ABSTRACT

Although extrafloral nectaries (EFNs) are reported as extremely important to understand ecological interactions, the diversity of EFN-bearing plants remains underappreciated. In Brazil, studies are concentrated in Cerrado and extremely poorly known in other phytogeographic domains, such as Atlantic forest. Thus, this study provides one of the first checklists of angiosperms with EFNs in a protected area of the Brazilian Atlantic forest, bringing information about the richness, location of EFNs on the plant’s organs, as well as plant’s habit and conservation status. A total of 93 EFN-bearing species belonging to 61 genera and 29 families is reported, which corresponds to 16.5 % of the local flora. The vast majority has not been evaluated for their conservation status, and twenty species are endemic to the Brazilian Atlantic forest. Vines and trees are the predominant plant habits and EFNs are mostly found on leaves. More than half of the species (53.7 %) comprises new additions to the world list of EFN-bearing plants, including the first record in Dilleniaceae. Our findings show a large number of EFN-bearing plant species from a single and small protected area in the Brazilian Atlantic forest. It reinforces the lack of studies of this nature, where further investigations are strongly recommended.

Keywords:
ant-plant interactions; checklist; nectar; plant morphology; taxonomy; ‘restingas’

Introduction

Nectaries are plant structures where the nectar, an aqueous solution compounded by sugar, water, and amino acids, is produced, secreted, and offered to interacting animals (Pacini et al. 2003Pacini E, Nepi M, Vesprini J L. 2003. Nectar biodiversity: a short review. Plant Systematics and Evolution 238 1: 7-21.; Heil, 2011Heil M. 2011. Nectar: generation, regulation and ecological functions. Trends in Plant Science 16 (4): 191-200. ; Nepi et al. 2018Nepi M, Grasso DA, Mancuso S. 2018. Nectar in plant-insect mutualistic relationships: from food reward to partner manipulation. Frontiers in Plant Science 9: 1063.). Nectaries are usually classified based on their location on the plant as well as on the ecological role they play. For instance, floral nectaries are found on flowers and generally have a nuptial function (i.e., pollination), while extrafloral nectaries (hereafter EFNs) occur on other organs aboveground (Elias 1983Elias TS. 1983. Extrafloral nectaries: their structure and distribution. In: Bentley BL, Elias TS (eds.) The biology of nectaries. New York, Columbia University Press. p. 174-203.) and are unrelated to pollination (Delpino 1874Delpino F. 1874. Rapporti tra insetti e nettari extranuziali nelle plante. Bollettino della Società Entomologica Italiana 6: 234-239.; Bentley 1977aBentley BL. 1977a. The protective function of ants visiting the extrafloral nectaries of Bixa orellana L. (Bixaceae). Journal of Ecology 65: 27-38.; bBentley BL. 1977b. Extrafloral nectaries and protection by pugnacious bodyguards. Annual Review of Ecology, Evolution, and Systematics 8: 407-428.; Heil 2011Heil M. 2011. Nectar: generation, regulation and ecological functions. Trends in Plant Science 16 (4): 191-200. ). EFNs can be found in different lineages of angiosperms with high diversity in their phylogenetic and biogeographic distribution, plant location, morphology, and ecology (Marazzi et al. 2013Marazzi B, Bronstein JL, Koptur S. 2013. The diversity, ecology and evolution of extrafloral nectaries: current perspectives and future challenges. Annals of Botany 111: 1243-1250. ).

For taxonomic and floristics purposes, the presence of EFN-bearing plants works as a reliable feature to determine the composition and the functional diversity of biological communities. Furthermore, EFNs also provide important ecological information once it highlights putative local inter- or intraspecific interactions between organisms (Heil 2011Heil M. 2011. Nectar: generation, regulation and ecological functions. Trends in Plant Science 16 (4): 191-200. ), which are primordial for modeling conservation strategies (Wang et al. 2015Wang HH, Wonkka CL, Treglia ML, Grant WE, Smeins FE, Rogers WE. 2015. Species distribution modelling for conservation of an endangered endemic orchid. AoB PLANTS 7: 1-12. ).

However, EFN-bearing plants richness is weakly reported and likely remains underappreciated (Marazzi et al. 2013Marazzi B, Bronstein JL, Koptur S. 2013. The diversity, ecology and evolution of extrafloral nectaries: current perspectives and future challenges. Annals of Botany 111: 1243-1250. ), once most of the detailed studies are focused on animal-plant ecology (Almeida & Figueiredo 2003Almeida AM, Figueiredo RA. 2003. Ants visit nectaries of Epidendrum denticulatum (Orchidaceae) in a Brazilian rainforest: effects on herbivory and pollination. Brazilian Journal of Biology 63: 551-558.; Schoereder et al. 2010Schoereder JH, Sobrinho TG, Madureira MS, Ribas CR, Oliveira OS. 2010. The arboreal ant community visiting extrafloral nectaries in the Neotropical cerrado savanna. Terrestrial Arthropod Reviews 3: 3-27. ; Lay et al. 2011Lay CR, Linhart YB, Diggle PK. 2011. The good, the bad and the flexible: plant interactions with pollinators and herbivores over space and time are moderated by plant compensatory responses. Annals of Botany 108: 749-763. ; Lange et al. 2013Lange D, Dáttilo W, Del-Claro K. 2013. Influence of extrafloral nectary phenology on ant-plant mutualistic networks in a neotropical savanna. Ecological Entomology 38: 463-469.; Câmara et al. 2017Câmara T, Almeida WR, Tabarelli M, Leal IR. 2017. Habitat fragmentation, EFN-bearing trees and ant communities: Ecological cascades in Atlantic forest of northeastern Brazil. Austral Ecology 42: 31-39. ), morphology and anatomy (Machado et al. 2008Machado SR, Morellato SPC, Sajo MG, Oliveira PS. 2008. Morphological patterns of extrafloral nectaries in woody plant species of the Brazilian cerrado. Plant Biology 10: 660-673. ; Melo et al. 2010aMelo Y, Machado SR, Alves M. 2010a. Anatomy of extrafloral nectaries in Leguminosae from dry-seasonal forest in Brazil. Botanical Journal of the Linnean Society 163: 87-98.; bMelo Y, Córdula E, Machado SR, Alves M. 2010b. Morfologia de nectários em Leguminosae senso lato em áreas de caatinga no Brasil. Acta Botanica Brasilica 24: 1034-1045. ; Aguirre et al. 2013Aguirre A, Coates R, Cumplido-Barragán G, Campos-Villanueva A, Díaz-Castelazo C. 2013. Morphological characterization of extrafloral nectaries and associated ants in tropical vegetation of Los Tuxtlas, Mexico. Flora-Morphology, Distribution, Functional Ecology of Plants 208: 147-156.; Pires et al. 2017Pires MS, Calixto ES, Oliveira DC, Del-Claro K. 2017. A new extrafloral nectary-bearing plant species in the Brazilian savanna and its associated ant community: nectary structure, nectar production and ecological interactions. Sociobiology 64: 228-236. ), phylogeny (Weber & Keller 2013Weber MG, Keeler KH. 2013. The phylogenetic distribution of extrafloral nectaries in plants. Annals of Botany 111: 1251-1261. ), or phylogeography (Schupp & Feener 1991Schupp EW, Feener DH. 1991. Phylogeny, lifeform, habitat dependence of ant-defended plants in a Panamanian forest. In: Cutler DF, Huxley CR. (eds.) Ant-Plant Interactions. Oxford, Oxford University Press. p. 175-197.; Del-Claro et al. 2013Del-Claro K, Stefani V, Lange D et al. 2013. The importance of natural history studies for a better comprehension of animal-plant interaction networks. Bioscience Journal 29: 438-448.; Krosnick et al. 2013Krosnick SE, Porter-Utley KE, MacDougal JM, Jørgensen PM, McDade LA. 2013. New insights into the evolution of Passiflora subgenus Decaloba (Passifloraceae): Phylogenetic relationships and morphological synapomorphies. Systematic Botany 38: 692-713. ; Marazzi et al. 2013Marazzi B, Bronstein JL, Koptur S. 2013. The diversity, ecology and evolution of extrafloral nectaries: current perspectives and future challenges. Annals of Botany 111: 1243-1250. ; Weber & Keeler 2013Weber MG, Keeler KH. 2013. The phylogenetic distribution of extrafloral nectaries in plants. Annals of Botany 111: 1251-1261. ). Nevertheless, extremely important, broader, and thorough ecological questions are strictly dependent on a solid floristic database and a correct taxonomic determination; otherwise, the answer proposed in such studies might not be validated. In this sense, knowing the geographical distribution of plants with EFNs in different vegetation types can provide a backbone that validates and might allow further ecological, evolutionary, and conservation studies.

EFNs can be widely found on plants in tropical and temperate zones, but available information is more commonly cited for the former (Oliveira & Leitão-Filho 1987Oliveira PS, Leitão-Filho HF. 1987. Extrafloral nectaries: their taxonomic distribution and abundance in the woody flora of cerrado vegetation in Southeast Brazil. Biotropica 19: 140-148.; Morellato & Oliveira 1991Morellato LPC, Oliveira PS. 1991. Distribution of extrafloral nectaries in different vegetation types of Amazonian Brazil. Flora 185: 33-38.; Koptur, 1992Koptur S. 1992. Extrafloral nectary-mediated interactions between insects and plants. In: Insect-plant Interaction 75: 39-50.; Fiala & Lisenmair 1995Fiala B, Linsenmair KE. 1995. Distribution and abundance of plants with extrafloral nectaries in the woody flora of a lowland primary forest in Malaysia. Biodiversity and Conservation 4: 165-182.; Rico-Gray & Oliveira 2007Rico-Gray V, Oliveira PS. 2007. The ecology and evolution of ant-plant interactions. Chicago, University of Chicago Press.; Schoereder et al. 2010Schoereder JH, Sobrinho TG, Madureira MS, Ribas CR, Oliveira OS. 2010. The arboreal ant community visiting extrafloral nectaries in the Neotropical cerrado savanna. Terrestrial Arthropod Reviews 3: 3-27. ). In tropical regions, plants bearing EFNs are remarkably rich and well distributed (Oliveira & Leitão-Filho 1987Oliveira PS, Leitão-Filho HF. 1987. Extrafloral nectaries: their taxonomic distribution and abundance in the woody flora of cerrado vegetation in Southeast Brazil. Biotropica 19: 140-148.; Koptur 1992Koptur S. 1992. Extrafloral nectary-mediated interactions between insects and plants. In: Insect-plant Interaction 75: 39-50.; Rico-Gray & Oliveira 2007Rico-Gray V, Oliveira PS. 2007. The ecology and evolution of ant-plant interactions. Chicago, University of Chicago Press.; Schoereder et al. 2010Schoereder JH, Sobrinho TG, Madureira MS, Ribas CR, Oliveira OS. 2010. The arboreal ant community visiting extrafloral nectaries in the Neotropical cerrado savanna. Terrestrial Arthropod Reviews 3: 3-27. ; Weber & Keeler 2013Weber MG, Keeler KH. 2013. The phylogenetic distribution of extrafloral nectaries in plants. Annals of Botany 111: 1251-1261. ). Nevertheless, in Brazil, most of EFNs records are given from Cerrado areas, where anatomical and ecological studies were vastly performed (Oliveira & Leitão-Filho 1987Oliveira PS, Leitão-Filho HF. 1987. Extrafloral nectaries: their taxonomic distribution and abundance in the woody flora of cerrado vegetation in Southeast Brazil. Biotropica 19: 140-148.; Oliveira & Pie 1998Oliveira PS, Pie MR. 1998. Interaction between ants and plants bearing extrafloral nectaries in cerrado vegetation. Anais da Sociedade Entomológica do Brasil 27: 161-176.; Del-Claro & Santos 2000Del-Claro K, Santos JC. 2000. A função de nectários extraflorais em plantas do cerrado. Tópicos atuais em botânica. Brasília, Embrapa.; Santos & Del-Claro 2001Santos JC, Del-Claro K. 2001. Interação entre formigas, herbívoros e nectários extraflorais em Tocoyena formosa (Cham. and Schlechtd.) K. Schum. (Rubiaceae) na vegetação do cerrado. Revista Brasileira de Zoociências (Juiz de Fora-MG) 3: 77-92.; Machado et al. 2008Machado SR, Morellato SPC, Sajo MG, Oliveira PS. 2008. Morphological patterns of extrafloral nectaries in woody plant species of the Brazilian cerrado. Plant Biology 10: 660-673. ; Nascimento & Del-Claro 2010Nascimento EA, Del-Claro K. 2010. Ant visitation to extrafloral nectaries decreases herbivory and increases fruit set in Chamaecrista debilis (Leguminosae) in a Neotropical savanna. Flora-Morphology, Distribution, Functional Ecology of Plants 205: 754-756. ; Schoereder et al. 2010Schoereder JH, Sobrinho TG, Madureira MS, Ribas CR, Oliveira OS. 2010. The arboreal ant community visiting extrafloral nectaries in the Neotropical cerrado savanna. Terrestrial Arthropod Reviews 3: 3-27. ; Anjos et al. 2017Anjos DV, Caserio B, Rezende FT, Ribeiro SP, Del-Claro K, Fagundes R. 2017. Extrafloral nectaries and interspecific aggressiveness regulate day/night turnover of ant species foraging for nectar on Bionia coriacea. Austral Ecology 42: 317-328. ; Pires et al. 2017Pires MS, Calixto ES, Oliveira DC, Del-Claro K. 2017. A new extrafloral nectary-bearing plant species in the Brazilian savanna and its associated ant community: nectary structure, nectar production and ecological interactions. Sociobiology 64: 228-236. ).

Even though plants bearing EFNs comprises a considerable fraction of plant species in tropical ecosystems (Schupp & Feener 1991Schupp EW, Feener DH. 1991. Phylogeny, lifeform, habitat dependence of ant-defended plants in a Panamanian forest. In: Cutler DF, Huxley CR. (eds.) Ant-Plant Interactions. Oxford, Oxford University Press. p. 175-197.; Fiala & Linsenmair 1995Fiala B, Linsenmair KE. 1995. Distribution and abundance of plants with extrafloral nectaries in the woody flora of a lowland primary forest in Malaysia. Biodiversity and Conservation 4: 165-182.; Morellato & Oliveira 1991Morellato LPC, Oliveira PS. 1991. Distribution of extrafloral nectaries in different vegetation types of Amazonian Brazil. Flora 185: 33-38.; 1994Morellato LPC, Oliveira PS. 1994. Extrafloral nectaries in the tropical tree Guarea macrophylla (Meliaceae). Canadian Journal of Botany 72: 157-160.; Blüthgen et al. 2000Blüthgen N, Verhaagh M, Goitía W, Jaffé K, Morawetz W, Barthlott W. 2000. How plants shape the ant community in the Amazonian rainforest canopy: the key role of extrafloral nectaries and homopteran honeydew. Oecologia 125: 229-240.), few studies surveyed EFN-bearing plants distribution in the Amazonian forest (Morellato & Oliveira 1991Morellato LPC, Oliveira PS. 1991. Distribution of extrafloral nectaries in different vegetation types of Amazonian Brazil. Flora 185: 33-38.; 1994Morellato LPC, Oliveira PS. 1994. Extrafloral nectaries in the tropical tree Guarea macrophylla (Meliaceae). Canadian Journal of Botany 72: 157-160.; Blüthgen et al. 2000Blüthgen N, Verhaagh M, Goitía W, Jaffé K, Morawetz W, Barthlott W. 2000. How plants shape the ant community in the Amazonian rainforest canopy: the key role of extrafloral nectaries and homopteran honeydew. Oecologia 125: 229-240.; Nogueira et al. 2020Nogueira A, Baccaro FB, Leal LC, Rey PJ, Lohmann LG, Bronstein JL. 2020. Variation in the production of plant tissues bearing extrafloral nectaries explains temporal patterns of ant attendance in Amazonian understorey plants. Journal of Ecology 108: 1578-1591.), Caatinga (e.g.Melo et al. 2010aMelo Y, Machado SR, Alves M. 2010a. Anatomy of extrafloral nectaries in Leguminosae from dry-seasonal forest in Brazil. Botanical Journal of the Linnean Society 163: 87-98.; bMelo Y, Córdula E, Machado SR, Alves M. 2010b. Morfologia de nectários em Leguminosae senso lato em áreas de caatinga no Brasil. Acta Botanica Brasilica 24: 1034-1045. ), and Atlantic forest (e.g.Almeida & Figueiredo 2003Almeida AM, Figueiredo RA. 2003. Ants visit nectaries of Epidendrum denticulatum (Orchidaceae) in a Brazilian rainforest: effects on herbivory and pollination. Brazilian Journal of Biology 63: 551-558.; Câmara et al. 2017Câmara T, Almeida WR, Tabarelli M, Leal IR. 2017. Habitat fragmentation, EFN-bearing trees and ant communities: Ecological cascades in Atlantic forest of northeastern Brazil. Austral Ecology 42: 31-39. ; Dutra 2019Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus.), a hotspot for conservation priorities (Myers et al. 2000Myers N, Mittermeier RA, Mittermeier CG, Fonseca GAB, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858. ). Under this point of view, this study focused on providing one of the first checklists of angiosperms with EFNs in a protected area of the Atlantic forest, bringing information about the taxonomic richness, their location on the plant’s organs as well as plant’s habit and conservation status.

Material and methods

The study was carried out in the State Park of Itaúnas (PEI), managed by ‘Instituto Estadual de Meio Ambiente e Recursos Hídricos’ (IEMA), located in the municipality of Conceição da Barra, on the shore region of northern Espírito Santo State, Brazil (Fig. 1). It has an area of approximately 3,500 ha, mean annual temperature ranges between 21.7º-26.7ºC, total annual precipitation of approximately 1,310 mm, and Köppen’s classification ‘Aw’ climate with rainy months during summer (Souza et al. 2016Souza WO, Machado JO, Tognella MM, Alves-Araújo A. 2016. Checklist of Angiosperms from Parque Estadual de Itaúnas, Espírito Santo, Brazil. Rodriguésia 67: 571-581. ). Most vegetation coverage is represented by 'restingas' (vegetation on sandy soils) and dunes, however, wetlands, 'Tabuleiro' forest (flat relief and soil from the tertiary formation), and mangroves are also recorded.

Figure 1
Study area. Geographic location of the State Park of Itaúnas.

The list herein provided is based on the previous angiosperm checklist published by Souza et al. (2016Souza WO, Machado JO, Tognella MM, Alves-Araújo A. 2016. Checklist of Angiosperms from Parque Estadual de Itaúnas, Espírito Santo, Brazil. Rodriguésia 67: 571-581. ). Vouchers were analyzed by observation of EFNs in order to investigate the taxonomic richness of EFN-bearing plants in the study area. In addition, field expeditions for collecting fresh material were performed from July/2017 to August/2019. Only species with visible secreting structures or secretions visited and defended by ants (not sampled) were included and, following Elias (1983Elias TS. 1983. Extrafloral nectaries: their structure and distribution. In: Bentley BL, Elias TS (eds.) The biology of nectaries. New York, Columbia University Press. p. 174-203.) and Delpino (1874Delpino F. 1874. Rapporti tra insetti e nettari extranuziali nelle plante. Bollettino della Società Entomologica Italiana 6: 234-239.), both extrafloral and extranuptial nectaries were considered. All fertile specimens were observed, collected, photographed, and processed according to traditional methods in plant taxonomy (Peixoto & Maia 2013Peixoto AL, Maia LC. 2013. Manual de Procedimentos para Herbários. INCT-Herbário virtual para a Flora e os Fungos. Recife, Editora Universitária UFPE.) for further incorporation in the Federal University of Espírito Santo herbarium, VIES (acronym follows Thiers (2021Thiers B. 2021. Index Herbariorum: A global directory of public herbaria and associated staff. New York, Botanical Garden’s Virtual Herbarium. http://sweetgum.nybg.org/science/ih/. 13 Aug. 2021.
http://sweetgum.nybg.org/science/ih/... )). All species with EFNs were thoroughly analyzed regarding characteristics such as plant habit, EFN location, and conservation status. After analyzing all samples, a careful literature search aiming to bring more information about EFN-bearing species was carried out. The World List of plants with EFNs (Weber et al. 2015Weber MG, Porturas LD, Keeler KH. 2015. World list of plants with extrafloral nectaries World list of plants with extrafloral nectaries www.extrafloralnectaries.org . 10 Oct. 2019.
www.extrafloralnectaries.org... ) was consulted and the list of specimen vouchers can be found in Table 1.

Family names followed the Angiosperm Phylogeny Group (APG IV (2016)APG IV. 2016. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society 181: 1-20. ) with some exceptions such as Passifloraceae and Turneraceae, herein recognized as different families. Author names follow the International Plant Names Index (IPNI), and conservation status was established according to the Centro Nacional de Conservação da Flora - CNCFlora (2021)CNCFlora - Centro Nacional de Conservação da Flora. 2021. http://www.cncflora.jbrj.gov.br/portal. 13 Aug. 2021.
http://www.cncflora.jbrj.gov.br/portal... .

Results

Of the 562 species previously surveyed in the study area, 93 species belonging to 61 genera and 29 families have EFNs (Tab. 1). Most of them (82 spp.) has no extinction risk assignments (NE - not evaluated), eight are categorized as Least Concern (LC), while Davilla cf. macrocarpa as Vulnerable (VU), Inga exfoliata as Nearly Threatened (NT), and Humiriastrum spiritu-sancti as Critically Endangered (CR) (Fig. 2A). Eudicotyledons are represented by 81 species, while 11 are monocotyledons and a single one is a basal angiosperm (Annona glabra). Moreover, 20 out of 93 species (21.5 %) are endemic to the Brazilian Atlantic forest, but none of them exclusive to the study area (Fig. 2B).

Most of the species was recorded in the ‘restingas’ formations (90 spp.), however Inga exfoliata, Laguncularia racemosa, and Ipomoea pes-caprae were exclusively found in the ‘Tabuleiro’ forest, mangroves, and dunes, respectively. The list brings a total of 50 species not previously cited as having EFNs, which represents new records for the World checklist of EFN-bearing plants.

The top five richest families and genera (Fig. 2C-D) hold more than half (54.8 %) and almost one third (30 %) of the species richness in the checklist, respectively. Considering plant habit, vines (woody or herbaceous) are predominant (Fig. 2E) with Passiflora (Passifloraceae) (nine spp.) and Smilax (Smilacaceae) (five spp.) as the top richest genera (Tab.1). On the other hand, herbs are the less species-rich group where Philodendron fragrantissimum and P. pedatum (Araceae) are hemiepiphytes, and Ctenanthe glabra (Marantaceae) and Epidendrum denticulatum (Orchidaceae) are terrestrial.

Figure 2
EFNs-bearing plants of the State Park of Itaúnas, Espírito Santo State, Brazil. A. Conservation status based on CNCFlora (2021)CNCFlora - Centro Nacional de Conservação da Flora. 2021. http://www.cncflora.jbrj.gov.br/portal. 13 Aug. 2021.
http://www.cncflora.jbrj.gov.br/portal... . B. Number of endemic species per family. C. Number of species per family. D. Number of species per genus. E. Percentage of plant habits. F. Percentage of EFNs location on plant’s body. CR= critically endangered; LC=least concern; NE=not evaluated; NT=nearly threatened; VU=vulnerable.

EFNs are more commonly found exclusively on the leaf blades (or leaflet blades + rachises) (65 species), followed by exclusively on the petioles with 20 species, and exclusively on calyx (five species). They are less frequently present on axillary buds, stipules, bracts or bracteoles, pedicels or flower buds, and fruits (Figs. 2F, 3, 4, 5). Some species show EFNs in different plant organs and not only in a restricted location, such as in Anacardium occidentale (Anacardiaceae), Philodendron species (Araceae), Canavalia rosea and Crotalaria incana (Fabaceae), and Guarea macrophylla (Meliaceae) (Tab. 1). Tocoyena formosa (Rubiaceae) has an ordinary nectary, forming a disk on the ovary apex that secretes nectar for pollination process, however it continues to exudate during fruit development.

Figure 3
EFNs of the State Park of Itaúnas, Espírito Santo State, Brazil. A. Amphilophium frutescens. B. Cynophalla flexuosa. C. Chrysobalanus icaco. D. Hirtella corymbosa. E. Ipomoea pes-caprae. F. Ipomoea fimbriosepala. G. Davilla cf. macrocarpa. H. Abarema filamentosa. I. Chamaecrista flexuosa. Bars= 1cm; EFNs= extrafloral nectaries; white arrows= EFNs (Photos A-D and G-H by Rafael S. Mathielo).

Figure 4
EFNs of the State Park of Itaúnas, Espírito Santo State, Brazil. A. Inga subnuda. B. Piptadenia adiantoides. C. Senna appendiculata. D. Humiria balsamifera. E. Humiriastrum spiritu-sancti. F. Stigmaphyllon ciliatum. G. Stigmaphyllon paralias. H-I. Talipariti pernambucensis. Bars= 1cm; EFNs= extrafloral nectaries; white arrows= EFNs (Photos A-B and D-G by Rafael S. Mathielo)

Figure 5
EFNs of the State Park of Itaúnas, Espírito Santo State, Brazil. A. Ouratea bahiensis. B. Epidendrum denticulatum. C. Passiflora alata. D-E. Passiflora mucronata. F. Tocoyena formosa. G. Turnera hermannioides. H. Turnera subulata. I. Stachytarpheta cayennensis. Bars= 1cm; EFNs= extrafloral nectaries; white arrows= EFNs (Photos A-F and H by Rafael S. Mathielo)

Discussion

EFNs taxonomic distribution

The species with EFNs in PEI corresponds to 16.5 % of the local flora which is somehow expected when compared to other studies, where the percentage values for EFN-bearing plants varies from 15 to 31 % depending on the type of vegetation (e.g.Madison 1979Madison M. 1979. Additional observations on ant-gardens in Amazonas. Selbyana 5: 107-115.; Oliveira et al. 1987Oliveira PS, Silva AF, Martins AB. 1987. Ant foraging on extrafloral nectaries of Qualea grandiflora (Vochysiaceae) in cerrado vegetation: ants as potential antiherbivore agents. Oecologia 74: 228-230.; Morellato & Oliveira 1991Morellato LPC, Oliveira PS. 1991. Distribution of extrafloral nectaries in different vegetation types of Amazonian Brazil. Flora 185: 33-38., 1994Morellato LPC, Oliveira PS. 1994. Extrafloral nectaries in the tropical tree Guarea macrophylla (Meliaceae). Canadian Journal of Botany 72: 157-160.; Del-Claro et al. 1996Del-Claro K, Berto V, Réu W. 1996. Effect of herbivore deterrence by ants increase fruit set in an extrafloral nectary plant, Qualea multiflora (Vochysiaceae). Journal of Tropical Ecology 12: 887-892. ; Oliveira & Pie 1998Oliveira PS, Pie MR. 1998. Interaction between ants and plants bearing extrafloral nectaries in cerrado vegetation. Anais da Sociedade Entomológica do Brasil 27: 161-176.; Blüthgen et al. 2000Blüthgen N, Verhaagh M, Goitía W, Jaffé K, Morawetz W, Barthlott W. 2000. How plants shape the ant community in the Amazonian rainforest canopy: the key role of extrafloral nectaries and homopteran honeydew. Oecologia 125: 229-240.; Del-Claro & Santos 2000Del-Claro K, Santos JC. 2000. A função de nectários extraflorais em plantas do cerrado. Tópicos atuais em botânica. Brasília, Embrapa.; Machado et al. 2008Machado SR, Morellato SPC, Sajo MG, Oliveira PS. 2008. Morphological patterns of extrafloral nectaries in woody plant species of the Brazilian cerrado. Plant Biology 10: 660-673. ; Schoereder et al. 2010Schoereder JH, Sobrinho TG, Madureira MS, Ribas CR, Oliveira OS. 2010. The arboreal ant community visiting extrafloral nectaries in the Neotropical cerrado savanna. Terrestrial Arthropod Reviews 3: 3-27. ; Nascimento & Del-Claro 2010Nascimento EA, Del-Claro K. 2010. Ant visitation to extrafloral nectaries decreases herbivory and increases fruit set in Chamaecrista debilis (Leguminosae) in a Neotropical savanna. Flora-Morphology, Distribution, Functional Ecology of Plants 205: 754-756. ; Del-Claro et al. 2013Del-Claro K, Stefani V, Lange D et al. 2013. The importance of natural history studies for a better comprehension of animal-plant interaction networks. Bioscience Journal 29: 438-448.; Lange et al. 2013Lange D, Dáttilo W, Del-Claro K. 2013. Influence of extrafloral nectary phenology on ant-plant mutualistic networks in a neotropical savanna. Ecological Entomology 38: 463-469.; Assunção et al. 2014Assunção MA, Torezan-Silingardi HM, Del-Claro K. 2014. Do ant visitors to extrafloral nectaries of plants repel pollinators and cause an indirect cost of mutualism? Flora-Morphology, Distribution, Functional Ecology of Plants 209: 244-249. ; Campos & Camacho 2014Campos RI, Camacho GP. 2014. Ant-plant interactions: the importance of extrafloral nectaries versus hemipteran honeydew on plant defense against herbivores. Arthropod-Plant Interactions 8: 507-512. ; Lange & Del-Claro 2014Lange D, Del-Claro K. 2014. Ant-plant interaction in a tropical savanna: may the network structure vary over time and influence on the outcomes of associations? PLOS ONE 9: e105574. https://doi.org/10.1371/journal.pone.0105574.
https://doi.org/10.1371/journal.pone.010... ; Anjos et al. 2017Anjos DV, Caserio B, Rezende FT, Ribeiro SP, Del-Claro K, Fagundes R. 2017. Extrafloral nectaries and interspecific aggressiveness regulate day/night turnover of ant species foraging for nectar on Bionia coriacea. Austral Ecology 42: 317-328. ; Dutra 2019Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus.). Specifically concerning our study site, located in the Atlantic forest, despite absolute richness is much higher compared to previous studies - 93 against 23 (Câmara et al. 2017Câmara T, Almeida WR, Tabarelli M, Leal IR. 2017. Habitat fragmentation, EFN-bearing trees and ant communities: Ecological cascades in Atlantic forest of northeastern Brazil. Austral Ecology 42: 31-39. ) and 46 species (Dutra 2019Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus.) - the relative values is approximate to the previously encountered, ranging between 12.2-17.1 % (e.g.Câmara et al. 2017Câmara T, Almeida WR, Tabarelli M, Leal IR. 2017. Habitat fragmentation, EFN-bearing trees and ant communities: Ecological cascades in Atlantic forest of northeastern Brazil. Austral Ecology 42: 31-39. ; Dutra 2019Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus.). However, unlike Dutra (2019)Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus., Câmara et al. (2017)Câmara T, Almeida WR, Tabarelli M, Leal IR. 2017. Habitat fragmentation, EFN-bearing trees and ant communities: Ecological cascades in Atlantic forest of northeastern Brazil. Austral Ecology 42: 31-39. included only trees in their analysis and the percentage likely would change if considered non-tree species.

Depending on environment, the habit predominance varies and, despite showing different phytophysiognomies, the proportion of EFN-bearing plants may converge. For instance, ‘restingas’ vegetation in PEI and semideciduous seasonal tropical forest (Dutra 2019Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus.), both in the Atlantic forest domain, are represented by vines as the richest habit of EFNs-bearing plant, followed by trees and shrubs. This pattern is different compared to other phytogeographic domains, such as the Cerrado and the Amazon forest where shrubs and lianas/epiphytes are the most common elements (Oliveira & Leitão-Filho 1987Oliveira PS, Leitão-Filho HF. 1987. Extrafloral nectaries: their taxonomic distribution and abundance in the woody flora of cerrado vegetation in Southeast Brazil. Biotropica 19: 140-148.; Oliveira & Oliveira-Filho 1991Oliveira PS, Oliveira-Filho AT. 1991. Distribution of extrafloral nectaries in the woody flora of tropical communities in western Brazil. In: Price PW, Lewinsohn TM, Fernandes G, Benson WW. (eds.) Plant-animal interactions: Evolutionary ecology in tropical and temperate regions. New York, John Wiley and Sons. p. 163-175.; Pires 2015Pires MS. 2015. The community extrafloral nectaries bearing plants in a Brazilian savanna: morphology, phenology and associated fauna. MSc Thesis. Universidade Federal de Uberlândia, Uberlândia.; Blüthgen et al. 2000Blüthgen N, Verhaagh M, Goitía W, Jaffé K, Morawetz W, Barthlott W. 2000. How plants shape the ant community in the Amazonian rainforest canopy: the key role of extrafloral nectaries and homopteran honeydew. Oecologia 125: 229-240.). Non-tree plants represent almost 70 % of the PEI’s flora with EFNs, however, they consist of a commonly neglected group of plants in broader surveys and the EFNs richness is possibly underestimated in several areas. According to Gentry & Dodson (1987Gentry AH, Dodson C. 1987. Contributions of nontrees to species richness of a tropical rainforest. Biotropica 19: 149-156.), herbs and shrubs, together, contribute with 33 to 52 % of the local diversity in tropical ecosystems. Thus, even with ephemeral life cycles, herbs have high importance in the vegetation dynamics (Lima 2011Lima EM. 2011. Influência do componente herbáceo da Caatinga na regeneração natural de plantas lenhosas em uma área de vegetação preservada e uma área de agricultura abandonada. PhD Thesis, Recife, Universidade Federal Rural de Pernambuco.) with EFNs and visitors establishing key ecological interactions.

The four richest families in PEI are in accordance with previous studies that listed them as the most common elements regardless phytophysiognomy and plant community composition (Oliveira & Leitão-Filho 1987Oliveira PS, Leitão-Filho HF. 1987. Extrafloral nectaries: their taxonomic distribution and abundance in the woody flora of cerrado vegetation in Southeast Brazil. Biotropica 19: 140-148.; Morellato & Oliveira 1991Morellato LPC, Oliveira PS. 1991. Distribution of extrafloral nectaries in different vegetation types of Amazonian Brazil. Flora 185: 33-38.; Díaz-Castelazo et al. 2005Díaz-Castelazo C, Rico-Gray V, Ortega F, Ángeles G. 2005. Morphological and secretory characterization of extrafloral nectaries in plants of coastal Veracruz, Mexico. Annals of Botany 96: 1175-1189. ; Machado et al. 2008Machado SR, Morellato SPC, Sajo MG, Oliveira PS. 2008. Morphological patterns of extrafloral nectaries in woody plant species of the Brazilian cerrado. Plant Biology 10: 660-673. ; Melo et al. 2010bMelo Y, Córdula E, Machado SR, Alves M. 2010b. Morfologia de nectários em Leguminosae senso lato em áreas de caatinga no Brasil. Acta Botanica Brasilica 24: 1034-1045. ; Urbanetz et al. 2010Urbanetz C, Tamashiro JY, Kinoshita LS. 2010. Chave de identificação de espécies lenhosas de um trecho de Floresta Ombrófila Densa Atlântica, no Sudeste do Brasil, baseada em caracteres vegetativos. Biota Neotropica 10: 349-398.; Weber et al. 2015Weber MG, Porturas LD, Keeler KH. 2015. World list of plants with extrafloral nectaries World list of plants with extrafloral nectaries www.extrafloralnectaries.org . 10 Oct. 2019.
www.extrafloralnectaries.org... ). Floristics comparisons are fundamental, however most of the plant checklists are essentially based on general information with no further biological aspects, such as the presence of EFNs themselves. By performing similarity analysis, for instance using those checklists, the results may not reflect the real richness of EFN-bearing plants by the inclusion of the well-known species only. More detailed data about the predominant plant habit, composition, proportion of EFN-bearing plants, and/or local phytophysiognomy may explain the distributional patterns of the vegetation. Even so, this issue has not been properly studied yet and available studies do not bring this specific data.

Although PEI has a huge vegetational heterogeneity (see Souza et al. 2016Souza WO, Machado JO, Tognella MM, Alves-Araújo A. 2016. Checklist of Angiosperms from Parque Estadual de Itaúnas, Espírito Santo, Brazil. Rodriguésia 67: 571-581. ), the ‘restingas’ are predominant and hold over 96 % of local EFN-bearing plant richness. These results corroborate the higher richness values for floras from open areas (Schupp & Feener 1991Schupp EW, Feener DH. 1991. Phylogeny, lifeform, habitat dependence of ant-defended plants in a Panamanian forest. In: Cutler DF, Huxley CR. (eds.) Ant-Plant Interactions. Oxford, Oxford University Press. p. 175-197.; Fiala & Lisenmair 1995Fiala B, Linsenmair KE. 1995. Distribution and abundance of plants with extrafloral nectaries in the woody flora of a lowland primary forest in Malaysia. Biodiversity and Conservation 4: 165-182.; Blüthgen & Reifenrath 2003Blüthgen N, Reifenrath K. 2003. Extrafloral nectaries in an Australian rainforest: structure and distribution. Australian Journal of Botany 51: 515–527.). The structure of the vegetation affects the distribution patterns and, according to Câmara et al. (2017Câmara T, Almeida WR, Tabarelli M, Leal IR. 2017. Habitat fragmentation, EFN-bearing trees and ant communities: Ecological cascades in Atlantic forest of northeastern Brazil. Austral Ecology 42: 31-39. ) and Díaz-Castelazo et al. (2020Díaz-Castelazo C, Martínez-Adriano CA, Dáttilo W, Rico-Gray V. 2020. Relative contribution of ecological and biological attributes in the fine-grain structure of ant-plant networks. PeerJ 8: e8314. http://doi.org/10.7717/peerj.8314.
http://doi.org/10.7717/peerj.8314... ), shaded habitats have a different pattern when compared to the open ones.

More than half of the herein listed species (53.7 %) comprises new additions to the world list of plants with EFNs, including the first record in the Dilleniaceae (see Weber et al. 2015Weber MG, Porturas LD, Keeler KH. 2015. World list of plants with extrafloral nectaries World list of plants with extrafloral nectaries www.extrafloralnectaries.org . 10 Oct. 2019.
www.extrafloralnectaries.org... ), and 21.5 % of them are endemic to the Atlantic forest. However, despite its importance as a basis for further ecological studies, especially on ant-plant interactions, information about species conservation status is vastly lacking with few assignments to a given CNCFlora’s category.

EFNs location and ecological insights

In PEI, most of EFNs (around 90 %) are found on leaves which is a common pattern (Melo et al. 2010aMelo Y, Machado SR, Alves M. 2010a. Anatomy of extrafloral nectaries in Leguminosae from dry-seasonal forest in Brazil. Botanical Journal of the Linnean Society 163: 87-98.; bMelo Y, Córdula E, Machado SR, Alves M. 2010b. Morfologia de nectários em Leguminosae senso lato em áreas de caatinga no Brasil. Acta Botanica Brasilica 24: 1034-1045. ; Pscheidt & Cordeiro 2012Pscheidt AC, Cordeiro I. 2012. Synopsis of the tribe Hippomaneae (Euphorbiaceae) in São Paulo State, Brazil. Hoehnea 39: 347-368. ; Silva 2015Silva DR. 2015. Efeitos da poluição atmosférica de Ipatinga-MG sobre Joannesia princeps Vell. (Euphorbiaceae) com ênfase nos nectários extraflorais. PhD Thesis, Universidade Federal de Viçosa, Viçosa.; Weber et al. 2015Weber MG, Porturas LD, Keeler KH. 2015. World list of plants with extrafloral nectaries World list of plants with extrafloral nectaries www.extrafloralnectaries.org . 10 Oct. 2019.
www.extrafloralnectaries.org... ; Dutra 2019Dutra SC. 2019. Angiospermas com nectários extraflorais e domácias em um fragmento de mata atlântica no norte do Espírito Santo, Brasil. MSc Thesis. Universidade Federal do Espírito Santo, São Mateus.). Those leaf EFNs are classified as both extrafloral and extranuptial, however, there are examples of floral and nuptial nectaries with extranuptial function (Santos & Del-Claro 2001Santos JC, Del-Claro K. 2001. Interação entre formigas, herbívoros e nectários extraflorais em Tocoyena formosa (Cham. and Schlechtd.) K. Schum. (Rubiaceae) na vegetação do cerrado. Revista Brasileira de Zoociências (Juiz de Fora-MG) 3: 77-92.). For example, Tocoyena formosa (Rubiaceae) and Epidendrum denticulatum (Orchidaceae) present floral nectaries with singular extranuptial activity (Santos & Del-Claro 2001Santos JC, Del-Claro K. 2001. Interação entre formigas, herbívoros e nectários extraflorais em Tocoyena formosa (Cham. and Schlechtd.) K. Schum. (Rubiaceae) na vegetação do cerrado. Revista Brasileira de Zoociências (Juiz de Fora-MG) 3: 77-92.; Almeida & Figueiredo 2003Almeida AM, Figueiredo RA. 2003. Ants visit nectaries of Epidendrum denticulatum (Orchidaceae) in a Brazilian rainforest: effects on herbivory and pollination. Brazilian Journal of Biology 63: 551-558.; Sousa-Lopes et al. 2020Sousa-Lopes B, Calixto ES, Torezan-Silingardi HM, Del-Claro K. 2020. Effects of ants on pollinator performance in a distylous pericarpial nectary-bearing Rubiaceae in Brazilian Cerrado. Sociobiology 67: 173-185. ). Tocoyena formosa has a nectar gland surrounding the apex of the ovary during blooming and, after pollination, the nectariferous disk on young fruits are fully functional and produce an exudate which is attractive for ants. Epidendrum denticulatum produces nectar on the apex of the peduncle on buds, flowers, and fruits that, by attracting ants, provide an efficient protection against herbivory (Calvo 1990Calvo RN. 1990. Inflorescence size and fruit distribution among individuals in three orchid species. American Journal of Botany 77: 1378-1381. ; 1993Calvo RN. 1993. Evolutionary demography of orchids, intensity and frequency of pollination and the cost of fruiting. Ecology 74: 1033-1042. ; Almeida & Figueiredo 2003Almeida AM, Figueiredo RA. 2003. Ants visit nectaries of Epidendrum denticulatum (Orchidaceae) in a Brazilian rainforest: effects on herbivory and pollination. Brazilian Journal of Biology 63: 551-558.; Ballarin et al. 2019Ballarin CS, Hachuy-Filho L, Sanz-Veiga PA, Amorim FW. 2019. The resource-mediated modular structure of a non-symbiotic ant-plant mutualism. Ecological Entomology 45: 121-129. ).

Five species belonging to Smilax (Smilacaceae) have inconspicuous EFNs only noticed due to ant visitation and secreted drops on the petiole. Our results provide new records for the genus adding up to the world list of plants with EFNs. This secreting activity was previously noticed to a native species from Cerrado, Smilax polyantha Griseb. which has EFNs composed of few cell layers with no external structure (Pires et al. 2017Pires MS, Calixto ES, Oliveira DC, Del-Claro K. 2017. A new extrafloral nectary-bearing plant species in the Brazilian savanna and its associated ant community: nectary structure, nectar production and ecological interactions. Sociobiology 64: 228-236. ). On the other hand, Ouratea bahiensis and O. parvifolia (Ochnaceae) have conspicuous EFNs on the stipules of the young branches (Fig. 5A). Those stipules are frequent and intensively visited by ants (pers. observ.) for a short time during the branch development, just before they fall off. After stipule’ senescence, signs of herbivory were noticed on mature leaves (pers. observ.).

In this study, we provide a relevant update for the world list of angiosperms with EFNs. Most importantly, it consists of a massive number of EFN-bearing plant species from a single and small protected area where we found high values of richness. It reinforces the lack of studies of this nature in the Brazilian Atlantic forest, where further investigations are strongly recommended. We realized that studies about ant-plant interactions have usually been the main source of detecting EFN-bearing plants (Calixto et al. 2018Calixto ES, Lange D, Del-Claro K. 2018. Protection mutualism: an overview of the ant-plant interactions mediated by extrafloral nectaries. Oecologia Australis 22: 410-425.) and, thus, they modulated our knowledge case-to-case about plant richness (e.g.Calixto et al. 2020Calixto ES, Lange D, Moreira X, Del-Claro K. 2020. Plant species specificity of ant-plant mutualistic interactions: Differential predation of termites by Camponotus crassus on five species of extrafloral nectaries plants. Biotropica, Early view: 1-9. ; 2021Calixto ES, Novaes LR, Santos DFB, Lange D, Moreira X, Del-Claro K. 2021. Climate seasonality drives ant-plant-herbivore interactions via plant phenology in an extrafloral nectary-bearing plant community. Journal of Ecology 109: 167-178.). EFNs are relevant mediators of the ant-plant mutualism (Calixto et al. 2018Calixto ES, Lange D, Del-Claro K. 2018. Protection mutualism: an overview of the ant-plant interactions mediated by extrafloral nectaries. Oecologia Australis 22: 410-425.), which can be an important enhancer of ecosystem biodiversity (Heil 2011Heil M. 2011. Nectar: generation, regulation and ecological functions. Trends in Plant Science 16 (4): 191-200. ). So, by knowing the richness of EFN-bearing flora, the chances of ensuring ecosystem conservation are greater. These results are part of ongoing studies on EFNs in areas of the Atlantic forest involving mainly cataloging of biodiversity. Here, we contribute with a thoroughly investigated EFN-bearing plant database that will allow the academic community to perform further studies regarding botanical, ecological, and evolutionary issues. A novel research avenue that could also be explored in future studies is the investigations of the functional attributes of EFN in plant communities. For example, two recent studies suggest that nectar volume and concentration in EFN are strongly related to ant-plant community structure and defensive behavior in ants that visit EFNs (Ballarin et al. 2019Ballarin CS, Hachuy-Filho L, Sanz-Veiga PA, Amorim FW. 2019. The resource-mediated modular structure of a non-symbiotic ant-plant mutualism. Ecological Entomology 45: 121-129. ; Calixto et al. 2021Calixto ES, Novaes LR, Santos DFB, Lange D, Moreira X, Del-Claro K. 2021. Climate seasonality drives ant-plant-herbivore interactions via plant phenology in an extrafloral nectary-bearing plant community. Journal of Ecology 109: 167-178.). These studies point out that other EFN traits (e.g., nectar volume and concentration) could be relevant for future surveys since they are closely related to ecological processes and patterns. Therefore, we point that not only novel inventories must be made in underappreciated ecosystems, but also meticulous studies of EFN traits should help novel studies to be developed.

Acknowledgements

We acknowledge to Fundação de Amparo à Pesquisa e Inovação do Espírito Santo-FAPES ((Edital FAPES Nº 21/2018 - Universal TO Nº 123/2019) (FAPES Nº 18/2018, TO 525/2018)), CNPq and Capes for providing financial support by fellowships to LG, VSM and SCD, UFES-Campus São Mateus and PEI for logistical support during fieldwork. Authors thank to Rafael Scherrer Mathielo for taking the images, and lastly to the anonymous reviewers who helped us to improve the manuscript.

References

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