Biota Neotropica 20(1): e20190782, 2020 www.scielo.br/bn ISSN 1676-0611 (online edition) Article Accessing the subterranean ant fauna (Hymenoptera: Formicidae) in native and modified subtropical landscapes in the Neotropics Mila Ferraz de Oliveira Martins*1 , Marcílio José Thomazini2, Dilmar Baretta3, George Gardner Brown2, Marcio Gonçalves da Rosa3 , Mauricio Rumenos Guidetti Zagatto5, Alessandra Santos2, Herlon Sérgio Nadolny2, Guilherme Borges Xarão Cardoso2, Cintia Carla Niva2,6, Marie Luise Carolina Bartz4 & Rodrigo Machado Feitosa1 1 Universidade Federal do Paraná, Avenida Francisco Heráclito dos Santos, s/n, Centro Politécnico, CEP: 81531980, Curitiba, PR, Brasil 2 Empresa Brasileira de Pesquisa Agropecuária, Estrada da Ribeira, km. 111, Guaraituba, CEP: 83411000, Curitiba, PR, Brasil 3 Universidade do Estado de Santa Catarina, Centro de Educação Superior do Oeste, Rua Beloni Trombeta Zanin, 680, Santo Antônio, CEP: 89815630, Chapecó, SC, Brasil 4 Universidade Positivo, Rua Professor Pedro Viriato Parigot de Souza 3841/3842, Cidade Industrial, CEP: 81280330, Curitiba, PR, Brasil 5 Escola Superior de Agricultura Luiz de Queiroz, Piracicaba, SP, Brasil 6 Empresa Brasileira de Pesquisa Agropecuária - Embrapa Cerrados Rodovia BR 020 Km18, Brasília, DF, Brasil *Corresponding author: Mila Ferraz de Oliveira Martins, e-mail: milafomartins@gmail.com MARTINS, M.F.O., THOMAZINI, M.J., BARETTA, D., BROWN, G.G., ROSA, M.G., ZAGATTO, M.R.G., SANTOS, A., NALDONY, H.S., CARDOSO, G. B.X., NIVA, C.C., BARTZ, M.L.C., FEITOSA, R.M. Accessing the subterranean ant fauna (Hymenoptera: Formicidae) in native and modified subtropical landscapes in the Neotropics. Biota Neotropica. 20(1): e20190782. http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Abstract: Several studies have characterized and delimited subterranean ant assemblages. Soil extraction, one of the methods employed to access this fauna, employs the removal of monoliths. One of the most widely used methods for the extraction of soil sampling is called TSBF (developed by the Tropical Soil Biology and Fertility Programme). This method provides relevant data about the species associated with the soil. In the present study we characterized assemblages of subterranean ants using the TSBF method in different subtropical areas of the Neotropics. We considered two sampling designs in different localities. The first design resulted in 315 TSBF samples obtained from layers at distinct depths. The second design resulted in 270 TSBF samples and 270 epigaeic pitfall trap samples. This material was used to delimit the species that occur exclusively in the subterranean stratum (TSBF) and that are not found on the soil surface. A total of 281 species were recorded. Of these, 57 can be considered subterranean, based on their occurrence in subterranean strata. We also verified that the highest occurrence of ants was in the first 10 cm of soil depth. Due to the importance of using methods that efficiently extract the subterranean ant fauna in studies, we suggest the TSBF method should be used to sample ants or to associate this method with epigaeic pitfall traps to delimit strictly subterranean assemblages in specific community stratification studies. Keywords: Soil, hypogaeic, Brazil, monoliths, pitfall. Avaliação da mirmecofauna subterrânea (Hymenoptera: Formicidae) em paisagens nativas e modificadas na região subtropical do Neotrópico Resumo: Vários estudos têm caracterizado e delimitado assembleias de formigas subterrâneas. A extração de solo, um dos métodos utilizados para amostragem dessa fala, implica na remoção de monólitos. Um dos métodos mais utilizados na extração de monólitos é chamado TSBF (desenvolvido pelo Programa Tropical Soil Biology and Fertility). Esse método fornece dados extremamente relevantes sobre as espécies associadas ao solo. No presente estudo, nós caracterizamos as assembleias de formigas subterrâneas usando o método TSBF em diferentes áreas subtropicais do Neotrópico. Utilizamos dois delineamentos amostrais distintos. O primeiro delineamento resultou em 315 amostras de TSBF obtidas a partir da estratificação do solo. O segundo resultou em 270 amostras de TSBF mais 270 provenientes de armadilhas pitfall epigeicas. Esse material foi utilizado para delimitar espécies que ocorrem exclusivamente no estrato subterrâneo (TSBF) e não são encontradas na superfície do solo. Registramos um total de 281 espécies. Destas, 57 podem ser consideradas subterrâneas (baseado na ocorrência no respectivo estrato). Também verificamos que a maior ocorrência de formigas se deu nos primeiros 10 cm de profundidade. Devido à importância do uso de métodos que coletem eficientemente formigas subterrâneas, sugerimos o uso do método TSBF em estudos de estratificação de comunidades ou sua associação a armadilhas do tipo pitfall epigeica para delimitar assembleias estritamente subterrâneas. Palavras-chave: Solo, hipogeicas, Brasil, monólitos, pitfall. http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 2 Martins, M.F.O. et al. Introduction Ants are conspicuous organisms present in most terrestrial environments (Folgarait 1998), from the arboreal to the subterranean strata (Lucky et al. 2013, Jacquemin et al. 2016). However, little is known about underground ant assemblages (Ryder Wilkie et al. 2007, Andersen & Brault 2010, Jacquemin et al. 2012), mainly because there are few efficient and specific methods for collecting this important fauna (Wong & Guénard 2017). Underground ants are the last biodiversity frontier to be explored in Formicidae (Rider Wilkie et al. 2007, Andersen & Brault 2010), and are also important for the understanding of the origin and evolution of Formicidae (Lucky et al. 2013). In addition, subterranean ants are considered bioturbators sensu strictu (Bottinelli et al. 2015), and because they respond to drastically changes in soil density, they can serve as bioindicators (Schmidt et al. 2013). Aiming to develop an efficient sampling method for subterranean ants, researchers have been testing useful sampling techniques that can be replicated. Wong & Guénard (2017) compiled the published research on the collecting methods of subterranean ants and classified them into three types: (1) subterranean baits, (2) soil sampling, and (3) direct sampling. Most of those publications (65%) described the use of baits or other attractive items (Wong & Guénard 2017), which favor the capture of predatory, generalist and mass-recruiting species (Longino 2000, Bestelmeyer et al. 2000, Schmidt & Solar 2010, Wong & Guénard 2017). Amongst the methods classified as “soil sampling” is the extraction of monoliths (Delabie & Fowler 1995, Fowler et al. 2000, Andersen & Brault 2010, Wong & Guénard 2017). The use of monoliths is promising because it enables us to determine the vertical stratification of species in the soil, among other advantages (see: Jacquemin et al. 2012, Jacquemin et al. 2016, Wong & Guénard 2017). A widely used technique of monolith extraction in soil macrofauna studies was proposed by the Tropical Soil Biology and Fertility Program (TSBF) (Anderson & Ingram 1993) and was standardized by the International Organization for Standardization (ISO) in the ISO norm 23611-5:2011. This technique, known as TSBF method, generates a substantial volume of ant specimens. It consists of the removal of soil monoliths that are 25 x 25 cm and are taken 20 or 30 cm deep in the soil, followed by hand sorting extraction of the associated fauna (Bartz et al. 2014a, Rosa et al. 2015, ISO 2018). Despite the massive volume of material that has been sampled using the TSBF method, published information on the ecological dynamics of the ants collected using it is insufficient. This is a function of the fact that Pedobiology studies (which employ the TSBF method with high frequencies) traditionally identify the collected material at high taxonomic levels (like Order, Family and Genera) and therefore do not give information on community composition and species ecology (see: Lourente et al. 2007, Klenk et al. 2009, Correia 2010, Lima et al. 2010, Rousseau et al 2010, Zagatto 2014, Benazzi et al. 2013, Pereira et al. 2012, Gutiérrez et al. 2017). The present work characterizes subterranean ant assemblages in three subtropical regions of the Neotropics using two sampling methods: TSBF and epigaeic pitfall traps. In situ extraction methods, like monoliths, can help to detect ecological, behavioral and morphological patterns, since the specimens collected using these methods are not attracted from other strata (from the surface, for example). Furthermore, http://www.scielo.br/bn TSBF is widely disseminated in studies that evaluate the impact of land use systems on the underground soil (micro, meso and macro fauna) (Lavelle et al 1997, Bartz et al 2014a, Rosa et al. 2015). In order to improve the scientific contribution of this study, we characterize the ant assemblages and compare the communities at the species level. Material and Methods 1. Study area The specimens came from three localities in the Southern Region of Brazil: (1) Ponta Grossa, Campos Gerais region, state of Paraná; (2) Western and (3) Plateau, regions of the state of Santa Catarina. In each region of Santa Catarina, the samples were collected from three municipalities: Xanxerê, Chapecó and São Miguel do Oeste in the West and Lages, Otacílio Costa and Campo Belo do Sul in the Plateau (Figure 1). Ponta Grossa is in the Second Plateau of Paraná, about 120 kilometers from the state capital, Curitiba. According to Maack (2012), the region is characterized by open grasslands with Araucaria angustifolia (Bertol.) Kuntze 1898 patches, within the Atlantic Forest domain. Climate is type Cfb (climatic classification of Köppen-Geiger) -subtropical humid, without defined dry periods throughout the year and with the average temperature of the warmest month lower than 22 °C. The sampled areas have mean elevation of 875 m and total annual rainfall between 1.300 and 1.800 mm throughout the year (IAPAR, 1978). Seven land use systems (LUSs) were sampled: (1) Integrated Crop-livestock (ICL); (2) Integrated Crop-livestock-forestry (ICLF); (3) Grazed native field (GN); (4) Eucalyptus sp. plantation (EP); (5) no-tillage (NT); (6) Campos Gerais (CG), and (7) Mixed Ombrophilous forest (F). The native areas, CG and F, were at Vila Velha State Park (25°14’17” S 50°0’39” W). The land use systems ICL, ICLF, and GN were in the Model Farm of the Instituto Agronômico do Paraná (IAPAR) (25° 5’ 11” S 50° 9’ 38” W) and the EU and NT systems were at the Experimental Field of the Empresa Brasileira de Pesquisas Agropecuárias (Embrapa) (25°08’17” S 50°04’47” W). The western region of Santa Catarina is characterized by humid subtropical Cfa climate, with no defined dry season, and the average temperature during the warmest month is above 22 °C (Bartz et al. 2014a). The native forest in the Western region of the state is a transition between mixed ombrophilous and semi-deciduous forest (Bartz et al. 2014b). In the Plateau region the climate is classified as Cfb (as described for Ponta Grossa), and native grasslands with mixed ombrophilous forest patches predominate (Rosa 2013). The municipalities of each region of Santa Catarina were chosen based on their geographic characteristics, soil type, LUSs, and management history. Five LUSs were sampled in each of the three municipalities: (1) Native forest (FN); (2) Eucalyptus sp. plantation (EP); (3) Pasture (PA); (4) No-tillage (NT) and (5) Integrated Crop-livestock integration (ICL) (for details on the location and characterization of the areas see: Rosa 2013, Bartz et al. 2014a, b, Rosa et al. 2015). All areas were part of a study of soil quality in different land use systems developed by the Embrapa Forestry (Colombo, PR), and Santa Catarina SisBiota Project, led by the Universidade Estadual de Santa Catarina (UDESC) at Chapecó. In these projects, soil fauna was evaluated as an indicator of soil quality in different LUS. http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 3 Biota Neotrop., 20(1): e20190782, 2020 Subterranean ants of southern Brazil Figure 1. Map of the sampled areas. 2. Sampling design The Tropical Soil Biology and Fertility Program organizes measures to monitor soil quality through the removal of monoliths in places where financial and technological resources are limited. The soil monoliths were 25 x 25 cm, and the depth at which they were collected was variable according to the extent of horizon A of the soil (Anderson & Ingram 1993, ISO 2018). This method can be applied to any soil type. The area for the removal of monoliths is delimited by a metallic quadrangular structure, followed by the excavation and removal of soil layers. Each layer is packed separately, and the associated fauna can be extracted either in the field or in the laboratory, with or whitout assistance of a stereomicroscope (Anderson & Ingram 1993). In the present study, all extractions were performed in the laboratory, without stereomicroscope. Our extractions, in Ponta Grossa, were made in layers up to 20 cm, delimited in three strata: 0 - litter; I – 0 to 10 cm and II – 10 to 20 cm. In Santa Catarina, extractions were not stratified. We used material from two different sample designs, according to the research group involved in the sampling. In Paraná, samples were collected on four different dates. In the areas of ICLF, ICL, NF, NT, and EU, samples were taken in October and November 2012 and in April and May 2013. In the Vila Velha State Park, samples were taken in September 2013 and in January 2014. All areas of Santa Catarina (West and Plateau) were sampled in the course of two seasons: winter (July and August 2011) and summer (December 2011 and January 2012). http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 In Paraná, for each area (seven LUSs) three plots of 50 x 100 m (0.5 hectares) were delimited per LUS, within which five monoliths were extracted, separated by at least 200 m. These samples were distributed in a central transect, 20 meters from the edge of the plot. A zigzag pattern was drawn with samples always at least 20 m apart and following the slope of the plot (Zagatto, 2014) (Figure 2). A total of 105 monoliths (seven LUSs x three plots x five monoliths) were collected, divided into the depth layers mentioned above (0, I and II), totalling 315 sample units per season of the year (630 total samples). In Santa Catarina, one area of each LUS (NF, EU, PA, ICL, and NT) was evaluated in each of the three municipalities per region (West and Plateau). Sampling grids, with nine points mounted in three lines of three points each, were sampled 20 m from the border and 30 m apart in each LUS, totaling one hectare. At each point a monolith was extracted and an epigaeic pitfall trap was installed (one meter distant from the monolith) (Figure 3). Each pitfall trap consisted of a 200 ml capacity recipient, half filled with water and detergent, buried with the opening at ground level and left in the field for 72 hours. A total of 270 samples of TSBF and 270 of epigaeic pitfalls (three municipalities x five LUSs x nine monoliths x two regions) were obtained per season (1080 total samples). The ants from all areas were conditioned in 80% alcohol and sent to the Laboratório de Sistemática e Biologia de Formigas of the Universidade Federal do Paraná (UFPR). The material was processed http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 4 Martins, M.F.O. et al. Figure 2. Schematic drawing of the sampling design in the state of Paraná. Adapted from Zagatto (2014). and identified to the specific level whenever possible. The identifications were generally based on the Guide for the Ant Genera of Brazil (Baccaro et al., 2015). In addition, UFPR specialists (Alexandre C. Ferreira, Gabriela P. Camacho, John E. Latke, Mayron Escárraga, Rodrigo M. Feitosa, and Thiago S. R. da Silva) were consulted to confirm species identifications. Finally, whenever necessary, we performed comparisons with the material deposited in the Coleção Entomológica Padre Jesus Santiago Moure at UFPR (DZUP), where voucher material was deposited. The species that were not possible to name were identified as morphospecies. 3. Data analysis Due to differences in the sampling design, the data were organized in two separate matrices that indicate presence and absence: one matrix with data from Paraná and the other with data from Santa Catarina. Since samples were taken at different seasons to increase the likelihood of finding different species, each species in a sampling unit was considered only once, regardless of the season when it was collected, as suggested by Brandão et al (2011). The observed richness (Sobs) obtained by each method and stratum were compared with the richness estimated by Jackniffe1 and Bootstrap. These estimators were chosen because they provide a satisfactory balance in the analyses, since they separately consider the effect of species occurring in a single sample (Jackniffe1) and of all species in the samples, without overweighting the presence of rare species (Bootstrap). To determine whether a species is epigaeic or hypogaeic, we used the exclusivity criterion: species collected only by the TSBF method or those present only in strata I and II were considered subterranean; conversely, the species collected only by pitfall traps or http://www.scielo.br/bn Figure 3. Schematic drawing of the sampling design in the state of Santa Catarina. Adapted from Bartz et al. (2014), and Rosa et al. (2015). those present only in stratum 0 of the TSBF method were considered epigaeic. We used the samples from Paraná to evaluate the sampling efficiency of the TSBF method, since only these samples were stratified (layer separation by depth). The stratification allowed us to evaluate the presence of ants in the different strata, providing data not only on ant richness but also on aspects related to their biology (stratum of occurrence). A generalized linear mixed model (GLMM) was used to verify if the presence of ants in strata was homogeneous (providing an equal probability of collection success between the layers). The model was elaborated from the proportion of ant occurrence in the plots (ranging from 0 to 5, in a half-hectare), with the strata as fixed variables and the LUSs as random variables. The model was compared by maximum likelihood and the data were evaluated for overdispersion, assuming a binomial distribution. The analyses and graphs were performed in the R environment (version 3.1.3, R-CORE-TEAM 2016) with the vegan, car, MASS, iNEXT and lme4 packages. The map of the localities was generated using Program QGis v. 2.16.3 and the coordinates provided in Zagatto (2014) and Rosa (2013) and the shape files obtained from the Brazilian Institute of Geography and Statistics data base - IBGE (2016). The figures were drawn in Adobe Illustrator (version CC 2018). http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Biota Neotrop., 20(1): e20190782, 2020 5 Subterranean ants of southern Brazil Results In Santa Catarina, 238 species were collected by both methods (epigaeic pitfall and TSBF). Pitfalls collected 202 species and generated 1,585 records of occurrence, while the TSBF collected 149 species, generating 769 records (Table 2). From the total, 89 species were collected only in pitfall (51.7% of them composed by the genus Pheidole) and 36 only in the TSBF (Table 1). The three most frequent species in the pitfalls were Pachycondyla striata Smith, 1858, Pheidole subarmata Mayr, 1884 and Linepithema micans (Forel, 1908), with 109, 97 and 71 occurrences, respectively. In TSBF the species with the greatest number of records were Hypoponera sp. 1, Hypoponera sp. 8 and Pheidole subarmata, with 60, 57 and 42 records, respectively. 1. Richness and species frequency In total, 281 ant species were collected (considering all counties sampled and the different methods – epigaeic pitfall trap and TSBF). These species were distributed in 49 genera and 10 subfamilies (Table 1). The subfamilies with the greatest number of species were Myrmicinae, with 175 (61.4% of the species collected); Formicinae, 39 (13.7%); and Ponerinae, with 32 species (11.2%). The richest genera were Pheidole, with 98 species (34%), Solenopsis and Hypoponera with 22 species (7.7%) each. Table 1. Species list by region (Campos Gerais, West and Plateau) and municipality. PG - Ponta Grossa, SMO - São Miguel do Oeste, CHP - Chapecó, XAN - Xanxerê, LGS - Lages, OTC - Otacílio Costa, and CBS - Campo Belo do Sul). The species sampled exclusively by a method (in Santa Catarina) are indicated by symbols: ♦ - TSBF, ♣ - Pitfall; and the exclusive species per stratum are indicated with superscript numbers: 0 - litter, I - stratum I (0-10 cm), and II - stratum II (10-20 cm). Paraná Species Santa Catarina Campos Gerais PG SMO Plateau West CHP XAN LGS OTC CBS Amblyoponinae Fulakora armigera (Mayr, 1887)♦ X Fulakora elongata (Santschi, 1912)♦ I X X X X X X X Dolichoderinae Dorymyrmex brunneus Forel, 1908 Dorymyrmex sp. 1♣ X X X Dorymyrmex sp. 2♣ X Linepithema gallardoi (Brèthes, 1914)♣ Linepithema humile (Mayr, 1868)♣ X X X Linepithema iniquum (Mayr, 1870) X X X Linepithema leucomelas (Emery, 1894)♣ X Linepithema micans (Forel, 1908) X Linepithema pulex Wild, 2007I X X X X X X X Dorylinae Labidus coecus (Latreille, 1802) X X Labidus praedator (Smith, 1858)♣ Neivamyrmex sp. 1♦ X II X Neocerapachys splendens (Borgmeier, 1957)♦ X X X Sphinctomyrmex stali Mayr, 1866♦ X X X Ectatomminae Ectatomma edentatum Roger, 1863♣ Ectatomma permagnum Forel, 1908I X X X Gnamptogenys rastrata (Mayr, 1866)♣ X Gnamptogenys regularis Mayr, 1870 X Gnamptogenys nr. striatula X X X X X X X X X X Gnamptogenys sp. n. A♦ X X Gnamptogenys striatula Mayr, 1884I Gnamptogenys sulcata (Smith, 1858)♣ Typhlomyrmex major Santschi, 1923♦ I Typhlomyrmex pusillus Emery, 1894 http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 X X X X X X X X X X X X X http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 6 Martins, M.F.O. et al. Continuation Table 1. Paraná Species Santa Catarina Campos Gerais PG SMO Plateau West CHP XAN LGS OTC CBS Formicinae Acropyga decedens (Mayr, 1887)♦ X Acropyga goeldii Forel, 1893 X I X Brachymyrmex pilipes Mayr, 1887♣ X Brachymyrmex coactus Mayr, 1887 Brachymyrmex pilipes gr. sp. 1♦ X X X X X Brachymyrmex sp. 1 X X X X X X Brachymyrmex sp. 2 X X X X X X X Brachymyrmex sp. 3 X X X X X X X Brachymyrmex sp. 4 X Brachymyrmex sp. 5 X X X X X Brachymyrmex sp. 6 X X X X Brachymyrmex sp. 7 X X X Brachymyrmex sp. 8♣ X X Brachymyrmex sp. 9 X Brachymyrmex sp. 10♣ X X X Camponotus balzani Emery, 1894 Camponotus blandus (Smith, 1858) Camponotus brasiliensis Mayr, 1862I X Camponotus crassus Mayr, 1862 X X X Camponotus melanoticus Emery, 1894♣I X X X Camponotus novogranadensis Mayr, 1870 X X X X X X X X X X X II X Camponotus renggeri Emery, 1894 Camponotus rufipes (Fabricius, 1775) X Camponotus sexguttatus (Fabricius, 1793) II X X X X X X X Camponotus sp. 1 X Camponotus sp. 2 X X X X X Camponotus sp. 4♣ X Camponotus sp. 5♣ X Camponotus sp. 6♣ X Myrmelachista catharinae Mayr, 1887♣ X Myrmelachista nodigera Mayr, 1887♣ I X X Nylanderia fulva (Mayr, 1862)I X X X X Nylanderia sp. 1 X X X X X X Nylanderia sp. 2 http://www.scielo.br/bn X X Camponotus zenon Forel, 1912♣ Myrmelachista gallicola Mayr, 1887 X X X Camponotus punctulatus Mayr, 1868 I X X X Camponotus nr. cingulatus♣ Nylanderia sp. 3♦ X X X X X http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Biota Neotrop., 20(1): e20190782, 2020 7 Subterranean ants of southern Brazil Continuation Table 1. Paraná Species Santa Catarina Campos Gerais PG SMO Plateau West CHP XAN LGS OTC CBS X X X Heteroponerinae Heteroponera dentinodis (Mayr, 1887) Heteroponera mayri Kempf, 1962 X Heteroponera microps Borgmeier, 1957♣ X X X Myrmicinae Acanthognathus ocellatus Mayr, 1887♦ X Acromyrmex aspersus (Smith, 1858)♣ Acromyrmex crassispinus (Forel, 1909) X I X X X X X Acromyrmex landolti (Forel, 1885)♣ X Acromyrmex lundii (Guérin-Méneville, 1838)♣ X Acromyrmex subterraneus (Forel, 1893)♣ X Apterostigma sp. 1 X Apterostigma sp. 2♣ Atta sexdens (Linnaeus, 1758) X Carebara sp. 1♦ X X X X X X X X X X X Carebara sp. 2♣ Carebara brasiliana Fernández, 2004♣ X X X X X Crematogaster ampla Forel, 1912♣ Crematogaster bruchi Forel, 1912 X Crematogaster corticicola Mayr, 1887♣ X X X Crematogaster quadriformis Roger, 1863 X Crematogaster sp. 1 X X Crematogaster sp. 2 X Cyphomyrmex rimosus (Spinola, 1851) Cyphomyrmex sp. 1 X X X X X X X X X X X X X X X Cyphomyrmex sp. 2 Cyphomyrmex sp. 3♣ X Hylomyrma balzani (Emery, 1894) I Hylomyrma reitteri (Mayr, 1887) X X X Megalomyrmex pusillus Forel, 1912 X Mycetophylax asper (Mayr, 1887)♣ X Mycetophylax lectus (Forel, 1911) X Mycetophylax olitor Forel, 1893 X Mycetophylax plaumanni Kempf, 1962 Mycocepurus goeldii (Forel, 1893) X X X Mycocepurus smithii (Forel, 1893) X X X Octostruma rugifera (Mayr, 1887) X Octostruma stenognatha (Brown & Kempf, 1960)♣ X X Oxyepoecus plaumanni Kempf, 1974I X Oxyepoecus reticulatus Kempf, 1974 X http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 X X X X X X Ochetomyrmex semipolitus Mayr, 1878♣ Oxyepoecus rosai Albuquerque & Brandão, 2009 X X X X X X X X X X X X http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 8 Martins, M.F.O. et al. Continuation Table 1. Paraná Species Pheidole aberrans Mayr, 1868 Santa Catarina Campos Gerais Plateau West PG SMO CHP XAN LGS OTC CBS X X X X X X X X X X X X X X X X X X X X X X X X X X X Pheidole acutidens (Santschi, 1922)♣ X Pheidole breviseta Santschi, 1919 Pheidole cavifrons Emery, 1906 X Pheidole fallax gr. sp. 10 X Pheidole gertrudae Forel, 18860 X Pheidole heyeri Forel, 1899 Pheidole oxyops Forel, 1908 X X X X X X x Pheidole nr. rosula Pheidole radoszkowskii Mayr, 1884I X Pheidole rosae Forel, 1901 X Pheidole subarmata Mayr, 1884 X X Pheidole transversostriata Mayr, 1887 X Pheidole triconstricta Forel, 1886 X Pheidole vafra Santschi, 1923 X X X X X Pheidole sp. n. A X Pheidole sp. n. II X Pheidole sp. n. NI X Pheidole sp. 1 X Pheidole sp. 2♦I X Pheidole sp. 3I X Pheidole sp. 4 X Pheidole sp. 5I X Pheidole sp. 6 X Pheidole sp. 7 X Pheidole sp. 8 X Pheidole sp. 9 X Pheidole sp. 10 X X Pheidole sp. 11 X Pheidole sp. 12 X Pheidole sp. 13 Pheidole sp. 14♣ X Pheidole sp. 15♣ X X Pheidole sp. 16♣ X X Pheidole sp. 17♣ X X Pheidole sp. 20 Pheidole sp. 21 X X X X X X X Pheidole sp. 18 Pheidole sp. 19 X X X X X X X X X X X X X X X X X X X Pheidole sp. 22♦ X X Pheidole sp. 23♣ X Pheidole sp. 24♣ X Pheidole sp. 25♣ X http://www.scielo.br/bn X X X X http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Biota Neotrop., 20(1): e20190782, 2020 9 Subterranean ants of southern Brazil Continuation Table 1. Paraná Species Santa Catarina Campos Gerais PG SMO CHP Pheidole sp. 26 X X Pheidole sp. 27 X Pheidole sp. 28♣ Pheidole sp. 29♣ XAN X X X X X X X X X Pheidole sp. 35♣ Pheidole sp. 36♣ X X X X X X X Pheidole sp. 38♣ Pheidole sp. 39♣ X Pheidole sp. 40♣ X Pheidole sp. 41 X X X X X X X X Pheidole sp. 44♣ X X Pheidole sp. 45♣ X X X X X X X X X Pheidole sp. 43♣ Pheidole sp. 46♣ X Pheidole sp. 48♣ X X X X X X X X X X X X X X X X X Pheidole sp. 50 X X Pheidole sp. 51 X X Pheidole sp. 52 Pheidole sp. 53♣ Pheidole sp. 54♣ X X X Pheidole sp. 57 X Pheidole sp. 58♦ X Pheidole sp. 59♣ X Pheidole sp. 60♣ X Pheidole sp. 61 X X Pheidole sp. 63♣ X X X X X X X Pheidole sp. 64♣ X Pheidole sp. 65♣ X Pheidole sp. 66♣ http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 X X Pheidole sp. 56♣ Pheidole sp. 68 X X Pheidole sp. 55♣ Pheidole sp. 67♣ X X Pheidole sp. 37♣ Pheidole sp. 62 X X Pheidole sp. 34 Pheidole sp. 49♣ CBS X X X Pheidole sp. 33 Pheidole sp. 47♣ OTC X Pheidole sp. 32♦ Pheidole sp. 42♣ LGS X Pheidole sp. 30 Pheidole sp. 31 Plateau West X X X X X http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 10 Martins, M.F.O. et al. Continuation Table 1. Paraná Species Santa Catarina Campos Gerais PG SMO Plateau West CHP XAN LGS Pheidole sp. 69♦ OTC CBS X Pheidole sp. 70♣ X Pheidole sp. 71♣ X Pheidole sp. 72♣ X Pheidole sp. 73♣ X Pheidole sp. 74♣ X X X X Pheidole sp. 75♣ X Pheidole sp. 76♣ X Pheidole sp. 77♣ X X X X X X Pheidole sp. 78♣ X Pheidole sp. 79♣ X Pheidole sp. 80♣ X Pogonomyrmex naegelli Emery, 1878 X X X Rogeria bruchi Santschi, 1922♦ X X Solenopsis gr. geminata sp. 1I X Solenopsis gr. geminata sp. 2 X Solenopsis gr. geminata sp. 3♣ Solenopsis invicta Buren, 1972 X X Solenopsis sp. 1♣ X X Solenopsis sp. 2 X X Solenopsis sp. 3I X Solenopsis sp. 4 X Solenopsis sp. 5 X II Solenopsis sp. 6 X Solenopsis sp. 7♣ X Solenopsis sp. 8I X Solenopsis sp. 9 X Solenopsis sp. 10 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Solenopsis sp. 11 X X X Solenopsis sp. 12 X X X X X X Solenopsis sp. 13 X X X X X X Solenopsis sp. 14♣ X X X X Solenopsis sp. 15 X X X X Solenopsis sp. 16 X X X X Solenopsis sp. 18♦ X Strumigenys appretiata (Borgmeier, 1954) X Strumigenys denticulata Mayr, 1887♦ X Strumigenys louisianae Roger, 1863 I X X Strumigenys eggersi Emery, 1890 X X Strumigenys oglobini Santschi, 1936♣ http://www.scielo.br/bn X X Solenopsis sp. 17 Strumigenys nr. epinotalisI X X X http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Biota Neotrop., 20(1): e20190782, 2020 11 Subterranean ants of southern Brazil Continuation Table 1. Paraná Species Santa Catarina Campos Gerais PG SMO Plateau West CHP XAN Strumigenys nr. louisianae sp. 1♣ LGS OTC CBS X Strumigenys nr. louisianae sp. 2 X X X X Strumigenys nr. louisianae sp. 3♣ X Mycetomoellerius holmgreni Wheeler, 1925 X X X Mycetomoellerius sp. 1 X X X Mycetomoellerius sp. 2♣ X Tranopelta gilva Mayr, 1866 X Wasmannia affinis Santschi, 1929 X X X X Wasmannia auropunctata (Roger, 1863) X X Wasmannia sulcaticeps Emery, 1894 X X X X X X X X X X X X X X X X X X X X X X X X X Ponerinae Centromyrmex brachycola (Roger, 1861) X Hypoponera foreli (Mayr, 1887) X Hypoponera opaciceps (Mayr, 1887) Hypoponera sp. 1 X X X X Hypoponera sp. 2 X X X X Hypoponera sp. 3 X Hypoponera sp. 4 X Hypoponera sp. 5♦ X Hypoponera sp. 6 I X Hypoponera sp. 7 X Hypoponera sp. 8 0 X Hypoponera sp. 9 X Hypoponera sp. 10I X Hypoponera sp. 11 X Hypoponera sp. 12II X Hypoponera sp. 13 X X X X X X X X X X X X X X X X X X X X X X X X X X Hypoponera sp. 14 X X X Hypoponera sp. 15 Hypoponera sp. 16♦ X Hypoponera sp. 17♦ X X X Hypoponera sp. 18♦ X X Hypoponera sp. 22♦ X X X Neoponera bucki (Borgmeier, 1927)♣ X Odontomachus chelifer (Latreille, 1802) X X X Pachycondyla harpax (Fabricius, 1804) X X X Pachycondyla striata Smith, 1858 X X X X X Rasopone ferruginea (Smith, 1858)♦ http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 X X Hypoponera sp. 21♦ Simopelta curvata (Mayr, 1887)♦ X X X Hypoponera sp. 19♦ Hypoponera sp. 23♦ X X X X X X X http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 12 Martins, M.F.O. et al. Continuation Table 1. Paraná Species Santa Catarina Campos Gerais PG SMO Plateau West CHP XAN LGS OTC CBS Proceratiinae Discothyrea sexarticulata Borgmeier, 19540 X Proceratium brasiliense Borgmeier, 1959♦ X Pseudomyrmecinae Pseudomyrmex flavidulus (Smith, 1858)♦ X I Pseudomyrmex gracilis (Fabricius, 1804) X Pseudomyrmex longus (Forel, 1912)I X X Pseudomyrmex termitarius (Smith, 1877)♦ X Pseudomyrmex sp. 1 X X Pseudomyrmex sp. 2♣ X Total number of species 101 Table 2. Observed (Sobs) and estimated (estimators: Jackknife 1 – Jack1 and Bootstrap - Boot) richness and number of exclusive species by method (Pitfall and TSBF) in Santa Catarina. Methods Sobs Jack1 Boot Exclusive Pitfall 202 260 227 89 TSBF 149 213 176 36 Total 238 294 323 - In Paraná, the TSBF method collected 101 species (Table 1), with 44, 88 and 52 species at strata 0, I and II (Table 3), and 97, 301 and 121 occurrence records, respectively. Four out of the 101 species were collected exclusively in stratum 0, 30 only in stratum I and five in stratum II (Table 1). The most frequent species were Hypoponera sp. 1, Brachymyrmex sp. 5 and Pheidole cavifrons Emery, 1906, with 53, 25 and 16 occurrence records. Species occurrences in strata were as follows: in litter (stratum 0), the three most frequent species were Hypoponera sp. 1, with 12 records; Brachymyrmex sp. 1, and Brachymyrmex sp. 5, with seven records each. In stratum I (0-10 cm), the most frequent species were Hypoponera sp. 1, Hypoponera sp. 3 and Brachymyrmex sp. 3, with 29, 17 and 14 records, respectively. In stratum II (10-20 cm), the highest frequency was recorded for Hypoponera sp. 1, Brachymyrmex sp. 5 and Pheidole cavifrons, with 12, 8 and 7 records. Table 3. Observed richness (Sobs) and estimated (estimators: Jackknife 1 – Jack1 E Bootstrap - Boot) and number of exclusive species by strata (0 - litter, I - 0-10 cm, and II- 10-20 cm). Sobs Jack1 Boot Exclusive 0 44 66 54 4 I 88 125 106 30 II 53 77 64 5 Total 101 129 114 - Strata http://www.scielo.br/bn 103 108 94 111 109 101 The collector curves constructed for each method demonstrate that a greater effort would be required to reach the asymptote (although this is apparently close), and that the number of accumulated species per sample unit was greater in pitfall than in TSBF (Figure 4a). Epigaeic pitfall collected 77% and TSBF 70% of the numbers estimated by Jackknife 1 and 89% and 84% of the numbers estimated by Bootstrap (Table 2). The collector curves for strata performed with data from Paraná show that under the same sampling effort there is a greater addition of species in stratum I, in relation to the others (Figure 4b). The numbers of species observed approximate the Bootstrap estimated values (Table 3). The species collected in each stratum correspond to 66%, 70% and 69% of the species estimated by Jackknife 1 (for strata 0, I and II respectively) and 81%, 83% and 83% of the species estimated by Bootstrap. The total number of species corresponds to 78% and 88% of the estimate by Jackknife 1 and Bootstrap, respectively. 2. Subterranean ant assembly characterization In the regions of Santa Catarina, we obtained 89 species exclusively in the epigaeic stratum and 36 species in the subterranean stratum. Seven species were recorded for the first time in the Plateau region of Santa Catarina and were collected only by TSBF: Acropyga decedens (Mayr, 1887), Fulakora armigera (Mayr, 1887), Gnamptogenys reichenspergeri (Santschi, 1929), Neocerapachys splendens (Borgmeier, 1957), Proceratium brasiliense Borgmeier, 1959, Sphinctomyrmex stali Mayr, 1866 and Typhlomyrmex major Santschi, 1923. No new records were found for the western region. Fifty-eight species were collected in Pitfall traps only once, while single species occurrences in TSBF samples totalled 64. These numbers correspond to different proportions (χ2 = 7.05, p = 0.007, gl = 1), with 28.7% and 42.9%, respectively, of the total species collected by each method. In Paraná, where the method utilised was exclusively TSBF, seven species were present only in stratum I: Fulakora elongata (Santschi, 1912), Gnamptogenys reichenspergeri, Oxyepoecus plaumanni Kempf 1974, Pheidole radozskowskii Mayr, 1884, Pseudomyrmex gracilis http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 13 Biota Neotrop., 20(1): e20190782, 2020 Subterranean ants of southern Brazil Figure 4. a - Collector’s curves for both methods (Pitfall and TSBF). Dashed line: Pitfall; and continuous line: TSBF. b - Collector’s curve for the strata. Continuous line: stratum 0; dashed line: stratum I; and dotted line: stratum II. (Fabricius, 1804), P. longus (Forel, 1912) and Tranopelta gilva Mayr, 1866. In stratum II, Neivamyrmex sp.1 was the only species (with only one occurrence). The number of species with only one record of occurrence in strata was 22 for stratum 0, 37 for stratum I and 24 for stratum II (Figure 5a, b, and c). These species correspond to 50%, 42.1% and 46.1% of the total species collected in each stratum. The graph of observed richness (Sobs) and frequency of occurrence per stratum (Figure 5d) shows that in stratum I there is a great number of species that occur more frequently. Stratum I had the highest number of occurrence frequencies, once this is the stratum with the highest number of occurrences of ants (GLMM, Binomial, z = 5,239, df = 4, p <0.05), with a probability of occurrence of 86.10 % in the sampled areas. Discussion The present study delimited and characterized subterranean ant assemblages in the subtropical regions of the Neotropics. In these regions, only two previous studies collected subterranean ants: (1) Silva & Silvestre (2004) in Western Santa Catarina, and (2) Schmidt & Diehl (2008) in Rio Grande do Sul. This is the first survey that evaluates subterranean ant assemblages in Paraná and the Plateau region of Santa Catarina. Despite the different sampling efforts, the aforementioned publications presented 32 and seven species belonging to the subterranean stratum, representing only 28% and 20% of the total species observed by those authors, respectively. Considering the number of species with restricted occurrence in the subterranean stratum in all sampled localities, our results were similar (in percentage): 57 species, or 20.2% of the total collected (Table 1). The biology of most subterranean species is unknown. TSBF samples provide data of occurrence and preference among strata, nesting, and species composition in the different soil layers. The manual extraction of ants in the stratified monoliths, characteristic of this http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 method, provides the opportunity for in loco observation of the collected organisms. Wong & Guénard (2017) emphasized the importance of the “observer bias” (represented by different levels of experience in active collections), in which a collector may underestimate the presence of subterranean ants (especially the small ones) in samples or even in the field. This error can be reduced if sorting is performed under a stereomicroscope. The total number of species observed in each municipality of Santa Catarina is extremely close to that observed in other studies already conducted in those regions (see Silva & Silvestre 2004, Ulysséa et al. 2011, Lutinski & Morais 2013). However, the number of species collected in Paraná is considerably high when compared with the richness found by other studies employing different methods for sampling subterranean ants (see Table 1 and Wong & Guénard 2017). This could indicate a higher efficiency of TSBF in sampling subterranean ants in these areas. However, additional studies comparing methods for collecting hypogaeic ants are indispensable to confirm this tendency. These comparisons should especially consider the most practical and widely employed method currently, the hypogaeic pitfall traps (Schmidt & Solar, 2010). When epigaeic pitfall traps and TSBF are compared, it becomes clear that pitfall traps capture a greater number of species that have a high frequency of occurrence. However, when evaluating the number of infrequent species, TSBF is superior (42.7% of the species sampled). This may be explained by the fact that pitfall traps remained in the field for 72 hours, increasing the likelihood of species occurring more than once and increasing the number of species captured (see Lasmar et al 2017). Another factor that can explain this difference is the higher ant activity in the ground surface due to nesting and foraging than in the hypogaeic stratum (Jacquemin et al. 2016). In addition, in Santa Catarina, 36 species were collected exclusively by TSBF. This means that 15% of the species sampled there in this study would not have been registered without an association between the pitfall and a specific sampling method for the subterranean stratum. http://www.scielo.br/bn Biota Neotrop., 20(1): e20190782, 2020 14 Martins, M.F.O. et al. Figure 5. a to c - Fisher graphs for each stratum sampled in Paraná; d - Comparative graph of observed richness (Sobs) and frequency of occurrence between the strata. The results obtained by the GLMM analysis of the Paraná data show that stratum I has more species than the other strata. A similar result was found by Andersen & Brault (2010) and Rider Wilkie et al. (2007), who also obtained a greater number of species in the topsoil and a great number of exclusive species (19 species) in this layer (up to 12,5 cm). These results indicate a tendency for greater richness and frequency of occurrence in this stratum, which consequently must be considered during the sampling design of projects focusing on subterranean ants or even in comprehensive inventories of the ant fauna. Sampling methods that record assemblages in different vertical strata allow for a more efficient evaluation of the association between ants and micro habitats. This association was effective for the delimitation of species that occur in different strata also in Jacquemin et al. (2016) and in the present work. The criterion of exclusive delimitation of http://www.scielo.br/bn occurrence used here proved to be efficient, as observed by Fowler et al. (2000). Most species identified by these authors belonged to a unique stratum (collected only in epigaeic traps or only in hypogaeic traps). Considering the exclusivity of occurrence in the strata, and collection methods (pitfall and TSBF), 33% of the total species are epigaeic and 20% are hypogaeic. More than a quarter (28.5%) of the species identified in this study are subterranean and most can be considered infrequent. We thus consider the criterion “exclusivity” (stratum and/or method) a good approach for delimiting the subterranean habit, as already mentioned in literature (e.g. Fowler et al. 2000). However, the delimitation of subterranean ant species was possible only with the association of epigaeic and hypogaeic sampling methods. In addition, we found that in stratified TSBF samples the 0-10 cm stratum has the greatest number of species http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Biota Neotrop., 20(1): e20190782, 2020 15 Subterranean ants of southern Brazil and highest frequency of occurrence. This fact, allied to the number of species obtained through sampling by the TSBF method, indicates that this is a useful method for sampling subterranean ants. The localities sampled in this study are under intense pressure for urbanization and agricultural production. Thus, TSBF is a promising tool in diversity assessments (because it is a widely used method in soil quality studies, facilitating the acceptance by producers for the development of research in their areas), especially to access the impact of human activities on subterranean species. In addition, it was demonstrated that the TSBF captures rarely sampled species or species that are still undiscovered. Acknowledgments We thank Alexandre C. Ferreira, Gabriela P. Camacho, John E. Latke, Mayron Escárraga and Thiago S. R. da Silva for species identification. Special thanks to Sebastian F. Sendoya for the support in the data analyses. We thank IAPAR and Vila Velha State Park for technical support, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Processes: 140260/2016; 563251/2010-7); Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC) (Process 6.309/2011-6/FAPESC) and Empresa Brasileira de Pesquisas Agropecuárias (Embrapa) (Project: 02.11.01.031.00.00) for the financial support. RMF was supported by CNPq (302462/2016-3). DB and GGB thank the CNPq for Research Productivity Grants (307162/2015-0, 304084/2010-7, 307486/2013-3). Collecting was done with permits from ICMBio (18128-1), IAP (463.12) and IBAMA (02027.002353/05-94). Author Contributions Mila Ferraz de Oliveira Martins: contributed substantially in the concept of the study, in data collection, data analysis and interpretation, and to prepare the manuscript. Marcílio José Thomazini: contributed in the concept and design of the study; data collection, and to critical revision, adding intellectual content. Dilmar Baretta: did a substantial contribution in the concept and design of the study, data collection, contributed to data analysis and interpretation and did a critical revision, adding intellectual content Marcio Gonçalves da Rosa: contributed in the concept and design of the study, data collection, data analysis and interpretation, and did a critical revision, adding intellectual content Maurício Rumenos Zagatto: contributed in the concept and design of the study, and data collection. Alessandra Santos: contributed in the concept and design of the study, and data collection. Herlon S. Nadolny: contributed in data collection Guilherme B. X. Cardoso: contributed in data collection Cintia C. Niva: contributed in data collection Marie L. C. Bartz: contributed in the concept and design of the study, and data collection. Rodrigo M. Feitosa: contributed substantially in the concept of the study, in data collection, data interpretation, and to prepare the manuscript. http://dx.doi.org/10.1590/1676-0611-BN-2019-0782 Conflicts of interest The authors declare that they have no conflict of interest related to the publication of this manuscript. References ANDERSEN, A. & BRAULT, A. 2010. Exploring a new biodiversity frontier: subterranean ants in northern Australia. Biodivers Cons 19(9): 2741-2750. ANDERSON, J.M. &INGRAM, J.S.I. (ed). 1993. Tropical soil biology and fertility: a handbook of methods. 2nd ed. C.A.B. International, Wallingford. BACCARO, F.F., FEITOSA, R.M., FERNÁNDEZ, F. et al. 2015. Guia para os Gêneros de formigas do Brasil. Editora INPA, Manaus. BARTZ, M.L.C., BROWN, G.G., ROSA, M.G. et al. 2014a. 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ZAGATTO, M.R.G. 2014. Fauna edáfica em sistemas de uso do solo no município de Ponta Grossa- PR. M. S. thesis, Universidade Federal do Paraná (UFPR). URL: http://dspace.c3sl.ufpr.br/dspace/bitstream/handle/1884/35923/R%20 -%20D%20-%20MAURICIO%20RUMENOS%20GUIDETTI%20 ZAGATTO.pdf?sequence=1 Received: 26/04/2019 Revised: 04/10/2019 Accepted: 16/10/2019 Published online: 02/12/2019 http://www.scielo.br/bn http://dx.doi.org/10.1590/1676-0611-BN-2019-0782