Rev. Biol. Trop., 45(4): 1635-1640, 1997

Fig wasps: mechanisms of pollen transfer in Malvanthera and Pharmacosycea figs (Moraceae)

William Ramírez B.I and Jollyanna Malavasi 2 I Escuela de Fitotecnia. Universidad de Costa Rica, 2060 Costa Rica. 2 Facultad de Medicina, Universidad de Costa Rica, 2060 Costa Rica.

Received 31-X-1996. Corrected I3-III-1997. Accepted 30-1X-1997.

Abstraet: Pollination of the pistillate fig flowers (Ficus spp.) has crucial effects for the figs and the pollinating wasps (Agaonidae). It a1lows nonnaldevelopment, of seeds and wasps. Sorne agaonid groups have evolved pockets and cor­ biculae to carry pollen, wlúle others carry it in the intersegmental membranes and other hidden areas of the body. Single female specimens of Pleistodontes rieki Wiebes, Robertsia mandibularis Boucek, Tetrapus americanus Mayr and Tetrapus sp., the first two from New Guinea and theTetrapus spp. from Costa Rica were studied with scanning electron microscopy. Sorne Pleistodontes species (Old World) have pollen cavities in the expanded scapes; theyalso carry pollen in the ridges and laminae of the mandib1es. Tetrapus species (NewWorld) carry pollen in cavities locat­ ed behind the mandibular appendages and in the lamellae, ridges and teeth of the mandibles. These ways of carrying pollen may be intennediate between pollen transport in the intersegmental membranes, and in pockets or corbiculae. The evolution of such mechanisms may preserve pollen from desiccation. These structures may have similar func­ tions to the pollinium of sorne plants that are a1so pollinated by specific insects. It is suggested that sorne fig wasps that become dusted with poli en in the syconia where they develop, and remove most of it once outside, fill up the "spu­ rÍous" pockets, and other elaborate pollen-carrying structures outside the syconia. Abundant pollen grains were also found among the mouth parts of Robertsia mandibularis (Sycoecinae) a non-pollinating figwasp.

Key words: Pollination, Ficus, fig wasps, Agaonidae, ultrastructure.

The Urticales are wind pollinated and suc­ groups (Ramírez 1969, Galil and Eisikowiteh cessful adaptation to inseet pollination is only 1969, Chopra and Kaur 1969). Other questions known in Ficus (Moraeeae) whieh has a about the Agaonidae-Ficus association are why pseudoearpous infloreseenee (Berg 1977). and how fig wasps transport pollen (Verkerke Aecording to Wiebes (1986) polI en transfer has 1987). It is known that unpollinated syconia do only been studied in a small number of agaonid not develop normally and that in the absenee of speeies, a situation which has not changed to pollination and fertilization of the "gall flow­ the present. ers" the nucelar tissue and the parthenocarpic The genus Ficus is exc1usively pollinated endosperm, if present, is a poor food source for by chaIcidoid wasps of the family Agaonidae. the wasp larvae and insuffieient for the devel­ The manners in which the minute, smooth and opment of most females (Galil and Eisikowiteh almost hairless wasps carry enough pollen to 1971, Galil 1973a). Ramírez (1969) noted that accomplish pollination and fertilization of the New World Tetrapus and sorne other Old hundreds, if not thousands, of female flowers World agaonids (e.g., Pleistodontes) carry of the receptive syconia had been in doubt for a pollen dusted over the body or in the digestive long time, until poli en pockets and corbiculae tract (see also Ramírez 1974), while aeeording to carry polIen were found in sorne agaonid to Wiebes (1977), "in many agaonid species, 1636 REVISTA DE BIOLOGIA TROPICAL pollen can sometimes be found in small quanti­ MATERIALS AND METHODS ties in several places of the body e.g., between the mouth parts, and under the hypogidium, One female specimen of each species studied and that, in the species of Pleistodontes the was dissected, treated and mounted (Table 1). axial, disto-dorsal excavation of the antennal The Tetrapus and P. plebejus wasps were scape may be such a place". originally preserved in 70% ethanol, while the Galil and Eisikowitch (1971) stated that R. mandibularis were pinned specimens. They "undoubtedly, the wide fluctuation in number were dissected under a stereoscope, c1eaned, of anthers in the syconia of different Ficus hydrated and inmersed in a fixative containing spp., reflects on the efficiency of pollination". 2% glutaraldehyde and 1 % formaldehyde in a Wiebes (1982a) stated that nothing about polli­ 0.1 M Sorensen phosphate buffer solution for 1 nation is known for the Pleistodontes species. hr. The wasps were post-fixed with buffered The purpose of this article is to explain how 1 % osmium tetroxide for 2 hr and, after a brief sorne Pleistodontes wasps (the pollinators of rinse in distilled water and dehydration through section Malvanthera) and Tetrapus (the polli­ a graded ethanol series, they were placed in nators of section Pharmacosycea) carry the isoamyl acetate, critical point-dried using C02' pollen and effect pollination. The presence of splutter-coated with platinum and observed in a abundant pollen grains among the mouth parts scanning electron microscope at 15 kV. . of Robertsia mandibularis (Sycoecinae), a non-pollinating wasp, is also reported.

TABLE 1

Wasps asedfor the SEM stady

Wasp species Geographic origin Fig hosts

Pleistodontes plebejus New Guinea, Ficas xylosycea Wiebes (1977) West, River Tor Die1s (1935)

Robertsia mandibalaris New Guinea, F. xylosycea Boucek (1988) Manki area

Tetrapas americanas Turrialba, F. maxima Mayr (1885) Costa Rica Miller (1768)

Tetrapas sp. San Isidro General, F. macbrideí Costa Rica Standley (1937)

RESULTS (Sycoecinae). a non-pollinating sycophilous wasp (van Noort 1994), that also develops in In P. rieki females there is a large cavity the syconia of F. xylosycia, was found to have with many pollen grains in the axial distodor­ numerous polIen grains among the mouth sal areas of the antennal scapes (Fig. 1). Pollen appendages (Fig. 3). grains were also found among the mandibular Tetrapus females, the pollinators of F. ridges and lamellae (Fig.2). P. rieki females do macbridei, had pollen grains lodged in the not have pollen pockets or corbiculae and the mandibular lamellae (Fig. 4) andin a cavity (Fig. axial surface of the coxa is rather smooth 5) on the rear partof the mandibular appendages. (Wiebes 1977). It beloflgs to the P. rieki group Tetrapus americanus females had ponen among (Wiebes 1991) characterized by the absence of the teeth of the mandibular appendage (Fig. 6). ponen pockets and corbiculae. P. rieki is the Tetrapus females of both species were also pollinator of F. xylosycia of subseries observed to eelose from the syconia completely Hesperidiiformes, a figfound in eastern New covered with pollen. which they scraped from Guinea (Comer 1965), Bouganville IsI. and their bodies with the legs while standing on the Papua (Wiebes 1991). Robertsia mandibularis syconia before flying away. Ramirez & Malavasi: Pollen transfer 1637

Fig. 1-6. Fig.l. Scape of Pleistodontes rieki with numerous grains of pollen in its cavity. Fig.2. Section of mandibles of Pleistodontes rieki with pollen grains lodged in the lamellae. Fig.3. Mouth parts of Robertsia mandibularis, a non-pollinat­ ing sycophilous wasp, with pollen grains lodged among mouth parts. Fig.4. Mandibular appendages of Tetrapus sp. the polli­ nator of Ficus macbridei, with pollen grains lodged on lamellae. Fig.5. Rear side of mandibular appendages of Tetrapus sp., the poIlinator of F. macbridei, with polIen grains lodged in cavity. Fig.6. Mandibular appendage of Tetrapus americanus with polIen grains lodged among the serrations of the mandibular appendage. 1638 REVISTA DE BIOLOGIA TROPICAL

Pollen in the scape of Pleistodontes: P. galvinus and P. regalis Grandi); 3. P. r;en­ Most, if not all the Pleistodontes females, have nellensis Wiebes, with a very vague trace of the scape several times longer than wide, stemal pockets. 4. Wiebes (1977) illustrated dorsoventrally depressed, with lateral and P. blandus and described P. mandibularis with sometimes apical extensions (e.g., P. rigisamos coxal corbiculae only. 5. Wasps with well Wiebes as illustrated by Wiebes (1991, Fig. 3). developed mesostemal pockets and smooth Grandi (1952) called the extension of the scape mesostema (e.g., P. froggatti, nigriventris and "lobo laminare de suo margine anteriore". The imperialis groups, (sensu Wiebes 1991). P. extension of the scape produces a flap over a imperialis Saunders also have a coxal corbicu­ large cavity in sorne of Pleistodontes females lae (Ramírez 1974, 1978, Wiebes 1977). None as illustrated for P. rieki (Fig. 1). Expanded of the wasps in categories 1-4 seem to have scapes were also observed by Ramírez in P. evolved conspicuous pockets or corbiculae to froggatti Mayr, P. blandus Wiebes, P. longi­ carry pollen, and they probably rely largely for caudus Wiebes, P. mandibularis Wiebes, P. pollination on the pollen carried in the cavities nigriventris (Girault), and P. rieki Wiebes, at of the scapes as in P. rieki and other hidden the Natural History Museum of London. The areas. Thus, in Pleistodontes there are wasps expanded scapes in other species of that probably accomplish topocentric pollina­ Pleistodontes may be used to carry pollen as in tion (those without pockets or corbiculae) and p. rieki and other wasps of the P. rieki and those which accomplish ethodynamic pollina­ rennellensis groups (sensu Wiebes 1977). tion those with pockets and or corbiculae (for Since the agaonid pollinating females are short pollination syndromes, see Galil 1973 b). A lived (Ramírez 1970) and do not feed as adults, similar trend is found in the pollinators of sec­ the pollen grains observed on the "spurious tion Conosycea (subgenus Urostigma) although pockets" of the wasps studied should be Ficus they do not carry pollen in the scapes (Ramfrez pollen. 1974, 1978). The expanded scapes in other Variety of locii in Pleistodontes: Pleistodontes species may be used to carry Pleistodontes wasps inhabit figs of the section pollen as in P. rieki and rennéllensis groups Malvanthera (subg. Urostigma). Malvanthera (sensu Wiebes 1977). Pleistodontes males do figs are found in Australasia (Comer 1965, not possess expanded scapes: Berg 1989). The syconia of this group have Pollen transport in Tetrapus: Tetrapus numerous large ("explosive") anthers from wasps are the exclusive pollinators of the which the pollen usually comes out naturally endemic New World Pharmacosycea figs after the deshiscence of the anthers (Ramírez (section Pharmacosycea). The syconia of 1974), and as observed by Pemberton (1921) this section have abundant long pedicellate for Ficus macrophylla Desf., and the wasps male flowers, with two or three stamens with emerge literally white with it; however, large "explosive" anthers with free long fila­ Pemberton (1921) did not mention wasp ments. The anthers dehisce and shed the cleansing; they have filiform (bifid or simple) pollen without the help of the wasps (Ramírez stigmata forming a synstigma. Pemberton 1970). Tetrapus females do not possess pock­ (1921) observed that the eclosing P. froggatti ets, corbiculae or expanded hollow scapes to Mayr females became dusted with pollen. carry pollen, and become completely dusted Wíebes (1991) divided the genus Pleistodontes with it when they eclose from the gall flow­ into several groups: the P. rieki and P. rennel­ ers. Once outside the syconium they remove lensis groups, that do not posses pollen pockets most of the pollen with the frontal and hind and the P. froggatti, P. nigriventris and P. legs. However, part of the pollen probably imperialis groups, which have them. In remains in sorne hidden body areas or spuri­ Pleistodontes we find a trend of pollen carry­ ous pockets (e.g., in a cavity between the ing mechanisms as follows: 1. Wasps with mandibular appendages (Fig. 4) on the lamel­ rather smooth mesostemum and axial surface lae (Fig. 5) and the teeth (Fig. 6) of the of the coxa (e.g., P. longicaudus; P. plebejus, mandibular appendages. Tetrapus wasps P. inmmaturus Wiebes, and probably P. galvi­ effect topocentric pollination as noted by nus Wiebes; 2. Wasps with an extensive field Galil and Meiri (1981) for the pollination of of setae on the axial edge of the fore coxa (e.g., the edible fig (F. carica L.) and by Berg Ramirez & Malavasi: Pollen transfer 1639

(1989, 1990) for section Pharmacosycea. ynamic pollination. Because of the mode of The ec10sing Pleistodontes and Tetrapus pollination and sorne morphological characters

females bocome completely dusted with pollen Le. g .• presence of sensilla linearia in the fourth inside the syconia where they develop, and segment, non-breakage of antennae and wings remove most of it with their legs from the body upon entrance of the syconium and the absence once outside the syconia; thus, the probability of pockets and corbiculae to carry pollen), exists that they fill up with pollen the body Tetrapus wasps seem to be the most primitive depressions (spurious pockets sensu Galil and group of symbiotic agaonids and may form a Neeman 1977) and pollen pockets while subfamily apart.. Section Pharmacosycea (the c1eansing the body. Consequently "loading" of host of Tetrapus) also is considered a primitive pollen may be not "passive". Galil and figgroup (Ramírez 1974, Berg 1990). That the Neeman (1977) also observed that the emerg­ pre-agaonids and the primitive agaonids proba­ irtgBlastophaga psenes L. (the pollinator of the bly carried the pollen in the mouth, and other edible fig F. carica L), a wasp without special natural cavities, as suggested by Ramírez structures to carry pollen, became abundantly (1974), is revealed by the presence of numer­ covered with pollen and then commenced the ous pollen grains among the mouth parts of prolonged task of c1eansing their bodies from Robertsia mandibularis (Sycoecinae) a non­ the surplus pollen once the wasps were outside pollinating wasp. The way the agaonid symbi­ the syconia (see also Galil and Meiri 1981). otic wasps carry pollen and the mode of polli­ Evolution of pollen transport: It is nation they effect may help to separate and assumed that Ficus species with numerous male establish the fig and agaonid taxa in more flowers and large anthers that open readily and meaningful biological and ethological ways. widely, from which pollen comes out naturally, Since the ec10sing females of Pleistodontes and and is "passively" carried on the wasps' bodies, Tetrapus become completely dusted with are more primitive than those with few male pollen inside the syconia where they develop, flowersfrom which the pollen is extracted from and remove most of it with their legs from the the anthers by the pollinating wasps (Ramírez body once outside the syconia, the probability 1974). The figspecies with topocentric pollina­ exists that they fill up. with polI en the body tion usually have long branched bifid stigmata depressions (spurious pockets sensu Galil and at least in the female flowers, usually forming a Neeman 1977) and pollen pockets while synstigmatic layer. The production of abundant c1eansing the body. Consequently "loading" of pollen by species of figs which have pollinators pollen may be not "passive". without pollen pockets or corbiculae is extreme­ ly wasteful since most of the pollen is removed from the wasps' body by their c1eansing move­ RESUMEN ments after leaving the syconium (Galil and Meiri 1981). Filling of "spurious" pockets or La polinización de las flores pistiladas de los higos other more elaborated structures outside the (Ficus spp.) tiene efectos positivos para los siconos y sus syconia may constÍtute another pollinating syn­ polinizadores agaónidos: pennite el desarrollo nonnal de drome in Ficus. semillas y de las avispas; especialmente, el de las hembras. Implications for pollinator systematics: Algunas avispas polinizadoras de los higos evolucionaron Pleistodondes and Tetrapus, as well as the pol­ sacos y corbículas poleníferos, mientras que otras . llevan el linators of the African fig section Galoglychia, polen en las membranas intersegmentales y otras áreas escon­ are considered to be related by Wiebes (1982b) didas del cuerpo .. Se infonna que algunas especies de avispas and inc1uded in subfarnily Agaoninae (see also Pleistodontes (Vi�o Mundo) poseen cavidades para acarrear Wiebes 1973, 1974, 1982 a, b and 1986). polen en los escapos y en los bordes y láminas mandibulares. However, because of their pollination syn­ Las avispas Tetrapus acarrean polen en cavidades localizadas dromes, their geographic distribution and the detrás de los ápendices mandibulares y en los dientes y lámi­ morphology of the head, these three agaonid nas de esos ápendices. Las formas de llevar pollen descritos groups do not seem to form a holophyletic parecen ser intermedios entre el transporte de polen en las group, as noted by Ramírez (1991). AH known membranas intersegmentales y aquella en sacos y corbículas. pollinators of Galoglychia figs have pockets La evolución de cavidades, sacos y corbículas para llevar and corbiculae to carry pollen and effect ethod- polen en los polinizadores de los higos parece ser mecanis- 1640 REVIsTA DE BIOLOGIA TROPICAL

mos que evitan que los granos de polen se sequen cuando las Galil, J. & L. Meiri. 1981. Number and structure of anthers avispas están buscando siconos receptivos para ovipositar, in figsyconia in relation lo behaviour of ¡he polIen vec­ torso New Phytol. 88: 83-87. estasestructuras pueden tener función similares a aquellas de las políneas de algunos otros grupos de plantas. Abundantes (JallÍ; 1. & G. Neeman. 1977. Pollen transport and granos de polen se encontraron también entre las piel1liS Pollination in Ihe common fig(Ficus carica L.). New Phytol. 79: 163-171. bucales de Robertsia mandibularis (Sycoecinae), UfilI avispa que no es polinizadora. Grandi, G. 1952. Insetti dei fichi messicani, malesi ed aus­ traliani. Boll. 1st. Ent. Bologna 19: 47-67.

ACKNOWLEDGMENTS Pemberton, C.E. 1921. The fig wasp in ils relation to the developrtiertl of fertile seed in the Morelon Bay Fig. This work was supported by grant No. H¡¡W1iH Planl. Rec. 24: 297-319. 31290034 from Vicerrectoría de Investigación, Rafufrez B., W. 1969. Fig wasps: mechanisms of pollen University of costa Rica. We thank PaUl transporto Science 163: 580-581. Hanson and Julián Monge for suggestions to Ranúrez B., W. 1970. Taxonornic and biological studies of improve an earlier draft, the Natural History Neotropical fig wasps (Hymenoptera: Agaonidae). Museum of Loudon staff for providiug the KansasSe. Bull. 49: 1-44. specimens of Pleistodontes and of Robertsia mandibularis, and J.T. Wiebes, Rijksmuseum R�� lt; W, 197it Cbevolution of Ficus aiid Agaonidáll. MIL MissiJUri Bol. Grd. 61: 770-780. van Natuurlijke, Leiden for lending the speci­ mens of Pleistodontes plebejus. Ramírez B., W. 1978. Evolution of mechanisms lo carry pollen by Agaonide (Hymenopera: Chalcidoidea). Tijdschr. Ent. 121: 279-293. REFERENCES Ramírez B .. W. 1991. Evolution of the mandibular appendagtílll flg Wasps (Hymenoptera: Agaonidae). Berg, C.C. 1977. Urticales, their differentiation and system­ Rev, Bi(jL Trop. 39: 87-95. atic position p. 349-374 In: K. Kubitzki, (ed.) F10wenng Plants, Evolution and Plant. Syst. Evo\. Supp. I \too Noort, S. 1994. Systematics of Sycoecinae fig wasps Classificationof Higher Categories. tPhilocaenus concJuded; generic key; check list. ProC. Kon. Ned. Akad.Wet 97: 341-375. Berg, C.C. 1989. Classification and distribution of Ficus: Experimentia 45: 605-611. Verkerke, W. 1987. Syconial anatomy of Ficus asperifolia (Mornceae) a gynodjo�cious tropical fig. ProC. Kon. Berg, C.C. 1990. Reproduction and Evolution in Ficus Ned. Akad. Wet 9tl: 461-492. (Moraceae): Traits Connected with the Adequate Rearing of Pollinators. Mem. New York Bot. Gard. 55: WieOO5; lI. 1973. Phylogenetic specificily of fig �d fig 169-185. . wasps p. 21-25 In: N.B.M. Brantjes (ed.) Polhnatlon and Dispersal. Depl. Bot. Nijrnegen. Comer, EJ.H. 1965. Check-list of Ficus in Asia Ilnd Australasia with a key to identificatíon. Gard. Bul\. Wiebes, lT. 1974. Nigeriella, a new genus ofWest African Singapore 21: 1-186. fig wnsps aIlied lo Elisábethiella Gftlndi. Zoo. Meded. Leiden 40: �25-233. Chopra, R.N. & H. Kaur. 1969. Pollination and fertilization in sorne Ficus species. Beitr. Bio\. Pflanzen 45: 441· Wiebes, J.T. 1977. Indo-Malayan and Papuan fig wasps 7. 446. Agaonidae, mainly caught at light. Zoo\. Med. Leiden. 52: 137-159. Galil, J. 1973a. Pollination in Dioecious Flgs Pollination of Ficus fistulosa by Ceratosolen hewitti. Gard. Bull. 26: Wjebes,J,'f.19�2a. The phylogeny of Agaonidae 303-311. (Hymenopera, Chalcidoidea). Nether. J. Zool. 32: 395- 411 Galil, J. 1973b. Topocentric and ethodynamic pollination p. 85-110 In: M.B.M. Brantjes and H.F. Linkens (ed.) Wiebes, J.T. 1982b, Fi� wnsps (Hymenoptera) p. 735-755. Pollination and Dispersal Universityof Nijmegen. In: J,L, Ol.'@8sítt (�d) Biogeography and ecology of New OUiMa. Monographiae Biologicae 42, W. Junk Galil, J. & D. Eisikowitch. 1969. Further studies on the pol­ Publishers, the Hague. lination ecology of Ficus sycomorus L. Tijdschr. Ent. 112: 1-13. Wiebes, J.T. 1986. Theassociation of figs and insects. Rev. Zool. afro 100: 63-71. Galil, J. & D. Eisikowitch. 1971. Studies on mutualistic symbiosis between syconia and sycophilous wasps in Wiebes, J.T. 1991. Agaonidae (Hymenoptera, Chalcidoidea) monoeciousfigs. New Phytol. 70: 773-787. and Ficus (Moraceae): fig wasps and their figs, VII (Pleistodontes) Proc. Kon. Ned. Akad. 94: 137-152. 10.07