A living fossil fig wasp (Hymenoptera, Agaoninae) developing in extant Neotropical fig species (Moraceae: Ficus, section Pharmacosycea)

William Ramírez-Benavides
Escuela de Agronomía, Facultad de Ciencias Agroalimentarias, Universidad de Costa Rica, San José, Costa Rica; william.ramirez.benavides@gmail.com

Received ……………		Corrected…………………		Accepted……………

Abstract: The aim object of this work was to study five Tetrapus wasps, whose females have two mandibular appendages and hexapodous males, their fig host association and phylogenetic position in the family Agaonidae. The question of which group of fig pollinating wasps and associated figs are the sister to the rest of the pollinating agaonids and figs respectively remain open. I report about a group of New World extant Tetrapus wasps (Agaonidae:Agaoninae) assigned to Hexapus subg. nov. ined. Morphologically, ecologically, geographically and historically Hexapus seems to be the most ancestral clade of the extant Agaoninae, including Tetrapus s.s. It develops in fig species of subsection Petenenses (section Pharmacosycea). In the know extant Tetrapus s.s., the females have one mandibular appendage and the males have reduced one or two short-lobe atrophied non-functional midlegs (tetrapodous). On the contrary, Hexapus females have two functional mandibular appendages; while the males have five segmented functional mid-legs (hexapodous). Molecularly Hexapus seems to be the most ancestral clade of extant Agaoninae; e.g., Hexapus sp. of Ficus crassivenosa was molecularly placed as the most ancestral clade of 101 species, representing 19 recognized world-wide Agaoninae genera including four Tetrapus s. s. species, by other author. Two females (T. apopnus and T. delclosi) preserved in Early Miocene (Burdigalian) amber from the Dominican Republic, also have two mandibular appendages and the general morphology of extant Hexapus. It is suggested that Hexapus stands up as a living fossil and as the sister clade of Tetrapus s.s. The presence of extinct and extant Hexapus; and extant Tetrapus s.s. and their fig host species, especially in South America supports a southern Gondwanaland origin for both of them, a non-trans-Pacific migrating connection with tropical America for Pharmacosycea, long oceanic dispersal, high levels of stem extinction of Tetrapus s.s. and Atlantic land connections, as proposed by other authors. However lastly, it has been assumed that figs and their pollinators arose simultaneously in Eurasia during early Tertiary and spread southwards.

Key words: Agaoninae; Tetrapus; Hexapus; Ficus; section Pharmacosycea; subsection Petenensis, living fossil wasps
Número total de palabras: 5.527

Ficus is a Pantropical group, it comprises about 35 species worldwide (Berg & Corner, 2005). It is found in all regions where frost does not occur (Corner, 1958), especially in areas of the Southern Hemisphere, including very small isolated oceanic islands (McKey, 1989; Yokoyama, 2003; Azuma, Harrison, Nakamura & Su, 2010; Ramírez-B. et al., 2011). New World Ficus has two endemic sections: Pharmacosycea (Miq.) Benth. & Hook. f. subgenus Pharmacosycea (Miq.) Miq., polllinated by Tetrapus s.l. wasps; and section Americanae Miq. subgenus Urostigma (Gasp.) Miq. , pollinated by Pegoscapus Cameron. Section Pharmacosycea is a relatively small group (Berg, 1989). However, Berg (2009) noted that this section comprises at least 25 species, of which 19 are found in Ecuador. Ficus is characterized by its unique flask-like urceolate inflorescence (fig or syconium) with internal staminate and pistillate flowers and a small circular or split-shaped terminal orifice, the ostiolum, barred by stiff bracts, through which the female wasps must pass to reach the syconial cavity, oviposit in some of the female florets that become galls where their larvae develop, and pollinate some of them that become seeds. Dispersal of Ficus is a double problem, since the pollinators must accompany the host plant (Corner, 1958; Ramírez-B. et al., 2011). Furthermore, Ficus pollen grains are very small (7-22 µm), with smooth exine (Berg & Corner, 2005) and probably lack the vacuolated stage (Ramírez-B., 2007); consequently, they must suffer dehydration and death during long distant transportation or emigration journals (Ramírez-B., 1989, 2007). Furthermore, fig seeds are small, 0.5-5 mm long (Corner, 1958), short-lived, and the seedlings initially grow very slowly (Ramírez-B. pers. obs.). New World section Pharmacosycea has free standing leptocaulous terrestrial trees, although Ficus crassiuscula Standl. (a host of Hexapus sp.) is usually hemi-epiphytic (Berg, 2009). According to Berg & Corner (2005), events of long-distance in Ficus dispersal will probably rarely result in reproduction and establishment by absence of pollinators and population of trees to allow establishment of the pollinators as well. However, Cruaud, Jabbour-Zahab, Genson, Couloux, Yan-Qiong, Da Rong, Ubaidillah, Pererira, Kjellberg, van Noort, Kerdelhué & Rasplus (2011) proposed fruit pigeons, in part, for long distal trans- oceanic dispersal for fig fruits. Ecologically Ficus provides one of the most important continuous food resources year around, e.g., Harrison (2005) noted that over 1200 vertebrate species feed on figs. Furthermore, the eclosing new generation of female wasps is eagerly consumed by ants, flying swallows and Anisoptera dragon flies; while the wingless males are consumed by ants and staphylinid beetles. Furthermore, the dead foundresses are internally consumed by Parasitodiplogaster nematodes (Ramírez-B. & Salazar, 2015). It has been suggested that the mutual relationship between Ficus and the pollinating Agaoninae was probably stablished before the break of Gondwana (Weiblen, 2002) in the Southern continent (Murray, 1985), while Berg & Corner (2005) noted that the general pattern of distribution and diversification suggest that the genus Ficus originated in eastern Gondwana. Interestingly, Corner (1967) suggested that subgenus Pharmacosycea seems to connect with an even earlier stock of Ficus.
Subgenus Pharmacosycea and its pollinators: Subgenus Pharmacosycea is monoecious with two sections (Berg & Corner, 2005): Pharmacosycea (Miq.) Benth & Hook. f. and Oreosycea Corner (Berg & Corner, 2005); both sections are pollinated by different Agaoninae genera: Pharmacosycea, by Tetrapus (Mayr, 1885), which are passive pollinators; Oreosycea, by Dolichoris (Wiebes), which are active pollinators, with the exception of one species (Wiebes, 1979). New World Section Pharmacosycea has ca 25 species; while Oreosycea, ca 45, ranging from Africa and Madagascar to Fiji and New Caledonia (Berg & Corner, 2005). Section Pharmacosycea has two subsections: Bergianae and Petenenses (Berg, 2006). Tetrapus s. l.; as well as, most other pollinating Agaoninae, are usually tiny (no more than 3mm in length), usually dark colored, diurnal, poor flyers, and do not feed as adults (Ramírez-B., Monge-Nájera, J. & Chavarría, 2009, and references therein).
The pollinators of section Pharmacosycea: Currently, Tetrapus s.s. has been assumed to be the exclusive pollinator of section Pharmacosycea (Corner, 1960; Ramírez-B., 1970; Bouček 1993; Ramírez-B., 1994; Wiebes, 1995). The other fig groups are pollinated by 20 recognized Agaoninae genera. Ramírez-B. (1978, 1991) noted, that Tetrapus seemed to be composed of two well defined groups: in one group (Tetrapus s.s.), the females have one mandibular appendage and tetrapodous males; in the other, the females have two mandibular appendages and hexapodous males. Bouček (1993) referred about three Tetrapus species with the female mandible split from the apex; while Wiebes (1995), mentioned two saw-like appendages. Tetrapus s.s., as well as Hexapus, are passive pollinators, and exclusively cleptoparasitized by Critogaster (Sycoryctinae). 
Geographical origin and dispersal of subgenus Pharmacosycea and Tetrapus: Corner (1967) noted that subgenus Pharmacosycea originated in the region that connected the Solomon Islands, New Hebrides and New Caledonia, it traveled via Caledonia to Asia, but very early a detachment went to America. He also noted that subgenus Pharmacosycea clearly has a trans-Pacific history, that was not of small islands, but of high forest, and that, North America could not have been the source supply of Pharmacosycea to South America. Rønsted, Weiblen, Cook, Salamin, Machado & Savolainen (2005) noted that, if their age estimates are correct, this could imply that long distance oceanic dispersal is an important process in the present distribution of Ficus; and that, the South American section Pharmacosycea would have separated from the rest of Ficus only 60 Myr ago, which postdates the separation of South America from Africa (about 90-100 Myr ago) during the break-up of Gondwana. Lopez- Vaamononde et al. (2009) noted that if their estimates and ancestral area correct reconstructions, it would imply that the current presence of Tetrapus and its associated Pharmacosycea figs in South America could be the result of long distance oceanic dispersal from Asia to the Neotropics; and that alternatively, high levels of stem lineage extinction could explain the observed young age for extant species of this lineage of Neotropical wasps (Tetrapus). Cruad et al. (2012) estimated 74.9 Ma for the stem Pharmacosycea/Tetrapus, and that biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance.
Time evolution of the Agaoninae-Ficus association with special reference to section Pharmacosycea and Tetrapus: The age, geographical origin and major periods of diversion of Ficus remain controversial (Lopez-Vaamonde, Wikström, Kjer, Weiblen, Rasplus, Machado & Cook., 2009). For the geographical origin of the association of Ficus-Agaonidae, it has been suggested that the pollinating wasps evolved during the Cretaceous period (Corner, 1958) in the Southern Hemisphere (Gondwanaland), Murray (1985). According to Machado, Jousselin, Kjellberg, Compton,. & Herre (2001), Ficus and its pollinating wasps seem to be of late Cretaceous origin. Ramírez-B. (1978, Fig. 21); (1991, Fig. 18), in his morphological cladistic analyses, about the evolution of the mandibular appendage, and the pollinating syndromes of the Agaoninae, placed Tetrapus s.s as the most basal clade to all other extant Agaoninae groups and section Pharmacosycea, as the most ancestral group of Ficus. Those cladistic positions were corroborated by molecular phylogenetic analyses by Herre, Machado, Bermingham, Nason, Windsor, McCafferty, Van Houten, & Bachma (1996), Rønsted, Weiblen, Clement, Zerega. & Savolainen (2008), Cruaud, Jabbour-Zahab, Genson, Cruaud, Couloux, Kjellberg, van Noort & Rasplus (2010, 2012), and Munro, Heraty, Burks, Hawks, Mottern, Cruaud, Rasplus & Jansta (2011). 
Machado et al. (2001) noted that the passively pollinating Tetrapus (s.s.) appears to be the most ancient pollinator genus associated with the monoecious Neotropical subgenus Pharmacosycea; and that, both morphological and molecular data, support New World section Pharmacosycea as the earliest derivation among existing figs. Furthermore, Machado, Herre, McCafferty & Bermingham (1996) noted that the ancestors of Neotropical Pharmacosycea and Tetrapus probably arose at the origin of their mutualism ca. 90 Myr ago in Gondwana. Rønsted et al. (2005) supported that section Pharmacosycea is the oldest group of Ficus. Lopez-Vaamonde et al. (2009) also confirmed that Tetrapus was consistently placed as a sister group of the rest of the extant pollinating fig wasps. Furthemore, Rønsted et al. (2005) obtained confidence intervals of 98-105 Myr for the age of the root node of Ficus, and 66-101 Myr for the age of the root node of the wasps. While Lopez-Vaamonde et al. (2009) estimated that the crown group of Agaonidae could have originated any time between 54 and 216 Myr ago. However, Zerega, Clement, Datwyler, & Weiblen (2004) suggested that the figs may have radiated more recently after the break-up of Gondwana, and gave a range of 40–51 Myr for the crown group age of Ficus. Furthermore, Zerega et al. (2005) results do not support the hypothesis of simultaneous diversification between figs and fig wasps and suggested that figs may have radiated more recently, during the Tertiary (cf. Cruaud et al. 2012) for codiversification of figs and fig-pollinating wasps). However, the molecular Tetrapus species analyzed by those authors, as well as others, correspond to extant Tetrapus s.s. (Herre et al., 1996; Machado et al., 1996; Jousselin, Rasplus & Kjellberg, 2003; Datwyler & Weiblen, 2004; Erasmus, van Noort, Jousselin & Greeff, 2007; Azuma et al., 2010; Jousselin et al., 2003; Jiang et al., 2006; Lopez-Vaamonde et al., 2009; Machado et al., 1996, 2001; Rønsted et al., 2005; Rønsted, Weiblen, Clement, Zerega, & Savolainen, 2008; Munro et al.; 2011; Cruad et al., 2012). Aims of the study: To study the association of five Tetrapus wasps whose females have two mandibular appendages and hexapodous males, explain their origin and morphological relationships to other pollinating wasps of subgenus Pharmacosycea and other pollinating genera, and study their taxonomic position and the general biology of Tetrapus s.l. and its origin.
MATERIALS AND METHODS
Syconia of F. crassiuscula Standl. were collected in Santa Cruz, Turrialba, Costa Rica (09°` 58´ 02” N, 83° 44´ 15” O, 1430m), fom F. macbridei Standl. in Tapantí National Park, Costa Rica (09° 46´ 13.2” N, 83° 47´ 59” O, 1239m) and of F. crassivenosa W. Burger, from near Quebrada Cataratitas, San Ramón, Alajuela, Costa Rica (10°14'3.26"N; 84°31'29.39"W, 755m). Naturally colonized syconia of Ficus crassiuscula and of F. macbridei were collected one day before they reached the male phase. Each syconium was individually enclosed in a tightly capped jar. Once the wasps emerged from the syconia, they were killed with acetone and preserved in ethanol 70% for further identification and dissection. Hexapus females and males were also collected by the author from the syconia of a cauliflorous riparian Pharmacosycea sp. in Jiménez, Biscucuy, Portuguesa State, Venezuela in 1966. The pollinators of F. petenensis Lundell was collected from Veracruz, Mexico, and described as Hexapus ramirezi by Xochitl M. Cuevas (2009, unpublished). A female of H. ramirezi was obtained from a preserved syconium of F. petenensis Lundell (=F. apollinaris Dugand); voucher number 2502 deposited at INBio Herbarium, Santo Domingo de Heredia, Costa Rica. The illustrations of two fossil female Hexapus are used with the permission of Enrique Peñalver (2006). The dissections of the wasps were made with the help of a dissecting microscope while the wasps were submerged in 70% ethanol in a Syracuse container, the body structures were disloged by mean of minute Nadeln hook and mounted in Hoyer’s fluid to be illustrated. The species of Ficus studied were identified by the author using the descriptions of Burger (1977) and Berg (2009). The scientific names of the figs were consulted in W³TROPICOS of the Missouri Botanical Garden and in Berg (2009). The term Agaoninae corresponds to the pollinating wasps, sensu Bouček (1988) or to Agaonidae sensu Rasplus, Kerdelhué, Le Clainche & Mondor (1998). It is assumed that the “first flagellomere” of Agaoninae groups represent the partial or complete fusion of three flagellomeres, and the gular bridge correspond to the post-oral bridge of Heraty et al. (2013) 
RESULTS
Females of five Hexapus species studied had two free mandibular appendages and males with functional mid legs (tetrapodous).They were the passive pollinators of five New World fig species of section Petenenses: subgenus Parmacosycea.The females are further characterized by an elongate head with a gular bridge without a sclerotized median carina or a felted line, mandibles with one gland only, absence of labrum, bilobed clypeus with a median setose carina, pedicel without axial spines, third segment with three completely fused anelli and a short unsegmented accuminated projection , maxillolabial complex with distinct stipes and galea, bacilliform process pointing backward, segmented labium, forewing closed costal cell, spureous veins and moderate pilosity, and a twelfth flagellomere. The males have two mandibular glands and antennae basically 4-segmented with an anellus, while the female has one gland. Two fossil Tetrapus females (T. apopnus and T. delclosi), described by Peñalver, Engel & Grimaldi (2006), preserved in Early Miocene ambar from the Dominican Republic, also have two mandibular appendages and the general morphology of extant Hexapus. The two Tetrapus species described by Peñalver, Engel & Grimaldi (2006), plus the five extant Hexapus species here studied are assigned to Hexapus gen. nov. ined., subfamily Tetrapusinae sensu Cruaud et al. (2009). There was a relationship between the presence of two mandibular appendages of extant Hexapus females and the ostiolar conformation of the five hosting fig species studied. Tetrapus s.s. and Hexapus subg. nov. ined. do not precisely follow the Prarmacosycea subdivisions Bergianae and Petenenses of Berg (2009). However, Hexapus is mainly associated with Petenenses species that predominate in submontane humid forests up to 2900 m of altitude, or more; see Berg (2009).
DISCUSSION
Hexapus subg.nov. ined. belongs to Tetrapus Mayr which has five extant nominal species (Wiebes, 1995). Tetrapus s.s. females have one mandibular appendage only (Fig. 1, A), open forewing costal cell, incomplete submarginal vein, no spureous veins and sparce pilosity; the males are tetrapodous. On the contrary, Hexapus females have two free mandibular appendages (Fig. 1, B; a-b), closed forewing costal cell, complete submarginal vein, spureous veins, moderate pilosity, short acuminate unsegmented projection in the first flagellomere, while the males have functional 5-segmented mid-legs (hexapodous). The five Hexapus species studied are the specific pollinators of five species of section Pharmacosycea: subsection Petenenses. Two fossil female Tetrapus (T. apopnus and T. delclosi), described by Peñalver et al. (2006), like those of the extant Hexapus, also have two mandibular appendages (Fig. 2, a-b), forewing closed costal cell, spurious veins and most of the general morphology (Peñalver et al. 2006, Fig.6). The ostiolum of fig species associated with extant Hexapus have a dual entrance morphology not found in other fig groups (Fig. 3) and are often protracted at the bottom of a cavity in the apex of the syconium (Berg, 2009). The ostiolum has several layers of superficial interlocking horizontal apressed bracts (Fig. 3, a); (spiral entrance), followed by a series of inflexed bracts pointing inwards into the syconial cavity (fig 3 b); (linear entrance). On the contrary, fig species associated with Tetrapus s. s. usually have the ostiolum without distinct superficial interlocking bracts, but a linear tubular entrance only. In Hexapus the internal appendage (Fig. 1, B, a) is wide, sub-rectangular, larger than the external one, and provided with lamellae, as in other female agaonines that enter syconia with a spiral ostiole; while the external appendage is narrower, with short lamellae ending in lateral teeth as in Tetrapus s.s. (Fig.1, B, b). Extant Hexapus females are characterized among other Agaoninae (but Tetrapus s. s.) because the following sympleisiomorphic characters: maxilla with distinct stipe and galea, pedicel without axial spines, flagellomeres 4-11 bearing narrow multiporous plate sensilla linaria, antennae 14-segmented, gular bridge without a medial carinated sclerite (cf. Rasplus et al. 1998, for Agaoninae). Tetrapus s.l. and Hexapus are uniquely characterized among all Agaoninae because the females have the bacilliform prossesess pointing backward instead of forward; have one mandibular glands, while the males two (Ramírez-B., 1991); the females do not break off the antennae and wings when they enter the syconium through the ostiolum (Ramírez-B, 1970) and may develop in unpollinated female fig florets . The fossil females of Hexapus apopnus and H. delclosi differ from extant Hexapus because they have an elongate sharp scale-like antennal process in the first flagellomere with an apical long spine, as depicted by Peñalver et al. (2006, Fig. 3ab), which seems to be a Blastophaginae character sensu Wiebes (1982). Both Tetrapus and Hexapus are exclusively cleptoparasited by New World Critogaster Mayr (Sycoryctinae), which Segar et al. (2012) considered as sister to other (Old World) Sycoryctinae. Hexapus, as well as Tetrapus s.s., are the exclusive pollinators of the New World section Pharmacosycea. Extinct and extant Hexapus females are uniquely characterized among most extant pollinating Agaoninae groups (taxa) because they have two independent mandibular appendages as depicted by Peñalver et al. (2006) for Tetrapus delclosi and T. apopnus dated 25-40 My (Poinar, 1993). The extinct and extant Hexapus seem to be the sister clade of extant Tetrapus s.s., the most ancestral clade of other extant Agaoninae and seems to represent a living fossil.
It is hypothesized that the association Tetrapus s.l., section Pharmacosycea, evolved in the Southern hemisphere as assumed by Murray (1985) for Agaoninae, before the Gondwanan break (Weiblen, 2002) and that the Agaoninae ancestor could be a Hexapus-like wasp; other way, the author hypothesize that there had been an unknown ancestral Agaoninae-Ficus association, that originated in eastern Gondwana, in agreement with Berg & Corner (2005); and that subgenus Pharmacosycea seems to connect with an even earlier stock of Ficus, in agreement with Corner (1967). According to Cruad et al. (2012) their ancestral recognition suggest that figs and their pollinators arose simultaneously in Eurasia and the major lineages of figs and pollinators split during the Tertiary; and that it appears, that then they spread southward from Eurasia. Furthermore, they noted that their, biogeography analyses indicate that Pharmacosycea and Tetrapus diverged before South America split from Antarctica (74.9-62.1 My); and that both lineages might have reached America across North Atlantic bridges (cf. Corner (1967). It is hypothesized that the ancestral Agaoninae that gave origin to Hexapus or their ancestors, and also had two mandibular appendages, as seem to be still found as remnants, in some extant Old World pollinating agaonines e.g., Eupristina (E.) grassi Grandi (1916), and Saunders (1883) for E. masoni (Saunders); furthermore, Eupritina (P.) koninsbergi Grandi, also seems to has two mandibular appendages, as illustrated by Chen et al. (1997, Fig. 12 C). Nigeriella excavata Compton (an African species) have a wide elongate mandibular appendage, with two lateral long sclerotized areas (Fig. 4, A, a-b), each section provided with lamellae and denticles separated by a medial membranous area; the two sclerotized mandibular areas of N. excavata, may be remnants of two mandibular appendages that became fused. Some Pleistodontes females, e.g. P. nitens and P. schizodontus of Australia, as illustrated by Lopez-Vaamonde et al. (2002, Figs. 21 and 44), also have a mandibular appendage that recalls the presence of two fused appendages. All other described agaonine females have one mandibular appendage (as in Fig. 4, B, a). It is suggested that the ancestral fig ostiolum had a superficial layer with superficial interlocking bracts followed by an internal linear passage as found in subsection Petenenses (cf. Jousselin et al., 2003). The two mandibular appendages of Hexapus females coevolved in response to the different ostiolar morphology of the two Pharmacosycea subsections, e.g. in Hexapus the lamellate internal appendage is used to displace the female through the superficial spiral ostiolar passage; while the external toothed appendage, to displace it though the internal linear passage. It is suggested that section Pharmacosycea and its pollinators may have evolved in submontane or montane humid tropical forest. By the way, most fig species associated with Hexapus are found from southern Mexico to Ecuador, especially in humid submontane or montane forest up to 3200 or more. Berg (2009) noted that Ecuador gives the impression to be a “melting pot” of section Pharmacosycea species; e.g., out the 25 species, 19 are found in Ecuador; furthermore, out of them, 9 have an ostiolar morphology that suggests their association with Hexapus species (pers. obs.). Based on the morphological, ecological characters and geographical distribution of extant Hexapus and its fig hosts; the author thinks that this group could be the earliest divergent lineage of extant fig pollinating groups. The probable early association of Hexapus–section Pharmacosycea in Gondwana, or even an earlier stock of Ficus that connected with subgenus Pharmacosycea , as suggested by Corner (1967); calls into question the ideas of a trans-Pacific fig route connection (Corner, 1967), north Atlantic land bridges (Cruaud et al. (2012) or long distance trans-oceanic dispersal, proposed for the association Tetrapus- Pharmacosycea in the New World, (cf. Corner, 1967; Rønsted et al., 2005; Lopez-Vaamonde et al., 2009; Azuma et al., 2010; Cruaud et al., 2011. Furthermore, it questions the radiation of Ficus during the Tertiary after the break-up of Gondwana proposed by Zerega et al. (2005) and Cruaud et al. (2011). Since the Agaoninae females are short-lived and do not feed as adults, long distance dispersal, or emigration, is intimately subject to ecological factors, e.g., the fig wasps must accompany the fig host in their migration (Corner, 1958; Ramírez-B. et al., 2011). Furthermore, the seeds of Ficus are small 0.5-5mm long (Corner, 1958), and short lived and the seedlings grow slowly (the author). According to Zerega et al. (2005) long distance dispersal over water seems unlikely for Moraceae, owing to the large short-living seeds. Furthermore, long distant transoceanic dispersal for particular pollinating wasps and other sycophylous invertebrates must occur inside the syconia of living fig trees (Ramírez-B. et al., 2011). It is suggested that for migration the suit of a fig tree or trees and associated invertebrates have to travel together on floating riparian or coastal rafs, or even on whole coral islands (atolls). Consequently, the author suggest that dispersion of Ficus seeds and its suits of sycophillous wasps and other associated organisms by wind, flying, or aquatic vertebrates displacing over long transoceanic distances, do not seem to have played a historical role in the intercontinental dispersion of the main groups of sycophillous wasps and other invertebrates (cf. Cruaud et al. (2011) for Sycophaginae dispersal.
Due to the presence of two mandibular appendages and a membranous un-carinated gular bridge, as pleisiomorphic Agaoninae characters, the author suggest that Hexapus is the most ancestral clade of all extant fig pollinator genera; as well as, the sister clade of extant Tetrapus s.s. This idea is reinforce by the molecular analysis of Cruaud et al. (2009, Fig. 4) who placed Tetrapus n. sp. (=Hexapus sp., pers. obs.) as the most basal clade of 101 Agaoninae species representing 20 world-wide pollinating genera. The author suggests that Hexapus may represent the supposed South America extinct stem agaonine lineage proposed by Lopez-Vaamonde et al. (2009). However, Cruad et al. (2012), concluded that neither their molecular study, nor previous ones, have a strong basis for inferring which agaonid group is sister to all other agaonids.
ACKNOWLEDGEMENTS
I thank Paul E. Hanson, Julián Monge-Nájera and William Eberhard, University of Costa Rica, for reading the manuscript and suggesting various changes; Gilbert Fuentes-González and the Organization for Tropical Studies for their assistance with literature and editing work; to Enrique Peñalver, Division of Invertebrate Zoology, American Museum of Natural History, for permission to use of some of the illustrations, and to Ana Cecilia Jinesta for the illustrations.
RESUMEN
Un fósil viviente de avispas de los higos (Hymenoptera, Agaoninae) que se desarrolla en especies de higos existentes del Neotrópico (Moraceae: Ficus, sección Pharmacosycea). El objetivo de este trabajo fue estudiar cinco especies de avispas Tetrapus, cuyas hembras tienen dos apéndices mandibulares y machos hexápodos; la asociación con sus higos hospederos y la posición filogenética en la familia Agaonidae. Continúa abierta la pregunta, cuál grupo de avispas polinizadoras de los higos constituye el grupo hermano de otras avispas polinizadoras de los higos (Agaonidae). Se informa sobre cinco especies de avispas (Agaoninae) Tetrapus s.s., asignado a Hexapus subgénero nuevo inédito. Morfológica, ecológica, e históricamente Hexapus parece ser el clado más ancestral de los Agaoninae existentes, incluyendo Tetrapus s.l., que se desarrollan en especies de higo de la subsección Petenenses (sección Pharmacosycea). En Tetrapus s.s., las hembras tienen un apéndice mandibular y los machos poseen las patas medias atrofiadas, con uno o dos lóbulos (tetrápodos); por el contrario, las hembras de Hexapus tienen dos apéndices mandibulares funcionales, mientras que los machos tienen las patas medias funcionales (hexápodos), con cinco segmentos. Molecularmente, Hexapus parece ser el clado más básico de los Agaoninae existentes, por ejemplo, Hexapus sp., de Ficus crassivenosa, fue localizado filogenéticamente, por otros autores, como el clado más ancestral de 101 especies de los 19 de géneros de Agaoninae de todo el mundo, e incluso cuatro especies de Tetrapus. Las hembras de T. apopnus y T. delclosi descritas del Mioceno Temprano (Burdigaliense), conservadas en ámbar de la República Dominicana, también tienen dos apéndices mandibulares y la morfología general de Hexapus existente. Se sugiere que Hexapus representa un fósil viviente y el grupo hermano de Tetrapus s.s. La presencia de Hexapus y Tetrapus, especialmente en Suramérica, apoya un origen en Gondwana meridional, para ambos grupos, y no parece apoyar una conexión migratoria a través del Pacífico con Suramérica, para los higos Pharmacosycea (los hospederos de Tetrapus y Hexapus), una larga dispersión transoceánica, o altos niveles de extinción antes de la evolución de Tetrapus, o conexiones terrestres Atlánticas, propuesto por otros autores. Los estudios morfológicos y moleculares de varios biólogos de los Agaoninae (excepto un estudio), no incluyeron a Hexapus spp., o a las especies de Ficus hospederas y han asumido que Tetrapus s.s. es el clado de agaónidos existente más ancestral. Sin embargo, finalmente se ha asumido que los higos y sus polinizadores surgieron simultáneamente en Eurasia en el Terciario Temprano y se difundieron hacia el sur.
Palabras clave: Chalcidoidea; Agaoninae; Tetrapus; Hexapus; sección Pharmacosycea; fósil viviente

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