Abstract:

Pteridium is a cosmopolitan genus of ferns that possess nectaries on its fronds (fern leaves), thereby attracting ants. Foliar (or extrafloral) nectaries are nectar-producing glands that are not related to pollination, but rather attract ants and other arthropods. Foliar nectaries are found in 101 fern species, belonging to 11 genera and six families. The aim of the study is to characterize the community of ants that visit the fronds of Pteridium esculentum subsp. arachnoideum, as well as daily and seasonal ant abundance in different frond development stages. The study was conducted in the Atlantic Forest of Rio de Janeiro state, Brazil. Bimonthly collections were established, where 30 expanding fronds and 30 fully expanded fronds were randomly marked. In each 1-hour shift starting at 8:30 am and ending at 5:30 pm, the fronds were observed for the presence of ants. Thirty three ant species were recorded on the Pteridium esculentum subsp. arachnoideum fronds, distributed into six subfamilies and 13 genera. The most abundant species were Solenopsis sp.1 and Ectatomma tuberculatum. Eight ant species were observed foraging the nectaries of tropical bracken fern fronds. Ectatomma tuberculatum has been observed feeding on the nectaries and patrolling the fronds. Ant activity peak was on mid-day during the rainy season. The tropical bracken fern Pteridium esculentum subsp. arachnoideum has a rich (the highest recorded until now on Pteridium species) and diverse ant community on its fronds, mainly on the expanding fronds. The presence of generalist predatory ants (Ectatomma tuberculatum and Solenopsis sp.1) during the entire study period suggests a positive interaction between ants and Pteridium esculentum subsp. arachnoideum.

Keywords
ant-plant interactions; Atlantic Forest; behavioral ecology; foliar nectaries; pteridophytes

Resumo:

Pteridium é um gênero cosmopolita de samambaias que possui nectários em suas frondes (folhas de samambaias), atraindo formigas. Nectários foliares (ou extraflorais) são glândulas produtoras de néctar que não estão relacionadas com a polinização, mas podem atrair formigas e outros artrópodes. Nectários foliares já foram registrados em 101 espécies de samambaias, pertencentes a 11 gêneros e seis famílias. O objetivo do estudo é caracterizar a comunidade de formigas que visitam as frondes de Pteridium esculentum subsp. arachnoideum, bem como a abundância diária e sazonal das formigas em diferentes estágios foliares. O estudo foi realizado na Mata Atlântica do estado do Rio de Janeiro, Brasil. Foram estabelecidas coletas bimestrais, onde foram marcadas aleatoriamente 30 frondes em expansão e 30 frondes totalmente expandidas. Em cada turno de 1 hora com início às 8h30 e término às 17h30, as frondes marcadas foram observadas quanto à presença de formigas. Trinta e três espécies de formigas foram registradas nas frondes do Pteridium esculentum subsp. arachnoideum, distribuídas em seis subfamílias e 13 gêneros. As espécies mais abundantes foram Solenopsis sp.1 e Ectatomma tuberculatum. Foram registradas oito espécies de formigas forrageando os nectários foliares da samambaia. Ectatomma tuberculatum foi observada se alimentando nos nectários e patrulhando as frondes. As formigas tiveram o pico de atividade ao meio-dia e na estação chuvosa. A samambaia tropical Pteridium esculentum subsp. arachnoideum tem uma elevada riqueza (a maior já registrada até o momento para espécies de Pteridium) e diversidade de formigas em suas frondes, com maior frequência nas frondes não totalmente expandidas. A presença de formigas predadoras generalistas (Ectatomma tuberculatum e Solenopsis sp.1) durante todo o período de estudo sugere uma interação positiva entre as formigas e Pteridium esculentum subsp. arachnoideum.

Palavras-chave
interações formiga-planta; Mata Atlântica; ecologia comportamental; nectários foliares; pteridófitas

Introduction

Pteridium (bracken fern) is a fern genus distributed world-wide. The circumscription of species is widely discussed in the literature, about 20 morphotypes have been recognized, and several infra-specific ranks have been adopted (PPG I 2016PPG I. 2016. A community-derived classification for extant lycophytes and ferns. J. Syst. Evol.54:563–603. https://doi.org/10.1111/jse.12229
https://doi.org/https://doi.org/10.1111/... , Thomson 2016THOMSON, J. A. 2016. Free Axial Lobes: An Important Diagnostic Character in Pteridium (Dennstaedtiaceae). Telopea 19:193–200. https://dx.doi.org/10.7751/telopea10722
https://doi.org/https://dx.doi.org/10.77... , Schwartsburd et al. 2018SCHWARTSBURD, P. B., YAÑEZ, A. & PRADO, J. 2018. Formal recognition of six subordinate taxa within the South American bracken fern, Pteridium esculentum (P. esculentum subsp. arachnoideum s.l.—Dennstaedtiaceae), based on morphology and geography. Phytotaxa 333:022–040. https://doi.org/10.11646/phytotaxa.333.1.2
https://doi.org/https://doi.org/10.11646... ). While some authors recognize only two species: Pteridium aquilinum (L.) Kuhn and P. esculentum (G. Forst.) Cockayne along their infraspecies (Thomson 2016THOMSON, J. A. 2016. Free Axial Lobes: An Important Diagnostic Character in Pteridium (Dennstaedtiaceae). Telopea 19:193–200. https://dx.doi.org/10.7751/telopea10722
https://doi.org/https://dx.doi.org/10.77... ). All the species of the genus have nectaries on their fronds (fern leaves) (Tryon & Tryon 1982TRYON, R. M. & TRYON, A. F. 1982. Ferns and allied Plants with Special reference to Tropical America. Springer-Verlag, New York. https://doi.org/10.1007/978-1-4613-8162-4
https://doi.org/https://doi.org/10.1007/... ), and the oldest record of nectaries in ferns was reported by Francis Darwin in 1877 for Pteridium aquilinum (L.) Kuhn (Lloyd 1901LLOYD, F. E. 1901. The extra-nuptial nectaries in the common brake, Pteridium aquilinum. Science 13:885–890. https://doi.org/10.1126/science.13.336.885
https://doi.org/https://doi.org/10.1126/... ).

Foliar (or extrafloral) nectaries are nectar-producing glands that are not related to pollination, but rather attract ants and other arthropods (Koptur et al. 2013KOPTUR, S., PALACIOS-RIOS, M., DÍAZ-CASTELAZO, C., MACKAY, W. P. & RICO-GRAY, V. 2013. Nectar secretion on fern fronds associated with lower levels of herbivore damage: field experiments with a widespread epiphyte of Mexican cloud forest remnants. Ann. Bot. 111:1277–1283. https://doi.org/10.1093/aob/mct063
https://doi.org/https://doi.org/10.1093/... ). Fern nectars contain a variety of amino acids and a large amount of sugar, particularly sucrose, fructose and glucose (Koptur 1992KOPTUR, S. 1992. Interactions between Insects and Plants Mediated by Extrafloral Nectaries, pp. 85–132. In E. Bernays (ed.), Insect/Plant Interactions. Press, Boca Raton., Mehltreter et al. 2022MEHLTRETER, K., TENHAKEN, R. & JANSEN, S. 2022. Nectaries in ferns: their taxonomic distribution, structure, function, and sugar composition. Am. J. Bot. 109:46–57. https://doi.org/10.1002/ajb2.1781
https://doi.org/https://doi.org/10.1002/... ). Foliar nectaries are found in 101 fern species, belonging to 11 genera and six families (Lloyd 1901LLOYD, F. E. 1901. The extra-nuptial nectaries in the common brake, Pteridium aquilinum. Science 13:885–890. https://doi.org/10.1126/science.13.336.885
https://doi.org/https://doi.org/10.1126/... , Koptur et al. 1982KOPTUR, S., SMITH, A. R. & BAKER, I. 1982. Nectaries in some neotropical species of Polypodium (Polypodiaceae): preliminary observations and analyses. Biotropica 14:108–113. https://doi.org/10.2307/2387739
https://doi.org/https://doi.org/10.2307/... , White & Turner 2012WHITE, R. A. & TURNER, M. D. 2012. The Anatomy and Occurrence of Foliar Nectaries inCyathea(Cyatheaceae). Am. Fern J. 102:91–113. https://doi.org/10.1640/0002-8444-102.2.91
https://doi.org/https://doi.org/10.1640/... , Mehltreter et al. 2022)MEHLTRETER, K., TENHAKEN, R. & JANSEN, S. 2022. Nectaries in ferns: their taxonomic distribution, structure, function, and sugar composition. Am. J. Bot. 109:46–57. https://doi.org/10.1002/ajb2.1781
https://doi.org/https://doi.org/10.1002/... . Some authors believe that the nectaries promote interactions between ferns and ants (Koptur et al. 1982KOPTUR, S., SMITH, A. R. & BAKER, I. 1982. Nectaries in some neotropical species of Polypodium (Polypodiaceae): preliminary observations and analyses. Biotropica 14:108–113. https://doi.org/10.2307/2387739
https://doi.org/https://doi.org/10.2307/... , 1998KOPTUR, S., RICO-GRAY, V. & PALACIO-RIOS, M. 1998. Ant protection of the nectaried fern Polypodium plebeium in Central Mexico. Am. J. Bot. 85:736–739. https://doi.org/10.2307/2446544
https://doi.org/https://doi.org/10.2307/... , Page 1982PAGE, C. N. 1982. Field observations on the nectaries of bracken, Pteridium aquilinum, in Britain. Fern Gaz. 12:233–240. https://doi.org/10.2307/2399541
https://doi.org/https://doi.org/10.2307/... ).

There are many records of ants and bees associated with the bracken fern nectaries (Page 1982PAGE, C. N. 1982. Field observations on the nectaries of bracken, Pteridium aquilinum, in Britain. Fern Gaz. 12:233–240. https://doi.org/10.2307/2399541
https://doi.org/https://doi.org/10.2307/... , Tempel 1983TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... , Heads & Lawton 1984HEADS, P. A. & LAWTON, J. H. 1984. Bracken, ants and extrafloral nectarines II. The effect of ants on the insect herbivores of bracken. J Animal Ecol 53:1015–1031., 1985HEADS, P. A. & LAWTON, J. H. 1985. Bracken, ants and extrafloralnectaries. III. How insect herbivores avoid ant predation. Ecol. Entomol. 10:29–42. https://doi.org/10.1111/j.1365-2311.1985.tb00532.x
https://doi.org/https://doi.org/10.1111/... , Olesen 1988OLESEN, J. M. 1988. Foraging behaviour of honeybees at the leaf nectaries of the fern, Pteridium aquilinum. J. Apic. Res. 27:22–25. https://doi.org/10.1080/00218839.1988.11100776
https://doi.org/https://doi.org/10.1080/... ; Rashbrook et al. 1992RASHBROOK, V. K., COMPTON, S. G. & LAWTON, J. H. 1992. Ant-herbivore interactions: Reasons for the absence of benefits to a fern with foliar nectaries. Ecology 73:2167–2174. https://doi.org/10.2307/1941464
https://doi.org/https://doi.org/10.2307/... , White & Turner 2012WHITE, R. A. & TURNER, M. D. 2012. The Anatomy and Occurrence of Foliar Nectaries inCyathea(Cyatheaceae). Am. Fern J. 102:91–113. https://doi.org/10.1640/0002-8444-102.2.91
https://doi.org/https://doi.org/10.1640/... ), though some authors reported no ants on the Pteridium nectaries for some bracken populations (Rumpf et al. 1994RUMPF, S., CROMEY, M. & WEBB, C. J. 1994. Ultrastructure and function of the nectaries of New Zealand bracken (Pteridium esculentum (Forst. f.) Cockayne). N. Z. J. Bot. 32:487–496. https://doi.org/10.1080/0028825X.1994.10412935
https://doi.org/https://doi.org/10.1080/... ). The function of nectaries remains controversial (Cooper-Driver 1990COOPER-DRIVER, G. A. 1990. Defense Strategies in Bracken, Pteridium aquilinum (L.) Kuhn. Ann. Mo. Bot. Gard. 77:281–286. https://doi.org/10.2307/2399541
https://doi.org/https://doi.org/10.2307/... ); some studies reported no benefits from ants that visited the foliar nectaries for Pteridium species (Tempel 1983TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... , Heads & Lawton 1984HEADS, P. A. & LAWTON, J. H. 1984. Bracken, ants and extrafloral nectarines II. The effect of ants on the insect herbivores of bracken. J Animal Ecol 53:1015–1031., Rashbrook et al. 1992RASHBROOK, V. K., COMPTON, S. G. & LAWTON, J. H. 1992. Ant-herbivore interactions: Reasons for the absence of benefits to a fern with foliar nectaries. Ecology 73:2167–2174. https://doi.org/10.2307/1941464
https://doi.org/https://doi.org/10.2307/... ), while others related benefits for ants and plants (Heads 1986HEADS, P. A. 1986. Bracken, ants and extrafloral nectaries. IV. Do wood ants (Formica lugubris) protect the plant against insect herbivores? J. Anim. Ecol. 55:795–809. https://doi.org/10.2307/44173
https://doi.org/https://doi.org/10.2307/... , Jones & Paine 2012). The highest density and frequency rates of ants have been observed on young fronds of Pteridium species in the rainy season (Tempel 1983TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... , Rashbrook et al. 1992RASHBROOK, V. K., COMPTON, S. G. & LAWTON, J. H. 1992. Ant-herbivore interactions: Reasons for the absence of benefits to a fern with foliar nectaries. Ecology 73:2167–2174. https://doi.org/10.2307/1941464
https://doi.org/https://doi.org/10.2307/... ).

Many studies have characterized arthropod fauna associated with Pteridium spp. fronds (Lawton 1976LAWTON, J. 1976. The structure of the arthropod community on bracken (Pteridium aquilinum (L.) Kuhn). Bot. J. Linn. Soc. 73:187–216., Balick et al. 1978BALICK, M. J., FURTH, D. G. & COOPER-DRIVER, G. 1978. Biochemical and evolutionary aspects of arthropod predation on ferns. Oecologia 35:55–89. https://doi.org/10.1007/BF00345541
https://doi.org/https://doi.org/10.1007/... , Hendrix 1980HENDRIX, S. D. 1980. An evolutionary and ecological perspective of the insect fauna of ferns. Am. Nat. 115:171–196. https://doi.org/10.1086/283554
https://doi.org/https://doi.org/10.1086/... , Winterbourn 1987WINTERBOURN, M. J. 1987. The arthropod fauna of bracken (Pteridium aquilinum) on the Port Hills, South Island, New Zealand. N. Z. Entomol. 10:99–104. https://doi.org/10.1080/00779962.1987.9722514
https://doi.org/https://doi.org/10.1080/... ), while others have reported ants visiting their nectaries (Lawton 1976LAWTON, J. 1976. The structure of the arthropod community on bracken (Pteridium aquilinum (L.) Kuhn). Bot. J. Linn. Soc. 73:187–216., Balick et al. 1978BALICK, M. J., FURTH, D. G. & COOPER-DRIVER, G. 1978. Biochemical and evolutionary aspects of arthropod predation on ferns. Oecologia 35:55–89. https://doi.org/10.1007/BF00345541
https://doi.org/https://doi.org/10.1007/... , Page 1982PAGE, C. N. 1982. Field observations on the nectaries of bracken, Pteridium aquilinum, in Britain. Fern Gaz. 12:233–240. https://doi.org/10.2307/2399541
https://doi.org/https://doi.org/10.2307/... ) and assessed the interactions between these two organisms (Page 1982PAGE, C. N. 1982. Field observations on the nectaries of bracken, Pteridium aquilinum, in Britain. Fern Gaz. 12:233–240. https://doi.org/10.2307/2399541
https://doi.org/https://doi.org/10.2307/... , Tempel 1983TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... , Heads & Lawton 1984HEADS, P. A. & LAWTON, J. H. 1984. Bracken, ants and extrafloral nectarines II. The effect of ants on the insect herbivores of bracken. J Animal Ecol 53:1015–1031., 1985HEADS, P. A. & LAWTON, J. H. 1985. Bracken, ants and extrafloralnectaries. III. How insect herbivores avoid ant predation. Ecol. Entomol. 10:29–42. https://doi.org/10.1111/j.1365-2311.1985.tb00532.x
https://doi.org/https://doi.org/10.1111/... , Rashbrook et al. 1992RASHBROOK, V. K., COMPTON, S. G. & LAWTON, J. H. 1992. Ant-herbivore interactions: Reasons for the absence of benefits to a fern with foliar nectaries. Ecology 73:2167–2174. https://doi.org/10.2307/1941464
https://doi.org/https://doi.org/10.2307/... ). However, few of these characterized the entire ant community present on the fronds of this fern (Douglas 1983DOUGLAS, M. M. 1983. Defense of bracken fern by arthropods attracted to axillary nectaries. Psyche 90:313–320. https://doi.org/10.1155/1983/86859
https://doi.org/https://doi.org/10.1155/... , Tempel 1983TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... , Heads & Lawton 1984HEADS, P. A. & LAWTON, J. H. 1984. Bracken, ants and extrafloral nectarines II. The effect of ants on the insect herbivores of bracken. J Animal Ecol 53:1015–1031., 1985HEADS, P. A. & LAWTON, J. H. 1985. Bracken, ants and extrafloralnectaries. III. How insect herbivores avoid ant predation. Ecol. Entomol. 10:29–42. https://doi.org/10.1111/j.1365-2311.1985.tb00532.x
https://doi.org/https://doi.org/10.1111/... ). These studies evaluated Pteridium species of USA, England, Scotland, South Africa, and New Zealand. For the Neotropical Region, Ávila & Otero (2013)ÁVILA, J. L. & OTERO, L. D. 2013. Insectos sobre cayados del helecho invasor Pteridium caudatum en Los Andes venezolanos. Entomotropica 28:99–102. recorded five ant species visiting the nectaries of the croziers of Pteridium caudatum from Venezuela [Brachymyrmex sp., Camponotus rufipes (Fabricius, 1775), Linepithema sp. Pheidole sp., and Solenopsis geminata (Fabricius, 1804)]. Martins et al. (1995)MARTINS, R. P., LEWINSOHN, T. M. & LAWTON, J. H. 1995. First survey of insects feeding on Pteridium aquilinum in Brazil. Rev. Bras. Entomol. 39:151–156. recorded one species (Atta sexdens rubropilosa Forel, 1908) cutting pinnae and rachis of the bracken fern fronds. Santos and Mayhé-Nunes (2007)SANTOS, M. G. & MAYHÉ-NUNES, A. J. 2007. Contribuição ao estudo das interações entre pteridófitas e formigas. Rev. Bras. Biocienc. 5:381–383. reported a single ant species [Dolichoderus attelaboides (Fabricius, 1775)] associated with the nectaries of P. arachnoideum [Pteridium esculentum subsp. arachnoideum (Kaulf.) Thomson].

Pteridium esculentum subsp. arachnoideum (Kaulf.) Thomson is a tropical bracken species widely distributed in South and Central America. Despite being a native species in these regions, it has a high invasive potential and can cause several environmental problems in natural and anthropic areas (Schwartsburd et al. 2017, Oliveira et al. 2018OLIVEIRA, V. M., SCHWARTSBURD, P. B., BRIGHENTI, A. M., D’OLIVEIRA, P. S. & MIRANDA, J. E. C. 2018. Plantas Tóxicas em Pastagens: Samambaia-do-campo (Pteridium esculentum subsp. arachnoideum (Kaulf.) Thomson, Família Dennstaedtiaceae). EMBRAPA, Juiz de Fora.). The present study aims to characterize the ant community on fronds of Pteridium esculentum subsp. arachnoideum (tropical bracken fern), a Neotropical fern species, as well as daily and seasonal ant abundance in different frond development stages.

Materials and Methods

1. Study area

The study was conducted in a population of Pteridium esculentum subsp. arachnoideum located in the Restinga (coastal sandy plain) of the Maricá Environmental Protection Area, in the municipality of Maricá, Rio de Janeiro state, Southeastern Brazil (22° 57’ 41.05”S, 42° 53’ 20.22” W). Restinga is a type of vegetation associated with the Atlantic Forest. Nimer (1972)NIMER, E. 1972. Climatologia da região Sudeste do Brasil. Rev. Bras. Geogr. 34:3–48. classified Maricá’s climate as hot tropical and super humid, with short dry periods. Studies that characterized Maricá’s climate between 1989 and 2000 indicate that the minimum temperature is lower in winter (8.6 °C to 15.2 °C), with July as the coldest month (always below 15 °C), while the highest temperatures are recorded in summer, with means between 29.2 °C and 37.3 °C, and February being the hottest month (temperatures always above 33 °C, reaching 37.7 °C) (Pereira et al. 2001PEREIRA, M. C. A., ARAÚJO, D. S. D. & PEREIRA, O. J. 2001. Estrutura de uma comunidade arbustiva da restinga de Barra de Maricá-RJ. Rev. Bras. Bot. 24:273–281. https://doi.org/10.1590/S0100-84042001000300005
https://doi.org/https://doi.org/10.1590/... ). In the study area there are only two seasons. According to Barbieri (2005)BARBIERI, P. R. B. 2005. Caracterização da estação chuvosa nas regiões sul e sudeste do Brasil associado com a circulação atmosférica. Dissertation, Instituto Nacional de Pesquisas Espaciais, Brazil., the rainy season in Southeastern Brazil occurs between October and March and the dry season between April and September.

2. Collection and laboratory procedures

Tempel (1983)TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... divided the frond development of Pteridium into 6 stages. In this study, the fronds in stages 1 to 5 were considered expanding fronds, and those in stage 6 fully expanded fronds (Figure 1). Since P. arachnoideum has subterraneous rhizomes, the fronds were used as a sampling unit. Thirty expanding fronds and thirty fully expanded fronds were randomly marked with red ribbon, at least 2m apart to each other. Throughout the day (8:30 am–5:30 pm), the fronds were inspected during each 1-hour shift and all ants on bracken fronds collected with an entomological aspirator, to obtain the abundance of each species. The ants which were feeding on the bracken fern nectaries were recorded. Collections were carried out every two months for one year, one day (without rain) per month, between February 2009 and February 2010. The ants were preserved in 70% alcohol, mounted in entomological boxes (Lattke 2003LATTKE, J. E. 2003. Subfamília Ponerinae. In: Fernández F. (ed.), Introducción a las hormigas de la región Neotropical. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Bogotá, Colombia, pp. 261–281.), and identified by Dr. Antônio Mayhé-Nunes and Dr. Rodrigo M. Feitosa. Vouchers were deposited in the herbarium of the Faculdade de Formação de Professores da Universidade do Estado do Rio de Janeiro (RFFP 20.281) and the Padre Jesus Santiago Moure Entomological Collection, Universidade Federal do Paraná, Department of Zoology (DZUP).

Figure 1.
Frond stages of Pteridium esculentum subsp. arachnoideum. A: Fully expanded frond. B-C: Expanding frond. All photos: Marcelo Guerra Santos.

3. Statistical analyses

To detect differences in ant frequencies (number of observations) between expanding and fully expanded fronds (categorical variables), we performed a Pearson’s chi-squared test (Gotelli and Ellison 2011GOTELLI, N. J., ELLISON, A. M. 2011. Princípios de estatística em ecologia. Artmed, Porto Alegre.). The expected richness of ants on bracken fronds was calculated using the estimator Chao 2. It provides a minimum estimate of richness in homogeneous samples using presence and absence (incidence) data, in just one or two samples (Magurran 2013MAGURRAN, A. E. 2013. Medindo a diversidade biológica. Editora da UFPR, Curitiba.). The statistical tests were conducted in PAST (PAleontological STatistics), version 3.10 (Hammer et al. 2001HAMMER, Ø., HARPER, D. A. T. & RYAN, P. D. 2001. PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontol. Electron. 4:1–9.).

Circular statistics were used to estimate the month of highest incidence of the ant species, time of intensity peak on fern fronds. Furthermore, the mean angle, Rayleigh test (p), and length of mean vector (r) were calculated. The program Oriana was used for the calculations of circular statistics (Kovach 2009KOVACH COMPUTING SERVICES. 2009. Oriana 2.0 http://www.kovcomp.co.uk.
http://www.kovcomp.co.uk... ).

Results

A total of 599 ants were observed on the fronds of Pteridium esculentum subsp. arachnoideum. Of this total, we managed to collect only 529 ants, and all were identified at least to the generic level. We recorded 33 ant species on the tropical bracken fern fronds, distributed among six subfamilies and 13 genera (Table 1). The observed richness was similar to the richness estimated by Chao 2 = 33.8 ± 6.0. Eight ant species were observed foraging the nectaries of tropical bracken fern fronds, Brachymyrmex sp.1 (Figure 2F), Camponotus crassus Mayr, 1862 (Figure 2B), Camponotus sp.1, Ectatomma tuberculatum (Olivier, 1792) (Figure 2A), Ectatomma brunneum (Fr. Smith, 1858) (Figure 2D), Pheidole sp.1 (Figure 2E), Solenopsis sp.1, and Wasmannia auropunctata (Roger, 1863) (Figure 2C). Ectatomma tuberculatum has also been observed patrolling the fronds.

The subfamilies with the highest ant richness were Formicinae and Myrmicinae (both with nine species). The species with the greatest abundance were Solenopsis sp.1 (165 individuals) and Ectatomma tuberculatum (151 individuals) (Figure 2A), and those with intermediate abundance were Brachymyrmex sp.1 (65 individuals), Camponotus sp.1 (26 individuals), and Wasmannia auropunctata (21 individuals). All other 28 ant species had an abundance of less than 10 individuals (Table 1).

Figure 2.
Ants on frond nectaries of Pteridium esculentum subsp. arachnoideum. A: Ectatomma tuberculatum feeding on the nectary. It’s possible to see a nectar droplet in their open jaws. B: Camponotus crassus. C: Wasmannia auropunctata. D: Ectatomma brunneum. E: Pheidole sp. F: Brachymyrmex sp. (arrow). *Nectaries. Photo A: Isabella Rodrigues Lancellotti. Photos B, C, E, F: Marcelo Guerra Santos. Photo D: Camille Santos Carraco.

The chi-squared test (X²) demonstrated a significant difference for ant abundance between the analyzed months (X² = 397.17, DF = 6, P < 0.000001), with the higher abundance in October (100 individuals) and December (226 individuals), both in the beginning of the rainy season (Table 1). October and December are the months most likely to find 25 of the 33 species recorded, among them the species with the highest abundance, Ectatomma tuberculatum and Solenopsis sp.1 (October and December respectively). April, June, and August (dry season), and February (end of the rainy season) are the months with the lowest probability of finding ant species on Pteridium esculentum subsp. arachnoideum (Table 1).

There was a significant difference in the frequency of ants (number of observations) visiting the expanding and fully expanded fronds of Pteridium esculentum subsp. arachnoideum in all the months observed, with ants showing a preference for the former (Table 2). Most ants (19 species) have their highest visitation intensity peak between 10:30 am and 1:30 pm, including the species with the highest abundance, Solenopsis sp.1 and Ectatomma tuberculatum (Table 3).

Discussion

The record of 33 ant species belonging to 13 genera is the highest ant richness recorded on Pteridium fronds until now, of this total, eight were observed foraging the nectaries. Tempel (1983)TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... listed 18 nectar feeding ants, distributed on 12 genera on Pteridium aquilinum, in USA. This author also found Myrmicinae as the richest subfamily (9 spp.). Douglas (1983)DOUGLAS, M. M. 1983. Defense of bracken fern by arthropods attracted to axillary nectaries. Psyche 90:313–320. https://doi.org/10.1155/1983/86859
https://doi.org/https://doi.org/10.1155/... reported nine ant species associated with the nectaries of P. aquilinum in USA. Ávila and Otero (2013)ÁVILA, J. L. & OTERO, L. D. 2013. Insectos sobre cayados del helecho invasor Pteridium caudatum en Los Andes venezolanos. Entomotropica 28:99–102. recorded five ant species visiting the nectaries of the croziers of Pteridium caudatum, in Venezuela. Heads and Lawton (1984HEADS, P. A. & LAWTON, J. H. 1984. Bracken, ants and extrafloral nectarines II. The effect of ants on the insect herbivores of bracken. J Animal Ecol 53:1015–1031., 1985HEADS, P. A. & LAWTON, J. H. 1985. Bracken, ants and extrafloralnectaries. III. How insect herbivores avoid ant predation. Ecol. Entomol. 10:29–42. https://doi.org/10.1111/j.1365-2311.1985.tb00532.x
https://doi.org/https://doi.org/10.1111/... ) reported three ant species on nectaries of P. aquilinum in England. In Brazil, Martins et al. (1995)MARTINS, R. P., LEWINSOHN, T. M. & LAWTON, J. H. 1995. First survey of insects feeding on Pteridium aquilinum in Brazil. Rev. Bras. Entomol. 39:151–156.reported a single ant species [Atta sexdens (Linnaeus, 1758)] cutting pinnae and rachis of the Pteridium fronds. Santos and Mayhé-Nunes (2007)SANTOS, M. G. & MAYHÉ-NUNES, A. J. 2007. Contribuição ao estudo das interações entre pteridófitas e formigas. Rev. Bras. Biocienc. 5:381–383. recorded Dolichoderus attelaboides (Fabricius 1775) as the only ant species associated with the foliar nectaries of P. arachnoideum [Pteridium esculentum subsp. arachnoideum] in the Atlantic Forest of Rio de Janeiro state.

Vargas et al. (2010)VARGAS, A. B., MARTINS, L., ARAÚJO, M. S., OLIVEIRA, M. J. P., LIMA, D. C. & MAYHÉ-NUNES, A. J. 2010. Composição, riqueza e diversidade de formigas de serapilheira da restinga da APA de Maricá, RJ. In I Simpósio de Pesquisa em Mata Atlântica (M.C.R.M. Santos, F.C.C. dos Santos, W.C. Rodrigues, coords.). Instituto Zoobotânico de Morro Azul/ Faculdade de Formação de Professores da Universidade do Estado do Rio de Janeiro, Engenheiro Paulo de Frontin, p.15–17. recorded 52 ant species in the litter of the Restinga of Maricá (restinga vegetation). So, the richness found on the P. esculentum subsp. arachnoideum fronds represents 63.5% of the ants registered for this region until now. For angiosperms species with foliar nectaries in Cerrado (Brazilian savanna), 34 ant species were found on Caryocar brasiliense Cambess. (Caryocaraceae), 24 on Ouratea hexasperma (A.St.-Hil.) Baill. (Ochnaceae), and 12 on Qualea grandiflora Mart. (Vochysiaceae) (Oliveira & Pie 1998OLIVEIRA, P. S. & PIE, M. R. 1998. Interaction Between Ants and Plants Bearing Extrafloral Nectaries in Cerrado Vegetation. An. Soc. Entomol. 27:161–176. https://doi.org/10.1590/S0301-80591998000200001
https://doi.org/https://doi.org/10.1590/... ). In mangrove vegetation, Talipariti pernambucense (Arruda) Bovini (Malvaceae) was visited by 19 ant species (Cogni & Freitas, 2002COGNI, R. & FREITAS, A. V. L. 2002. The ant assemblage visiting extrafloral nectaries of Hibiscus pernambucensis (Malvaceae) in a Mangrove Forest in Southeast Brazil (Hymenoptera: Formicidae). Sociobiology 40:373–383.).

The ant visitation on Pteridium esculentum subsp. arachnoideum fronds was greater in October and December (both in the beginning of the rainy season). Tempel (1983)TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... conducted a study in New Jersey (EUA) and found that nectar secretion by Pteridium aquilinum and ant visitation were more frequent from May to August, that is, in summer, with high rates of precipitation. In South Africa, the period in which ants have the greatest impact on Pteridium herbivores occurs between November and December, corresponding to the rainy season in this region (Rashbrook et al. 1992RASHBROOK, V. K., COMPTON, S. G. & LAWTON, J. H. 1992. Ant-herbivore interactions: Reasons for the absence of benefits to a fern with foliar nectaries. Ecology 73:2167–2174. https://doi.org/10.2307/1941464
https://doi.org/https://doi.org/10.2307/... ). These data indicate that the change in ant visitation of bracken fronds is highly associated with the summer season.

The most abundant ant species (Brachymyrmex sp.1, Camponotus sp.1, Ectatomma tuberculatum, Solenopsis sp.1, and Wasmannia auropunctata) on tropical bracken fern have their highest visitation intensity peak near to mid-day, between 12:00 and 2:00 pm. According to Kaspari (2003)KASPARI, M. 2003. Introducción a la Ecologia de las Hormigas. In: Fernández, F. (Ed.), Introducción a las hormigas de la región Neotropical. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Bogotá, Colombia, pp. 97–117., temperature is an important factor that regulates the activities of ant populations. Increased visitation may be attributed to the natural rise in the daily activities of ants as a function of their ectothermy and exudation of foliar nectaries. Ectatomma tuberculatum was one of the ant species with highest abundance, and visited the fronds of Pteridium esculentum during 9:30 am to 4:30 pm with the intensity peak at 1:07 pm. (Table 3). This ant species presents high activity at night, with massive exiting of workers at end of the day (sunset) and massive entry in the nest at beginning of the morning (sunrise), or high foraging activity in the morning and no mass exit at sunset (Wheeler 1986WHEELER, D. E. 1986. Ectatomma tuberculatum, foraging biology and association with Crematogaster (Hymenoptera, Formicidae). Ann. Entomol. Soc. Am. 79:300–303. https://doi.org/10.1093/aesa/79.2.300
https://doi.org/https://doi.org/10.1093/... ). According to Valenzuela-González et al. (1995)VALENZUELA-GONZÁLEZ, J., LÓPEZ-MÉNDEZ, A. & LACHAUD, J. P. 1995. Activity patterns and foraging activity in nests of Ectatomma tuberculatum (Hymenoptera: Formicidae) in cacao plantations. Southwest. Entomol. 20:507–515., E. tuberculatum foraging was mainly nocturnal during the dry season and diurnal in the rainy season. Page (1982)PAGE, C. N. 1982. Field observations on the nectaries of bracken, Pteridium aquilinum, in Britain. Fern Gaz. 12:233–240. https://doi.org/10.2307/2399541
https://doi.org/https://doi.org/10.2307/... reported that the foliar nectaries of Pteridium aquilinum were more active in the morning. Mehltreter et al. (2022)MEHLTRETER, K., TENHAKEN, R. & JANSEN, S. 2022. Nectaries in ferns: their taxonomic distribution, structure, function, and sugar composition. Am. J. Bot. 109:46–57. https://doi.org/10.1002/ajb2.1781
https://doi.org/https://doi.org/10.1002/... , analyzing 16 fern species (Aglaomorpha and Campyloneurum genera), observed that nectar secretion occurred mainly during the night and early morning, but could continue until the afternoon, depending on air humidity conditions. In fact, in Restinga of Maricá it was possible to observe a few tropical bracken ferns secreting nectar during the period close to noon (Figure 2A). But we did not measure local climatic data in these days. The nighttime period was not assessed here and probably a different ant community may be visiting the foliar nectaries of Pteridium esculentum subsp. arachnoideum at this period. However, Tempel (1983)TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... evaluated populations of Pteridium aquilinum Kuhn in New Jersey (USA) at night and found no evidence of nocturnal ant activity.

Ectatomma tuberculatum and Solenopsis sp.1, both generalist predator ants (Wheeler 1986WHEELER, D. E. 1986. Ectatomma tuberculatum, foraging biology and association with Crematogaster (Hymenoptera, Formicidae). Ann. Entomol. Soc. Am. 79:300–303. https://doi.org/10.1093/aesa/79.2.300
https://doi.org/https://doi.org/10.1093/... , Valenzuela-González et al. 1995VALENZUELA-GONZÁLEZ, J., LÓPEZ-MÉNDEZ, A. & LACHAUD, J. P. 1995. Activity patterns and foraging activity in nests of Ectatomma tuberculatum (Hymenoptera: Formicidae) in cacao plantations. Southwest. Entomol. 20:507–515., Wojcik et al. 2001WOJCIK, D. P., ALLEN, C. R., BRENNER, R. J., FORYS, E. A., JOUVENAZ, D. P. & LUTZ, R. S. 2001. Red Imported Fire Ants: Impact on Biodiversity. Am. Entomol. 47:16–23., see also comments by Tolofo 2011 on other Ectatomma species), were present during the entire period of observations, and were the most abundant species on the fronds of Pteridium esculentum subsp. arachnoideum. E. tuberculatum has been observed feeding on the nectaries, and patrolling the fronds, whereas Solenopsis sp.1 was only feeding on nectaries. However, other generalist predator ants were also present in lower abundance, as follows: Dorymyrmex brunneus (Forel, 1908), Dorymyrmex sp., Ectatomma brunneum, Neoponera villosa (Fabricius, 1804), Pheidole sp.1, Pheidole sp.2, Pseudomyrmex sp.1 and Pseudomyrmex sp.2 (Table 4). Douglas (1983)DOUGLAS, M. M. 1983. Defense of bracken fern by arthropods attracted to axillary nectaries. Psyche 90:313–320. https://doi.org/10.1155/1983/86859
https://doi.org/https://doi.org/10.1155/... observed that Camponotus pennsylvanicus (De Geer, 1773), Formica obscuriventris (Mayr, 1870), and Formica subsericea (Say, 1836) defend the nectaries of the developing croziers, while other smaller species such as Tapinoma sessile (Say, 1836), Temnothorax rugatulus (Emery, 1895), Leptothorax muscorum (Nylander, 1846), and Lasius alienus (Foerster, 1850) (not predatory ant species) only utilize nectar and do not seem to defend the fern croziers. The establishing mutualistic relationship between ants and foliar nectaries seems to depend on the occurrence and abundance of large or aggressive ants visiting the plants (Koptur 1984KOPTUR, S. 1984. Experimental evidence for defence of Inga saplings (Mimosoideae) by ants. Ecology 65:1787–1793. https://doi.org/10.2307/1937775
https://doi.org/https://doi.org/10.2307/... , Heads 1986HEADS, P. A. 1986. Bracken, ants and extrafloral nectaries. IV. Do wood ants (Formica lugubris) protect the plant against insect herbivores? J. Anim. Ecol. 55:795–809. https://doi.org/10.2307/44173
https://doi.org/https://doi.org/10.2307/... ).

The frequency of ants (number of observations) was greater on expanding fronds than on fully expanded fronds of P. esculentum subsp. arachnoideum. Marquis (2012)MARQUIS, R. J. 2012. Uma abordagem geral das defesas das plantas contra a ação dos herbívoros. In: Del-Claro K, Torrezan-Silingardi HM (org.), Ecologia das interações plantas-animais: uma abordagem ecológico-evolutiva. Technical Books Editora, Rio de Janeiro, pp. 55–65. reports that phenology is vital in determining herbivore attack intensity, since the plant life cycle can allow plants to evade attack or be exposed in its most vulnerable phase, when not fully mature. Tempel (1983)TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... found that nectar secretion is significantly higher in the first developmental stages of Pteridium aquilinum, and becomes practically inactive in the final stages. As such, the activity pattern of ants is correlated to foliar nectar exudation. Rashbrook et al. (1992)RASHBROOK, V. K., COMPTON, S. G. & LAWTON, J. H. 1992. Ant-herbivore interactions: Reasons for the absence of benefits to a fern with foliar nectaries. Ecology 73:2167–2174. https://doi.org/10.2307/1941464
https://doi.org/https://doi.org/10.2307/... observed a larger number of ants on young fronds, which have more active nectaries (greater nectar exudation). Thus, it can be inferred that in the most vulnerable stage (juvenile), higher nectar production acts as a lure for ants, which sometimes protect individuals that supply nectar. In the Restinga of Maricá we observed very few full expanded fronds of the tropical bracken fern secreting nectar during the day (Figure 2A). However, almost all observed fronds (expanding and full expanded) were not secreting nectar. Even though, the ants still remained scraping the nectaries (Figures 2B–F).

According to Tempel (1993)TEMPEL, A. S. 1983. Bracken fern (Pteridium aquilinum) and nectar-feeding ants: a nonmutualistic interaction. Ecology 64:1411–1422. https://doi.org/10.2307/1937495
https://doi.org/https://doi.org/10.2307/... , most of the significant damage to Pteridium aquilinum occurs before maturity, demonstrating no need for the additional protection provided by ants in the final stage of development, and in turn, the low nectar production. Furthermore, studies conducted by Santos et al. (2005)SANTOS, M. G., CARVALHO, C. E. M., KELECOM, A., RIBEIRO, M. L. R. C., FREITAS, C. V. C., COSTA, L. M. & FERNANDES, L. V. G. 2005. Cianogênese em esporófitos de pteridófitas avaliada pelo teste do ácido pícrico. Acta Bot. Brasílica 19:783–788. https://doi.org/10.1590/S0102-33062005000400014
https://doi.org/https://doi.org/10.1590/... found cyanogenesis in all young Pteridium arachnoideum fronds but in only 9.1% of the mature fronds. Cyanogenesis is a defensive process in which the plant releases hydrocyanic acid when injured by herbivores (Vetter 2000VETTER, J. 2000. Plant cyanogenic glycosides. Toxicon 38:11–36. https://doi.org/10.1016/S0041-0101(99)00128-2
https://doi.org/https://doi.org/10.1016/... ). The levels of phenolic substances, which may also act defensively, increase significantly during the maturation of P. esculentum subsp. arachnoideum fronds (unpublished data). Furthermore, the fronds exhibit a norsesquiterpene denominated ptaquiloside, which is responsible for toxic, mutagenic and cancerigenous action in Pteridium species (Yamada et al. 2007YAMADA, K., OJIKA, M. & KIGOSHI, H. 2007. Ptaquiloside, the major toxin of bracken, and related terpene glycosides: Chemistry, biology and ecology. Nat. Prod. Rep. 24:798–813. https://doi.org/10.1039/b614160a
https://doi.org/https://doi.org/10.1039/... ).

The tropical bracken fern Pteridium esculentum subsp. arachnoideum has a remarkedly rich (the highest recorded until now on Pteridium species) and diverse ant community on its fronds, mainly on the expanding fronds, which peaks at the mid of the day in the rainy season. The presence of generalist predatory ants (Ectatomma tuberculatum and Solenopsis sp.1) during the entire study period suggests that there may be a positive interaction between ants and Pteridium esculentum subsp. arachnoideum. Future studies are needed to understand the relationship between these ants and the tropical bracken fern, especially those that interact with leaf nectaries.

Acknowledgments

We are indebted to Bianca da Silva (in memoriam), Rafael Pontes, André Siqueira, and Luiz José Soares Pinto for their help in collecting and screening the material. Thanks to Márcio Mendes Bento da Silva for his help with the illustrations. MGS and RMF thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; grants 308045/2017-3 and 301495/2019-0, respectively), FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro grant E-26/203.236/2017), and PROCIÊNCIA (Programa de Incentivo à Produção Científica, Técnica e Artística) of UERJ (Universidade do Estado do Rio de Janeiro) for financial support.

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