Annals of Botany 111: 1277–1283, 2013 doi:10.1093/aob/mct063, available online at www.aob.oxfordjournals.org HIGHLIGHT ON ECOLOGY AND EVOLUTION OF EXTRAFLORAL NECTARIES Nectar secretion on fern fronds associated with lower levels of herbivore damage: field experiments with a widespread epiphyte of Mexican cloud forest remnants Suzanne Koptur1,*, Mo´nica Palacios-Rios2, Cecilia Dı´az-Castelazo3, William P. Mackay4 and Vı´ctor Rico-Gray5 1Department of Biological Sciences, Florida International University, Miami, FL 33199, USA, 2Posgrado INBIOTECA, Universidad Veracruzana, Xalapa, Veracruz 91190, Me´xico, 3Departamento de Interacciones Multitro´ficas, Instituto de Ecologı´a, A.C., Xalapa, Veracruz 91070, Me´xico, 4Department of Biological Sciences, University of Texas, El Paso, TX 79968, USA and 5Instituto de Neuroetologı´a, Universidad Veracruzana, Xalapa, Veracruz 91190, Me´xico Downloaded from * For correspondence. E-mail kopturs@fiu.edu Received: 13 August 2012 Revision requested: 25 September 2012 Accepted: 7 February 2013 Published electronically: 21 April 2013 † Background and Aims The oldest group of plants in which nectar secretions have been observed are the http://aob.oxfordjournals.org/ Polypodiopsida (ferns sensu lato). Nectaries have been reported in a dozen extant genera. The function of these nec- taries has been investigated in several fern species, and in some circumstances has been demonstrated to have an antiherbivore role, attracting and maintaining biotic defence (ants and/or other predatory arthropods). This study documents foliar nectaries in Pleopeltis crassinervata, a widespread Central American epiphyte growing on a variety of trees in cloud forest areas of Veracruz, Mexico. This is a new record for this genus and species. † Methods As previous experimental work on epiphytic species of Polypodium has demonstrated a protective role of ants for developing fronds, we conducted similar experiments (using nylon nail polish to cover nectaries rather than excluding ants with bands of sticky resin as in earlier work). The fronds of Pl. crassinervata developed over 6 weeks, at which time damage was assessed. The experiment was simultaneously conducted on a sympatric at Florida International University on May 24, 2013 species lacking nectaries, Polypodium furfuraceum. Herbivore placement experiments were conducted with large and small caterpillars on both of these ferns. † Key Results Fronds with nectaries covered suffered greater damage from herbivores over the course of their development, compared with fronds that had uncovered nectaries functioning normally. The parallel experiment on Po. furfuraceum showed no difference between manipulated and control fronds. Six species of ants (Brachymyrmex minutus, Crematogaster formosa, Paratrechina longicornis, Solenopsis geminata, S. picea and Wasmannia auropunctata) were observed visiting nectaries of Pl. crassinervata; most were effective in removing herbivore larvae placed on the fronds. † Conclusions The long evolutionary history of ferns may explain why some previous studies of fern nectaries have shown little or no benefit to ferns from nectary visitors, as any coevolved herbivores are those resistant to ant defence. The results suggest that ants protect Pl. crassinervata fronds against herbivory. The presence of nectaries, and the relationship with ants, may contribute to this fern’s widespread occurrence and persistence in the face of disturbance, though many other factors also play a role. Ant defence may be more likely to benefit a widespread species of disturbed habitats that encounters a wide range of non-adapted herbivores. Key words: Extrafloral nectar, extrasoral nectar, ferns, pteridophytes, nectaries, ant protection, Formicidae, herbivory, antiherbivore defence, Mexico, field experiment, epiphyte, cloud forest, Pleopeltis crassinervata, Polypodium furfuraceum. INTRODUCTION (Koptur et al., 1982; Koptur, 1992), perhaps because no ferns yet have been found to have nectaries in the sori on No consideration of extrafloral nectaries in plants would be their leaves. complete without including the non-flowering plants that Ferns are the most primitive plants possessing nectaries. also have nectaries. In this paper, we briefly review the occur- Modern classification of the ferns sensu lato (Pryor et al., rence of nectaries in ferns, and then report our experimental 2001; Smith et al., 2006, 2008) recognizes three lineages studies investigating the ecological role of nectaries in the leading to the Ophioglossales (grape ferns), Psilotales (whisk biology of a species recently discovered to bear them. Fern ferns) and Equisetales (horsetails). None of these lineages nectaries have sometimes been called ‘extrasoral has nectaries. The remaining ferns comprise two clades, the nectaries’(Wagner and Gomez, 1983; Cooper-Driver 1990), Marattiales (the marattioid ferns) and the leptosporangiate contrasting sori (clusters of sporangia that are the reproductive ferns, which include all the remaining extant ferns. Nectaries structures of ferns) with flowers (the reproductive structures of are found in both of these lineages, which date to the angiosperms). But the term ‘extrasoral’ has not caught on Paleozoic (Schuettpelz and Pryer, 2008). # The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 1278 Koptur et al. — Fern nectaries and anti-herbivore defence in Pleopeltis crassinervata Among the Marattiales, nectaries are found in some tree-ferns Nepi et al. (2009) suggested that the nectars of ferns and gne- of the genus Angiopteris (Marattiaceae) (Bonnier, 1879). Among tophytes are more similar to the extrafloral nectars of angios- the leptosporangiate ferns, nectaries are found in the Cyatheaceae, perms, while pollination drops of gymnosperms are more in the genus Cyathea [into which Cnemidaria and Hemitelia, also similar in composition to floral nectars. This similarity in com- with nectaries (Bonnier, 1879; Arens and Smith, 1998) have been position suggests that there may also be similarity in function, subsumed]. Within Cyathea, some species ‘primitively lack and new insights gained from molecular biology and proteomics nectaries’ and the presence or absence of nectaries corresponds of the two secretion types is reviewed by Nepi et al. (2009). to recent molecular phylogenies (White and Turner, 2012). Extrafloral nectar appeared many millions of years before Nectaries also appear in the Dennstaedtiaceae, specifically in floral nectar (Nepi et al., 2009), and apparently long before bracken fern (Pteridium aquilinum)(Darwin, 1877; Bonnier, ants appeared in the fossil record (Ward, 2007); therefore, 1879; Figdor, 1891; Lloyd, 1901; Lu¨ttge, 1961; Schremmer, their initial function in plants did not involve ants. This gap pro- 1970). Nectaries have been observed in the Dryopteridaceae vides some support for the ‘leaky phloem’ hypothesis of the (Polybotrya; Koptur et al., 1982) and in many genera of origin of nectar (De la Barrera and Nobel, 2004), where nectaries Polypodiaceae: Aglaomorpha, including Photinopteris (Lu¨ttge, are ‘sap valves’, and release excess sugar. 1961; Potes, 2010), Merinthosorus and Holostachyum (Zamora In more recent times, extrafloral nectar is involved in a variety Downloaded from and Vargas, 1974), Drynaria (Zamora and Vargas, 1974; of interactions, particularly mutualisms, between plants and Koptur et al., 1982; Potes, 2010), Platycerium (Du¨mmer, 1911; beneficial insects (Koptur, 2005). Ferns do not have flowers, Lu¨ttge, 1961), Polypodium (Koptur et al., 1982, 1998)and and so their nectaries cannot correctly be described as ‘extraflor- Pleopeltis (the present study). Nectaries have evidently arisen al’; they are always on the fronds (leaves), and so are best independently multiple times within ferns. described as ‘foliar nectaries’ or simply ‘nectaries’. The http://aob.oxfordjournals.org/ A B at Florida International University on May 24, 2013 C D F IG. 1. (A) Epiphytic ferns growing on tree trunks: Pleopeltis crassinervata; (B) Polypodium furfuraceum; (C) nectar droplet on nectary on adaxial surface of frond lamina base (×24), Pleopeltis crassinervata; (D) abaxial surface of lamina lobe of Polypodium furfuraceum with hydathodes (×40), two of which are indicated by arrows. Scale bars: (A, B) ¼ 1 cm; (C, D) ¼ 1 mm. Koptur et al. — Fern nectaries and anti-herbivore defence in Pleopeltis crassinervata 1279 occurrence and anatomy of nectaries in ferns have been lead many ferns to produce new fronds; the herbivore place- described much more often than their ecological role has been ment experiments in early June at the beginning of the rainy studied. season. Both study species occurred together on trunks of Earlier work in relatively undisturbed fragments of lower trees at the forest edges and throughout the nursery. montane wet forest in Banderilla (near Xalapa, Veracruz, Mexico) was undertaken as some of the species of Is it nectar? Polypodium known (from the literature and from cultivation) to have nectaries occurred as epiphytes there. Exclusion To verify that the sooty mould we had observed was experiments conducted during the period of leaf expansion growing due to the presence of nectar and not arthropod exu- demonstrated that ants visiting the nectaries reduced herbivore dates, we carefully examined many fronds, and found no larval damage to the developing fronds of Polypodium plebeium insects whose presence might produce honeydew. On several