., 2007). The main methods used to estimate the total nuclear genome size are image cytometry, flow cytom- etry (FCM), and complete genome sequencing (Gregory, 2005b). Image cytometry was the first method used to determine genome size estimates. Basically, it operates by statically imaging a large number of cells stained with specific chemicals or fluorochromes, using optical microscopy (Torresan et al., 1994; Basiji et al., 2007). In contrast, flow cytometry evaluates the relative fluores- cence intensity of suspended nuclei, also stained with specific fluorochromes, and presents the data in a typi- cal histogram with a higher peak relative to the nuclei in the G0/G1 phase of cell cycle, and a lower peak, rela- tive to the nuclei in G2 phase (Price et al., 2000; Dolezel and Bartos, 2005). The complete genome sequencing method, on the other hand, provides the complete DNA sequence of the genome of an organism at a single time with the precise order of the nucleotides and an estimate of the genome size after its assembly (Klug et al., 2014). A fourth less common technique known as biochemi- cal analysis (BCA) was used during the early studies of genome size. It includes ‘the chemical extraction and quantification of DNA combined with cell counts to give an average DNA amount per nucleus or the reasso- ciation kinetics, in which the DNA molecule was dena- tured and then the time taken for the strands to rena- ture is used to calculate the amount of DNA (Gregory, 2005b). Among the methods, flow cytometry has been shown to be the least cost and time expensive technique when compared to other molecular tools and provides rapid generation of accurate results (Merkel et al., 1987; Doležel et al., 2007). According to Gregory (2018), haploid DNA contents (C–values, in picograms - pg) are currently available for 6,222 species of animals (3,793 vertebrates and 2,429 invertebrates), with insects representing 21.6% of this total. Li and Heinz (2000) performed the first DNA con- tent estimation of an ant by mean of biochemical analy- sis (BCA), to quantify the genome of Solenopsis invicta Buren, 1972. Subsequently, Johnston et al. (2004) also estimated the genome size of S. invicta but now using flow cytometry. Yet, in 2008, Tsutsui et al. (2008) carried out the first comprehensive study regarding the evolu- tion of the genome size in ants, reporting genome size estimates for 40 species from nine subfamilies. This was the last inclusion of a large number of ant species esti- mates to the genome size database that was followed by the study of Ardila–Garcia et al. (2010), which added a further 29 species. These two studies raised differ- ent questions about genome size, being the first a study of genome size evolution in Formicidae and the second a study of correlation between genome size with para- sitism and eusociality in the order Hymenoptera as a whole. It is important to note that they applied different methodologies in genome size estimation: in Tsutsui et al. (2008) the DNA content was estimated by using only flow cytometry, while Ardila–Garcia et al. (2010) also performed the FIAD method (Feulgen image analysis densitometry) to estimate the DNA content, and then compared the results from both techniques. Later, others studies explored the DNA content of ants, however in some cases covering only one species through complete genome sequencing (e.g. Nygaard et al., 2011) or, in other cases, considering specifically an ant genus through flow cytometry. The genome size of the genus Mycetophylax Emery, 1913 (sensu Klingen- berg and Brandão, 2009) was estimated by Cardoso et al. (2012) that explored the data placing them in a phyloge- netic context, also correlating it with chromosome num- ber of fungus–growing ants; and Aguiar et al. (2016) that evaluated three Camponotus Mayr, 1861 species, exploring their correlation with the karyotype of the studied species. Despite the importance of genome size, little is known about the ecological and evolutionary conse- quences of DNA amount in ants. Yet, the biological sig- 31Genome size in ants nificance and evolution of the genome size diversity in other groups has received much more attention over the last decades (Dufresne and Jeffery, 2011; Alfsnes et al., 2017; Pellicer et al., 2018). The diversity of genome size in plants has been shown to correlate with several pheno- typic features of cells and ultimately the organisms. For instance, plant species with larger genomes are adapted to xeric and higher elevation environments (e.g. Bottini et al., 2000). Here, we evaluate the available information about the genome size of ants, assembling the DNA con- tent estimated so far, in order to provide insights into the distribution, evolution and possible consequences of ant genome size diversity. We have also investigated and verified the needs of a re–evaluation in the genome size data (DNA C–value) for ants, as well the technique used in the estimation of the DNA content in respect of methodological issues such as: the internal standard and tissues used in the analysis. The basic information about ant genomes analyzed here may improve our knowledge about the evolution and diversification regarding this diverse group of insects and may help as a baseline and guidance for future studies about ant genome biology. MATERIALS AND METHODS To evaluate the knowledge about nuclear DNA con- tent on ants, we compiled the haploid genome size esti- mates for ants and other insect groups from the Ani- mal Genome Size Database (Gregory, 2018) and from the literature by searching in the publication databases Scopus® and ISI Web Science KnowledgeTM, by using the terms: “genome size”, “DNA amount”, “C–value” and “ants”. Based on the seven manuscripts found on ant genome size, we evaluated the method used to measure genome size, the type of tissue and the internal standard used to obtain the total content of DNA. To examine the genome size variation over Formi- cidae subfamilies we compiled the estimates in a Table of all the values available in the literature, expressed in picograms of DNA (pg) and mega base pairs (Mbp). Then we manually placed them in the phylogenetic tree proposed by Moreau and Bell (2013) by collapsing branches with equal names (same Operational Units - OTUs) and separating the subfamilies by color. General linear models were built to check for differences between the averaged genome sizes of the sampled subfamilies. The differences in genome size average for each subfam- ily were assessed by variance analysis of the GLM. When the p-value of ANOVA was significant (p < 0.05), a con- trast analysis at 5% level was then performed to deter- mine which mean was different. All the statistical analy- sis was performed in R v2.15.1 software (R Core Team, 2013) and GLM was submitted to residual analysis to evaluate adequacy of normal error distribution (Crawley, 2013). RESULTS AND DISCUSSION Overview: number of estimates, methods, tissues and inter- nal standards used The compiled data resulted in 86 specimens of ants whose genome size had been estimated, comprising 69 different species (Table 1). This number represents 0.52% of the total number of 13,369 ant species accepted until now, covering only 40 genera from 333 accepted (Bol- ton, 2018). From 17 existing subfamilies, we only found estimates for nine, with Myrmicinae having the largest number of species evaluated (32 spp.) (Figure 1). The number of estimates may reflect the richness of this sub- family that is the most diverse within Formicidae. Yet, Formicinae and Dolichoderinae together bear 20 spp. with DNA content estimates available. These three sub- families represent 65% of DNA content estimates on ants. The two main methods used to estimate DNA con- tent in ants were FCM and FIAD. A third method, biochemical analysis (BCA), was used in a pioneering work from Li and Heinz (2000) in order to estimate the genome size sole for Solenopsis invicta. It is impor- tant to mention that S. invicta has the genome size esti- mates by all three methods listed above and different values were obtained in each estimate: 0.60 pg by BCA (Li and Heinz, 2000), 0.47 pg by FIAD (Ardila-Garcia et al., 2010) and 0.77 pg by flow cytometry (Johnston et al., 2004). Such huge variation in genome sizes may be explained by the occurrence of different ploidy levels in S. invicta or even outcomes due the different techniques employed in the studies. Cytogenetical evidence suggests that there may be different levels of ploidy in S. invicta. All genome sizes are estimated by mean of com- parison with nuclei of reference standard, whose genome size is known that is called the “internal standard”. In the genome size estimation Drosophila melanogaster Meigen, 1830 (0.18 pg), Scaptotrigona xantotricha Moure, 1950 (0.43 pg) and Tenebrio molitor Linnaeus, 1758 (0.52 pg) are the internal standards most commonly used con- sidering Hymenoptera as a whole. Most of the estimates were obtained using D. melanogaster as internal stand- ard (76%), while FCM was the most common method used (83.5%). Generally, brain tissue is used to estimate nuclear genome size, but cells (hemocytes) obtained through hemolymph smears have also been tested (Ardi- 32 Mariana Neves Moura et al. Table 1. Overview of the genome size data available in literature for Formicidae species. Subfamily Species 1C-value (pg) 1C-value (Mbp) Method Cell type Standard References Amplyoponinae Amblyopone pallipes (Haldeman, 1844)* 0.34 332.52 FCM BR DM Tsutsui et al., 2008 Amblyopone pallipes (Haldeman, 1844)* 0.37 361.86 FCM BR DM Ardila-Garcia et al., 2010 Dolichoderinae Dolichoderus mariae (Forel, 1885) 0.18 176.04 FCM BR DM Ardila-Garcia et al., 2010 Dolichoderus taschenbergi (Mayr, 1866) 0.23 224.94 FCM BR DM Ardila-Garcia et al., 2010 Dorymyrmex bicolor Wheeler, 1906 0.25 244.5 FCM BR DM Tsutsui et al., 2008 Dorymyrmex bureni (Trager, 1988) 0.18 176.04 FIAD HE TM Ardila-Garcia et al., 2010 Forelius pruinosus (Roger, 1863) 0.22 215.16 FIAD HE TM Ardila-Garcia et al., 2010 Linepithema humile (Mayr, 1868) 0.26 254.28 FCM BR DM Tsutsui et al., 2008 Linepithema humile (Mayr, 1868) 0.26 250.8 Genome sequencing NS NS Smith et al., 2011 Liometopum occidentale Emery, 1895 0.29 283.62 FCM BR DM Tsutsui et al., 2008 Tapinoma sessile (Say, 1836) 0.37 361.86 FCM BR DM Ardila-Garcia et al., 2010 Tapinoma sessile (Say, 1836) A 0.38 371.64 FCM BR DM Tsutsui et al., 2008 Tapinoma sessile (Say, 1836) B 0.61 596.58 FCM BR DM Tsutsui et al., 2008 Dorylinae Cerapachys edentata 0.22 215.16 FCM BR DM Tsutsui et al., 2008 Eciton burchelli (Westwood, 1842) 0.27 264.06 FCM BR DM Tsutsui et al., 2008 Labidus coecus (Latreille, 1802) 0.37 361.86 FCM BR DM Tsutsui et al., 2008 Ectatomminae Ectatomma tuberculatum (Olivier, 1792) 0.71 694.38 FCM BR DM Tsutsui et al., 2008 Formicinae Camponotus castaneus (Latreille, 1802) 0.31 303.18 FCM BR DM Tsutsui et al., 2008 Camponotus crassus Mayr, 1862 0.29 283.62 FCM BR SX Aguiar et al., 2016 Camponotus floridanus (Buckley, 1866) 0.23 224.94 FIAD HE TM Ardila-Garcia et al., 2010 Camponotus floridanus (Buckley, 1866) 0.245 240 Genome sequencing NS NS Bonasio et al., 2010 Camponotus pennsylvanicus (De Geer, 1773) 0.33 322.74 FCM BR DM Tsutsui et al., 2008 Camponotus renggeri Emery, 1894 0.29 283.62 FCM BR SX Aguiar et al., 2016 Camponotus rufipes (Fabricius, 1775) 0.29 283.62 FCM BR SX Aguiar et al., 2016 Formica pallidifulva Wheeler, 1913 0.39 381.42 FCM BR DM Tsutsui et al., 2008 Lasius (Acanthomyops) latipes (Walsh, 1863) 0.27 264.06 FCM BR DM Ardila-Garcia et al., 2010 Lasius alienus (Foerster, 1850) 0.31 303.18 FCM BR DM Tsutsui et al., 2008 Lasius minutus Emery, 1893 0.23 224.94 FCM BR DM Ardila-Garcia et al., 2010 Paratrechina longicornis (Latreille, 1802) 0.18 176.04 FIAD HE TM Ardila-Garcia et al., 2010 Prenolepis imparis (Say, 1836) 0.30 293.4 FCM BR DM Tsutsui et al., 2008 Myrmeciinae Myrmecia varians Mayr, 1876 0.28 273.84 FCM BR DM Tsutsui et al., 2008 Myrmicinae Acromyrmex echinatior (Forel, 1899) 0.36 335 FCM BR CRBC Sïrvio et al., 2006 Acromyrmex echinatior (Forel, 1899) 0.32 313 Genome sequencing NS NS Nygaard et al., 2011 Aphaenogaster rudis (texana group N16) Enzmann, 1947 0.43 420.54 FCM BR DM Ardila-Garcia et al., 2010 Aphaenogaster rudis (texana group N17) Enzmann, 1947 0.46 449.88 FCM BR DM Ardila-Garcia et al., 2010 Aphaenogaster rudis (texana group N22b) Enzmann, 1947 0.44 430.32 FCM BR DM Ardila-Garcia et al., 2010 Aphaenogaster fulva Roger, 1863 0.42 410.76 FCM BR DM Ardila-Garcia et al., 2010 Aphaenogaster treatae Forel, 1886 0.50 489 FCM BR DM Ardila-Garcia et al., 2010 Apterostigma dentigerum Wheeler, 1925 0.65 635.7 FCM BR DM Tsutsui et al., 2008 Atta cephalotes (Linnaeus, 1758) 0.31 303.18 FCM BR DM Tsutsui et al., 2008 Atta cephalotes (Linnaeus, 1758) 0.30 290 Genome sequencing NS NS Suen et al., 2011 Atta colombica Guérin-Méneville, 1844 0.31 303.18 FCM BR DM Tsutsui et al., 2008 Atta texana (Buckley, 1860) 0.27 264.06 FCM BR DM Ardila-Garcia et al., 2010 33Genome size in ants Subfamily Species 1C-value (pg) 1C-value (Mbp) Method Cell type Standard References Crematogaster hespera Buren, 1968* 0.28 273.84 FCM BR DM Tsutsui et al., 2008 Eurhopalothrix procera (Emery, 1897) 0.39 381.42 FCM BR DM Tsutsui et al., 2008 Messor andrei (Mayr, 1886)* 0.26 254.28 FCM BR DM Tsutsui et al., 2008 Monomorium viridum Brown, 1943 0.50 489 FIAD HE TM Ardila-Garcia et al., 2010 Mycetophylax conformis (Mayr, 1884) 0.32 312.96 FCM BR SX Cardoso et al., 2012 Mycetophylax morschi (Emery, 1888) 0.32 312.96 FCM BR SX Cardoso et al., 2012 Mycetophylax simplex (Emery, 1888) 0.39 381.42 FCM BR SX Cardoso et al., 2012 Myrmecina americana Emery, 1895 A 0.26 254.28 FCM BR DM Tsutsui et al., 2008 Myrmecina americana Emery, 1895 B 0.31 303.18 FCM BR DM Tsutsui et al., 2008 Pheidole dentata Mayr, 1886 0.24 234.72 FIAD HE TM Ardila-Garcia et al., 2010 Pheidole floridana Emery, 1895 0.21 205.38 FIAD HE TM Ardila-Garcia et al., 2010 Pheidole hyatti Emery, 1895 0.33 322.74 FCM BR DM Tsutsui et al., 2008 Pogonomyrmex badius (Latreille, 1802) 0.27 264.06 FCM BR DM Tsutsui et al., 2008 Pogonomyrmex barbatus (Smith, 1858) 0.24 235 Genome sequencing NS NS Smith et al., 2011 Pogonomyrmex californicus (Buckley, 1867) 0.25 244.5 FCM BR DM Tsutsui et al., 2008 Pogonomyrmex coarctatus Mayr, 1868 0.29 283.62 FCM BR DM Tsutsui et al., 2008 Pyramica rostrata (Emery, 1895) 0.28 273.84 FCM BR DM Tsutsui et al., 2008 Sericomyrmex amabilis Wheeler, 1925 0.45 440.1 FCM BR DM Tsutsui et al., 2008 Solenopsis invicta Buren, 1972 0.62 606.36 BCA BR NS Li and Heinz 2000 Solenopsis invicta Buren, 1972 0.77 753.06 FCM BR DM Johnston et al., 2004 Solenopsis invicta Buren, 1972 0.47 459.66 FIAD HE TM Ardila-Garcia et al., 2010 Solenopsis invicta Buren, 1972 0.49 482 Genome sequencing NS NS Wurm et al., 2011 Solenopsis molesta Emery, 1895 0.38 371.64 FCM BR DM Ardila-Garcia et al., 2010 Solenopsis xyloni McCook, 1880 0.48 469.44 FCM BR DM Tsutsui et al., 2008 Temnothorax ambiguus (Emery, 1895) 0.31 303.18 FCM BR DM Ardila-Garcia et al., 2010 Temnothorax texanus (Wheeler, 1903) 0.32 312.96 FCM BR DM Ardila-Garcia et al., 2010 Tetramorium caespitum 0.26 254.28 FCM BR DM Tsutsui et al., 2008 Tetramorium caespitum (Linnaeus, 1758) 0.27 264.06 FCM BR DM Ardila-Garcia et al., 2010 Trachymyrmex septentrionalis (McCook, 1881) 0.25 244.5 FIAD HE TM Ardila-Garcia et al., 2010 Ponerinae Dinoponera australis Emery, 1901 0.57 557.46 FCM BR DM Tsutsui et al., 2008 Harpegnathos saltator Jerdon, 1851 0.34 330 Genome sequencing NS NS Bonasio et al., 2010 Odontomachus bauri Emery, 1892 0.49 479.22 FCM BR DM Tsutsui et al., 2008 Odontomachus brunneus (Patton, 1894) 0.33 322.74 FIAD HE TM Ardila-Garcia et al., 2010 Odontomachus brunneus (Patton, 1894) 0.44 430.32 FCM BR DM Tsutsui et al., 2008 Odontomachus Cephalotes Smith, 1863 0.43 420.54 FCM BR DM Tsutsui et al., 2008 Odontomachus chelifer (Latreille, 1802) 0.54 528.12 FCM BR DM Tsutsui et al., 2008 Odontomachus clarus Wheeler, 1915 0.42 410.76 FCM BR DM Tsutsui et al., 2008 Odontomachus haematodus (Linnaeus, 1758) 0.51 498.78 FCM BR DM Tsutsui et al., 2008 Ponera pennsylvanica Buckley, 1866 0.55 537.9 FCM BR DM Ardila-Garcia et al., 2010 Ponera pennsylvanica Buckley, 1866 0.60 586.8 FCM BR DM Tsutsui et al., 2008 Pseudomyrmicinae Pseudomyrmex ejectus (Smith, 1858) 0.29 283.62 FIAD HE TM Ardila-Garcia et al., 2010 Pseudomyrmex gracilis (Fabricius, 1804) 0.35 342.3 FCM, FIAD BR, HE DM, TM Ardila-Garcia et al., 2010 Pseudomyrmex gracilis (Fabricius, 1804) 0.40 391.2 FCM BR DM Tsutsui et al., 2008 Method: FCM = Flow cytometry, FIAD = Feulgen image analysis densitometry; Cell type: BR = Brain tissue, HE = Haemocyte; Standard: DM = Drosophila melanogaster, CRBC = Chicken Red Blood Cells, SX = Scaptotrigona xantotricha, TM = Tenebrio molitor, NS = not specified. *Valid names: Stigmatomma pallipes (Haldeman, 1844); Crematogaster laeviuscula Mayr, 1870; Veromessor andrei (Mayr, 1886), respectively. 34 Mariana Neves Moura et al. la-Garcia et al., 2010). Considering S. xantotricha, this internal standard was started to be used in studies com- prised stingless bees, and after with ants by the same research group (Tavares et al. 2010, Cardoso et al. 2012, Aguiar et al. 2016). Since no genome size histograms are available in either Ardila-Garcia et al. (2010) or Tsut- sui et al. (2008), it is impossible to compare the useful- ness of one or another internal standard considering the other two studies (Cardoso et al. 2012 and Aguiar et al. 2016) used S. xantotricha. In studies with plants, the choice of an appropriate internal standard considers the genome size magnitude of standard and studied group, mainly to avoid superposition of picks. Concerning the methods employed in genome size estimation, the study from Ardila-Garcia et al. (2010) is the only one that multiple species in the same work had the genome measured by two methods. They evaluated by FIAD and FCM the genome size on Odontomachus brunneus, Pseudomyrmex gracilis, and Solenopsis invicta and showed that the estimates using the first method tended to be smaller. The authors argue that the values from both techniques do not differ statistically. However, it is difficult to say that this difference is solely due to the technique itself, since both the tissue and the inter- nal standard used during the analysis were different. The nuclear DNA content of some ants has also been measured using a fourth method, which utilized complete genome sequencing techniques in species such as Acro- myrmex echinatior (Forel, 1899) (Nygaard et al., 2011), Atta cephalotes (Linnaeus, 1758) (Suen et al., 2011), Cam- ponotus floridanus (Buckley, 1866) (Bonasio et al., 2010), Harpegnathos saltator Jerdon, 1851  (Bonasio et al., 2010), Linepithema humile (Mayr, 1868) (Smith et al., 2011), Pogonomyrmex barbatus (Smith, 1858) (Smith et al., 2011) and Solenopsis invicta (Wurm et al., 2011) (Table 1). The genome size of Ac. echinatior was 313 Mbp (or 0.32 pg considering 1 pg = 978 Mbp; (Doležel et al., 2003)) obtained with complete genome sequencing (Nygaard et al., 2011) and 335 Mbp (0.36 pg) by FCM (Sïrvio et al., 2006). This difference can be attributed to the loss of repetitive regions and some chromosomal regions, such as telomeres, through genome sequencing techniques (Gregory, 2005b). The same was observed in A. cepha- lotes, whose genome size estimated by complete genome sequencing was 290 Mbp (approximately 0.30 pg) (Suen et al., 2011) and by FCM was 303.18 Mbp (approximately 0.31 pg) (Tsutsui et al., 2008). The differences were great- er in S. invicta, whose genome size was obtained with all four different techniques (BCA, FIAD, FCM, and Genome Sequencing): 606 Mbp (0.62 pg) (Li and Heinz, 2000) by BCA, 459 Mbp (0.47 pg) (Ardila-Garcia et al., 2010) by FIAD, 753 Mbp (0.77 pg) (Johnston et al., 2004) by FCM and 482 Mbp (0.49 pg) (Wurm et al., 2011) by genome sequencing. Values obtained with FIAD and genome sequencing are more similar. So, considering the loss of certain repetitive regions of DNA by the complete genome sequencing and the difficulties in using other techniques such as BCA and FIAD (mainly due to the low number of repetitions available to estimate de DNA amount) the use of FCM has proven to be the most efficient methodology to obtain accurately the total DNA content. Genome size evolution The reported DNA C–value of insects range from 0.07 pg (Clunio tsushimensis Tokunaga, 1933 – Diptera) to 16.93 pg (Podisma pedestris Linnaeus, 1758 – Orthop- tera) and out of 1344 estimates found, 1224 (91%) were comprised of values between 0.07 to 2.00 pg (Gregory, 2018). From 27 orders of insects, 24 currently have esti- mates of genome size, with Diptera accounting for the largest number of measurements (386 specimens, 29% of the total), followed by Coleoptera (278 specimens, 21% of the total) and Hymenoptera (240 specimens, 18% of the total). The average genome size for the Formicidae (Hymenoptera) was 0.36 pg (± 0.13), with values rang- ing from 0.18 pg (the smallest value, found in Dolicho- derinae and in Formicinae) to 0.77 pg in S. invicta (Myr- micinae) (Table 1; Figure 2), being always less than 1 pg. This is in accordance with the pattern already observed for others eukaryotes that most of the distribution of genome size is skewed towards smaller values (Oliver et al., 2007), since it is evident that the number of species declines as the genome doubles in size. As can be seen in Figure 2 the variation of genome size among species of a subfamily is similar to the varia- Figure 1. Distribution of the number of species across Formicidae subfamilies with published genome size estimates. The list of species is presented in Table 1. 35Genome size in ants Figure 2. Phylogeny of the extant Formicidae. Phylogenetic tree redrawn from Moreau and Bell (2013). The figure highlights the subfamilies containing species with estimated genome size. Aside of each terminal on the tree the genome size is shown in picograms (pg) of DNA and also the mean genome size per Formicidae subfamilies. 36 Mariana Neves Moura et al. tion found between subfamilies. Significant differences in genome size were observed between the subfamilies sam- pled (ANOVA, p-value < 0.01). Through contrast analy- sis, most of the subfamilies grouped statistically (group average = 0.34 pg, p-value > 0.05) except for Ponerinae, whose average was different from the others (average = 0.47, p-value < 0.01). The subfamilies Ectatomminae (Ectatomma tuberculatum (Olivier, 1792), 0.71 pg) and Myrmeciinae (Myrmecia varians Mayr, 1876, 0.28 pg) were not considered in the analysis because only one value for each was available, so it was not possible to calculate a mean for the comparison test (Figure 2). Dif- ferences in the genome size were also observed between genera within the sampled subfamilies and mainly between species of the same genus, as observed in Atta Fabricius, 1804 spp. (e.g. Atta cephalotes = 0.31 pg and Atta texana (Buckley, 1860) = 0.27 pg), Camponotus spp. (e.g. Camponotus floridanus = 0.23 pg and Camponotus pennsylvanicus (De Geer, 1773) = 0.33 pg) and Odontom- achus spp. (e.g. Odontomachus brunneus (Patton, 1894) = 0.33 pg and Odontomachus chelifer (Latreille, 1802) = 0.54 pg) (Table 1, Figure 2). These differences in genome size among closely related species have been associated in several studies with the amount of heterochromatin in the chromosomes (Lopes et al., 2009; Tavares et al., 2010; Cardoso et al., 2012), transposable elements (Kidwell, 2002; Vieira et al., 2002) and other repetitive genome sequences (Gregory and Hebert, 1999; Petrov, 2001). In some species, as Ectatomma tuberculatum and Apterostig- ma dentigerum Wheeler, 1925 the differences in genome size was correlated with whole genome duplication events given the large genome size of this both species when compared with the others of Formicidae (0.71 pg and 0.65 pg, respectively) (Tsutsui et al., 2008). The correlation between genome size and chromo- some number has been reported in some studies for ants, for example, Cardoso et al. (2012) within fungus– growing ants. In their study, they found a relationship between these two characteristics being Sericomyrmex amabilis Wheeler, 1925 the species with the highest number of chromosomes and also the largest genome size and other two species with the lowest number of chromosomes also had the smallest genome size. Cor- relation between chromosome and genome size has been reported for some insects. For instance, Ardila–Garcia and Gregory (2009) also found this positive correla- tion among species of damselflies, but not in dragon- f lies (Insecta: Odonata). Lack of correlation between genome size and chromosome number has been shown in the highly eusocial stingless bees of Meliponini tribe (Hymenoptera: Apidae) (Tavares et al., 2012). Yet, body size was correlated with genome size among dragonflies and damselflies (Ardila–Garcia and Gregory, 2009), but not among stingless bees (Tavares et al., 2010) or ants (Tsutsui et al., 2008). These contradictory observations remain the issue whether genome size is shaped by neu- tral or natural selection. It has been proven that changes in genome size are related to the addition and deletion of heterochromatin and that species with low amounts of heterochroma- tin also have lower DNA content per haploid nucleus, likewise the reverse is also true (Tavares et al., 2017). Although conclusion remarks still unlike due the limited availability of data and sampling representing more gen- era and species, important question could be addressed when more data became available. Considering the assembled data e evidences from other social insects, as bees, we propose that the differences in DNA content among ant species may also be related to the different amount of heterochromatin in the chromosomes. Nev- ertheless, we emphasize that this can only be confirmed after a detailed study of chromosomal structure and chromosome counts across genera and subfamilies. CONCLUSIONS AND PERSPECTIVES The compilation of the genome size data currently available in the literature for ants has highlighted the scarcity of estimates for this hyper–diverse family (with only 0.52% of known species having been estimated). Little is known about the methodologies employed and the lack of standardization of the works makes it problematic to compare the different estimates (Ardi- la–Garcia et al., 2010; Doležel and Greilhuber, 2010), especially regarding the buffer to isolate the nuclei, tis- sue and internal standard used. Also, the mechanisms involved in the evolution of the genome in ants are still unknown, especially those related to the total amount of heterochromatin in chromosomes and their relationship with genome size; the whole–genome duplication events, which could explain the large variation of the genome of some species, such as Ectatoma tuberculatum and Apter- ostigma dentigerum (Tsutsui et al., 2008); and polyploidy events as in Solenopsis invicta males (Glancey et al., 1976; Lorite and Palomeque, 2010). Our analysis high- light the importance and accuracy of the use of FCM to estimate the genome size of species and the possibility of obtaining robust results, since a large number of nuclei (10.000 or more per sample) are analyzed to determine the DNA content. Therefore, the standardization of the techniques used and a large–scale study of the ant genome size are urgently required, given the ecological and economic importance of this group contributing to 37Genome size in ants our knowledge on ant evolution by using another genetic diversity and independent dataset. CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ACKNOWLEDGMENTS This study was carried out as part of the PhD thesis of the first author. Author is grateful to CAPES for the scholarship. The authors thanks FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and UFOP (Universidade Federal de Ouro Preto) for their financial support. Author received a Research Pro- ductivity Fellowship from FAPEMIG (PPM–00126–15). REFERENCES Aguiar HJAC Barros LAC Soares FAF Carvalho CR Pom- polo SG (2016) Estimation of nuclear genome size of three species of Camponotus (Mayr 1861) (Hyme- noptera: Formicidae: Formicinae) and their cytoge- netic relationship. Sociobiology 63: 777–782. http:// dxdoiorg/1013102/sociobiologyv63i2948 Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2007) Molecular biology of the cell fifth ed. Gar- land Science New York. Alfsnes K, Leinaas HP, Hessen DO (2017) Genome size in arthropods, different roles of phylogeny habitat and life history in insects and crustaceans. Ecology and Evolution 7: 5939–5947. https://doi.org/10.1002/ ece3.3163 Ardila–Garcia AM, Gregory TR (2009) An exploration of genome size diversity in dragonflies and damselflies (Insecta: Odonata). Journal of Zoology 278: 163–173. https://doi.org/10.1111/j.1469-7998.2009.00557.x Ardila–Garcia AM, Umphrey GJ, Gregory TR (2010) An expansion of the genome size dataset for the insect order Hymenoptera with a first test of parasitism and eusociality as possible constraints. Insect Molecular Biology 19: 337–346. https://doi.org/10.1111/j.1365- 2583.2010.00992.x Basiji DA, Ortyn WE, Liang L, Venkatachalam V, Morris- sey P (2007) Cellular image analysis and imaging by flow cytometry. Clinics in Laboratory Medicine 27: 653–70. https://doi.org/10.1016/j.cll.2007.05.008 Bolton, B. 2018. An online catalog of the ants of the world. Available from http://antcat.org. [accessed 04 March 2018]. Bonasio R, Zhang G Ye C, Mutti NS, Fang X, Qin N, Donahue G, Yang P, Li Q, Li C, Zhang P, Huang Z, Berger SL, Reinberg D, Wang J, Liebig J (2010) Genomic comparison of the ants Camponotus flori- danus and Harpegnathos saltator. Science 329: 1068– 1071. https://doi.org/10.1126/science.1192428 Brandão CRF (1999) Família Formicidae in: Brandão CRF, Cancello EM (Eds) Invertebrados terrestres. ed. Biodiversidade do Estado de São Paulo: síntese do conhecimento ao final do século XX. (Joly CA Bicudo CEM (orgs)) FAPESP São Paulo pp 215– 223. Cardoso DC, Carvalho CR, Cristiano MP, Soares FAF, Tavares MG (2012) Estimation of nuclear genome size of the genus Mycetophylax Emery 1913: evi- dence of no whole–genome duplication in Neoat- tini. Comptes Rendus Biologies 335: 619–624. http:// dx.doi.org/10.1016/j.rvi.2012.09.012 Crawley MJ (2013) The R Book second ed. John Wiley & Sons Ltd. London. Doležel J, Bartoš J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany 95: 99–110. https://doi.org/10.1093/aob/mci005 Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry. Part A 51: 127–128. https://doi. org/10.1002/cyto.a.10013 Doležel J, Greilhuber J (2010) Nuclear genome size: are we getting closer? Cytometry. Part A 77A: 635–642. https://doi.org/10.1002/cyto.a.20915 Doležel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytom- etry. Nature Protocols 2 2233–2244. https://doi. org/10.1038/nprot.2007.310 Dufresne F, Jeffery N (2011) A guided tour of large genome size in animals: what we know and where we are heading. Chromosome Research 19: 925–938. https://doi.org/10.1007/s10577-011-9248-x Eddy SR (2012) The C–value paradox junk DNA and ENCODE. Current Biology 22: 898–899. https://doi. org/10.1016/j.cub.2012.10.002 Françoso ML, Brandão CRF (1993) Classificação superi- or dos Formicidae. Biotemas 6: 121–132. https://doi. org/10.5007/%25x Glancey BM, Romain MKS, Crozier RH (1976) Chromo- some numbers of the red and the black imported fire ants Solenopsis invicta and S. richteri. Annals of the Entomological Society of America 69: 469–470. htt- ps://doi.org/10.1093/aesa/69.3.469 38 Mariana Neves Moura et al. Goodisman MAD, Kovacs JL, Hunt BG (2008) Function- al genetics and genomics in ants (Hymenoptera: For- micidae): The interplay of genes and social life. Myr- mecological News 11: 107–117. Gregory TR (2001) Coincidence coevolution or causa- tion? DNA content cell size and the C–value enig- ma. Biological Reviews 76: 65–101. https://doi. org/10.1111/j.1469-185X.2000.tb00059.x Gregory TR (2005a) The C–value enigma in plants and animals: a review of parallels and an appeal for partnership. Annals of Botany 95: 133–146. https:// dx.doi.org/10.1093%2Faob%2Fmci009 Gregory TR (2005b) Genome Size Evolution in Animals in: Gregory TR (Ed) The Evolution of the Genome. Elsevier San Diego pp 3–87. Gregory TR (2018) Animal genome size database. Avail- able online: http://wwwgenomesizecom. [accessed 04 March 2018]. Gregory TR, Hebert PDN (1999) The modulation of DNA content: proximate causes and ultimate conse- quences. Genome Research 9: 317–324. https://doi. org/10.1101/gr.9.4.317 Hölldobler B, Wilson EO (1990) The ants. Harvard Uni- versity Press Cambridge. Johnston JS, Ross LD, Beani L, Hughes DP, Kathiritham- by J (2004) Tiny genomes and endoreduplication in Strepsiptera. Insect Molecular Biology 13: 581–585. https://doi.org/10.1111/j.0962-1075.2004.00514.x Kidwell MG (2002) Transposable elements and the evo- lution of genome size in eukaryotes. Genetica 115: 49–63. https://doi.org/10.1023/A:1016072014259 Klingenberg C, Brandão CRF (2009) Revision of the fun- gus–growing ant genera Mycetophylax Emery and Paramycetophylax Kusnezov rev stat and description of Kalathomyrmex n gen (Formicidae: Myrmicinae: Attini). Zootaxa 2052: 1–31. Klug WS, Cummings MR, Spencer CA, Palladino MA (2014) Concepts of genetics eleventh. Ed. Pearson. New Jersey Li J, Heinz KM (2000) Genome complexity and organi- zation in the red imported fire ant Solenopsis invicta Buren. Genetical Research 75: 129–135 Lopes DM, Carvalho CR, Clarindo WR, Praça MM, Tavares MG (2009) Genome size estimation of three stingless bee species (Hymenoptera: Meliponinae) by flow cytometry. Apidologie 40: 517–523. https://doi. org/10.1051/apido/2009030 Lorite P, Palomeque T (2010) Karyotype evolution in ants (Hymenoptera: Formicidae) with a review of the known ant chromosome numbers. Myrmecological News 13: 89–102. Merkel DE, Dressler LG, McGuire WL (1987) Flow cytometry cellular DNA content and progno- sis in human malignancy. Journal of Clinical Oncology 5: 1690–1703. https://doi.org/10.1200/ JCO.1987.5.10.1690 Moore GP (1984) The C–value paradox. BioScience 34: 425–429. https://doi.org/10.2307/1309631 Moreau CS, Bell CD (2013) Testing the museum ver- sus cradle biological diversity hypothesis: Phylog- eny diversification and ancestral biogeographic range evolution of the ants. Evolution 67: 2240–2257. htt- ps://doi.org/10.1111/evo.12105 Nygaard S, Zhang G, Schiøtt M, Li C, Wurm Y, Hu H, Zhou J, Ji L, Qiu F, Rasmussen M, Pan H, Haus- er F, Krogh A, Grimmelikhuijzen CJP, Wang J, Boomsma JJ (2011) The genome of the leaf–cut- ting ant Acromyrmex echinatior suggests key adap- tations to advanced social life and fungus farming. Genome Research 21: 1339–1348. https://dx.doi. org/10.1101%2Fgr.121392.111 Oliver MJ, Petrov D, Ackerly D, Falkowski P, Schofield OM (2007) The mode and tempo of genome size evolution in eukaryotes. Genome Research 17: 594– 601. https://dx.doi.org/10.1101%2Fgr.6096207 Pellicer J, Hidalgo O, Dodsworth S, Leitch IJ (2018) Genome Size Diversity and Its Impact on the Evo- lution of Land Plants. Genes 9: 88. https://doi. org/10.3390/genes9020088 Petrov DA (2001) Evolution of genome size: new approaches to an old problem. Trends in Genet- ics 17: 23–28. https://doi.org/10.1016/S0168- 9525(00)02157-0 Price HJ, Hodnett G, Johnston S (2000) Sunflower (Heli- anthus annus) leaves contain compounds that reduce nuclear propidium iodide fluorescence. Annals of Botany 86: 929–934. https://doi.org/10.1006/ anbo.2000.1255 R Core Team (2013) R Foundation for statistical com- puting. R: a language and environment for statisti- cal computing. http://wwwR–projectorg [accessed 15 May 2017]. Sirviö A, Gadau J, Rueppell O, Lamatsch D, Boomsma JJ, Pamilo P, Page RE Jr (2006) High recombination fre- quency creates genotypic diversity in colonies of the leaf–cutting ant Acromyrmex echinatior. Journal of Evolutionary Biology 19: 1475–1485. Smith CD, Zimin A, Holt C, Abouheif E, Benton R, Cash E, Croset V, Currie CR, Elhaik E, Elsik CG, Fave M-J, Fernandes V, Gadau J, Gibson JD, Graur D, Grubbs KJ, Hagen DE, Helmkampf M, Holley JA, Hu H, Vin- iegra ASI, Johnson BR, Johnson RM, Khila A, Kim JW, Laird J, Mathis KA, Moeller JA, Muñoz-Torres MC, Murphy MC, Nakamura R, Nigam S, Overson 39Genome size in ants RP, Placek JE, Rajakumar R, Reese JT, Robertson HM, Smith CR, Suarez AV, Suen G, Suhr EL, Tao S, Torres CW, Wilgenburg EV, Viljakainen L, Walden KKO, Wild AL, Yandell M, Yorke JA Tsutsui ND (2011) Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile). Pro- ceedings of the National Academy of Sciences of the United States of America 108: 5673–5678. https://doi. org/10.1073/pnas.1008617108 Smith CR, Smith CD, Robertson HM, Helmkampf M, Zimin A, Yandell M, Holt C, Hu H, Abouheif E, Benton R, Cash E, Croset V, Currie CR, Elhaik E, Elsik CG, Favé M-J, Fernandes V, Gibson JD, Graur D, Gronenberg W, Grubbs KJ, Hagen DE, Viniegra ASI, Johnson BR, Johnson RM, Khila A, Kim JW, Mathis KA, Munoz-Torres MC, Murphy MC, Mus- tard JA, Nakamura R, Niehuis O, Nigam S, Overson RP, Placek JE, Rajakumar R, Reese JT, Suen G, Tao S, Torres CW, Tsutsui ND, Viljakainen L, Wolschin F, Gadau J (2011) Draft genome of the red harvest- er ant Pogonomyrmex barbatus. Proceedings of the National Academy of Sciences of the United States of America 108: 5667–5672. https://doi.org/10.1073/ pnas.1007901108 Suen G, Teiling C, Li L, Holt C, Abouheif E, Bornberg– Bauer E, Bouffard P, Caldera EJ, Cash E, Cavanaugh A, Denas O, Elhaik E, Favé MJ, Gadau J, Gibson JD, Graur D, Grubbs KJ, Hagen DE, Harkins TT, Helm- kampf M, Hu H, Johnson BR, Kim J, Marsh SE, Moe- ller JA, Muñoz–Torres MC, Murphy MC, Naughton MC, Nigam S, Overson R, Rajakumar R, Reese JT, Scott JJ, Smith CR, Tao S, Tsutsui ND, Viljakainen L, Wissler L, Yandell MD, Zimmer F, Taylor J, Slater SC, Clifton SW, Warren WC, Elsik CG, Smith CD, Weinstock GM, Gerardo NM, Currie CR (2011) The genome sequence of the leaf–cutter ant Atta cephalotes reveals insights into its obligate symbi- otic lifestyle. PLoS Genetics 7: e1002007. https://doi. org/10.1371/journal.pgen.1002007 Tavares MG, Carvalho CR, Soares FAF (2010) Genome size variation in Melipona species (Hymenoptera: Apidae) and sub–grouping by their DNA content. Apidologie 41: 636–642. https://doi.org/10.1051/api- do/20010023 Tavares MG, Carvalho CR, Soares FAF, Campos LAO (2012) Genome size diversity in stingless bees (Hymenoptera: Apidae, Meliponini). Apidologie 43: 731–736. https://doi.org/10.1007/s13592-012-0145-x Tavares MG, Lopes DM, Campos LAO (2017) An over- view of cytogenetics of the tribe Meliponini (Hyme- noptera: Apidae). Genetica 145 241–258. https://doi. org/10.1007/s10709-017-9961-2 Torresan F, Zanella L, Mattarozzi A, Quiroga A, Bacchini P, Bertoni F, Gandolfi L (1994) DNA analysis with flow cytometry and image cytometry in colorectal polyps. Surgical Endoscopy 8: 1412–1416. Tsutsui ND, Suarez AV, Spagna JC, Johnston JS (2008) The evolution of genome size in ants. BMC Evolu- tionary Biology 8: 64. https://doi.org/10.1186/1471- 2148-8-64 Vieira C, Nardon C, Arpin C, Lepetit D, Biémont C (2002) Evolution of genome size in Drosophila Is the invader’s genome being invaded by transposable ele- ments? Molecular Biology and Evolution 19: 1154– 1161. https://doi.org/10.1093/oxfordjournals.molbev. a004173 Wurm Y, Wang J, Riba–Grognuz O, Corona M, Nygaard S, Hunt BG, Ingram KK, Falquet L, Nip- itwattanaphon M, Gotzek D, Dijkstra MB, Oettler J, Comtesse F, Shih CJ, Wu WJ, Yang CC, Thomas J, Beaudoing E, Pradervand S, Flegel V, Cook ED, Fab- bretti R, Stockinger H, Long L, Farmerie WG, Oakey J, Boomsma JJ, Pamilo P, Yi SV, Heinze J, Goodis- man MAD, Farinelli L, Harshman K, Hulo N, Cerutti L, Xenarios I, Shoemaker DW, Keller L. (2011) The genome of the fire ant Solenopsis invicta. Proceedings of the National Academy of Sciences of the United States of America 108: 5679–5684. 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