Taxonomic revision and molecular phylogenetics of the Idarnes incertus species-group (Hymenoptera, Agaonidae, Sycophaginae)

Sycophaginae is a group of non-pollinating fig wasps considered closely related to the fig pollinators (Agaoninae, Tetrapusiinae, and Kradibiinae) in the most recent phylogenetic analyses. They occur in all tropical regions and are associated with Ficus subgenera Urostigma and Sycomorus. There are six described genera of Sycophaginae, and two are native and confined to the Neotropics, namely Idarnes Walker, 1843 and Anidarnes Bouček, 1993. Genus Idarnes is divided into three morphologically distinct groups that were proven to be monophyletic by recent molecular phylogenetic analyses. In this paper we reviewed the Idarnes incertus species-group and provide detailed morphological descriptions and illustrations for the species belonging to this group. Three previously described species were redescribed: I. brasiliensis (Mayr, 1906) comb. nov., I. hansoni Bouček, 1993, and I. incertus (Ashmead, 1900). Seventeen new species are described by Farache and Rasplus: I. amacayacuensis sp. n., I. amazonicus sp. n., I. americanae sp. n., I. badiovertex sp. n., I. brevis sp. n., I. brunneus sp. n., I. comptoni sp. n., I. cremersiae sp. n., I. dimorphicus sp. n., I. flavicrus sp. n., I. flaviventris sp. n., I. gibberosus sp. n., I. gordhi sp. n., I. maximus sp. n., I. nigriventris sp. n., I. pseudoflavus sp. n. and I. ramirezi sp. n. We provided keys for the identification of the species as well as for recognising the different species-groups of Idarnes and a closely related genus (Sycophaga Westwood, 1840). Additionally, phylogenetic relationships among 13 species of the I. incertus species-group were inferred using four molecular markers and discussed in the light of Ficus taxonomy and host specificity.

Two genera of Sycophaginae are associated with Ficus in the Neotropical region, namely Anidarnes Bouček, 1993and Idarnes Walker, 1843(Bouček, 1993Rasplus & Soldati, 2005). They are strictly associated with Ficus section Americanae. Idarnes is the most diverse NPFW genus in the Neotropics. Twenty-three species are recognised as belonging to Idarnes (Bouček, 1993;Cruaud et al., 2011b;Gordh, 1975), but the overall diversity of the genus is estimated to nearly 300 species (Cruaud et al., 2011b). Some Old-World species were classified under Idarnes; however, they all belong to Sycophaga Westwood, 1840(= Apocryptophagus Ashmead, 1904 (Bouček, 1993;Cruaud et al., 2011b;Gordh, 1975) and consequently, Idarnes is restricted to the Neotropics. Concerning nomenclature, Idarnes should be treated as masculine as well as Anidarnes and other derived names (Farache et al., 2013). The name probably refers to an eminent Persian commander, Hydarnes, who was given command of the ''Immortals'' and fought the Greeks in the battle of Thermopylae, 480 BC.
Three morphological species-groups of Idarnes are recognised, namely I. carme, I. flavicollis and I. incertus species-groups. They exhibit clear morphological differences (Bouček, 1993) and contrasted life history traits. Species belonging to the I. incertus species-group are gall-makers and oviposit before pollination. Idarnes flavicollis species-group species are also gall-makers but oviposit at the same time as pollinators. The species belonging to the I. carme species-group oviposit after pollination and are probably cleptoparasites associated with pollinator's larvae (Elias, Menezes Jr & Pereira, 2008;Elias et al., 2012).
The purpose of this paper is to provide a taxonomic revision of the Idarnes incertus species-group. Three species are re-described and 17 species are described from samples Illustrations. Images of specimens were produced with a Leica MZ16 stereoscope connected to a digital camera and a computer workstation running Leica Application Suite (LAS) V3.6 imaging software. Image series comprising about 15-20 focal planes were merged to produce a single image with increased depth of field. Some specimens were dissected, mounted and sputter-coated with gold for scanning electron microscopy (SEM), which was performed with a Zeiss EVO 50 microscope. SEM images of species with few specimens were obtained with a low vacuum protocol.
Pictures include details comparing Sycophaga and Idarnes species groups (Fig. 1). Images of species belonging to Idarnes incertus species-group include: habitus in lateral view (Figs.

Molecular protocols and phylogenetic analyses
In this study, we amplified one nuclear protein-coding region (F2 copy of elongation factor-1α, EF-1α), two mitochondrial protein-coding regions (Cytochrome Oxydase I-COI and Cytochrome B-CytB), and two regions of the rRNA 28S (D2-D3 and D4-D5 expansion regions). DNA extraction, PCR conditions, and sequencing protocols follow Cruaud et al. (2010) andCruaud et al. (2011a). Forward and reverse strands for each fragment were assembled using the software Geneious v.6.1.8. All the sequences were deposited in GenBank (accession numbers in Supplemental Information 2). Our dataset consisted of 33 terminals, comprehending 25 specimens for 13 ingroup species belonging to the Idarnes incertus species-group and eight outgroup species, representing all other Idarnes species-groups, all known Sycophaginae genera, and an Epichrysomallinae genus (Pteromalidae). Sequence alignment for all markers was performed in MAFFT v. 7 (Katoh & Standley, 2013) using the L-INS-i algorithm, and visually inspected. In protein coding genes, we checked protein translations to detect frameshift mutations and premature stop codons using MEGA 4 (Kumar et al., 2008). The most appropriate model of sequence evolution for each data subset most likely to have experienced similar evolutionary processes (mitochondrial genes, EF-1α, rRNA 28S) was identified using Akaike information criterion (Akaike, 1973) as implemented in jModeltest v. 2.1.7 (Darriba et al., 2012;Guindon & Gascuel, 2003). Since we used multiple loci to infer phylogenetic relationships, we established different partitions for each locus included in the analyses, allowing parameters to vary among partitions. Phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian methods, conducted in the CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010).
Partitioned ML analyses were performed using RAxML v 8 (Stamatakis, 2014), and the GTRCAT approximation was used for performing associated bootstrapping (1,000 replicates). Bootstrap percentage (ML BP ) > 95% was considered as strong support and a ML BP < 70% as weak.
Bayesian phylogenetic analyses were conducted using MrBayes v. 3.2.2 (Ronquist et al., 2012). We assumed across-partition heterogeneity in model parameters by considering the parameter m. Parameter values were initiated with default priors; branch lengths were estimated using default exponential priors. The optimization of the posterior probability was achieved using Metropolis-coupled Markov chain Monte Carlo (MCMC). To improve mixing of the cold chain and avoid converging on local optima, we executed two independent runs including a cold chain and three incrementally heated chains for each run. The heating parameter was set to 0.02 in order to allow more frequent swapping between cold and heated chains. The runs were executed for 10 million generations, and values were sampled every 1,000 generations. A NEXUS file including gene alignment and MrBayes block is included as Supplemental Information 3. We also ensure the convergence between parameters from the two chains by analysing estimates and frequency distributions of each parameter using Tracer v. 1.5 (Rambaut et al., 2013). We examined the plot of overall model likelihood against generation number to find the point where the likelihood started to fluctuate around a constant value, and applied a 10% relative burn-in. The results were based on the pooled samples from the stationary phases of the two independent runs. Posterior probabilities (PP) >0.95 were considered as strong support.

Morphological definition; key to genera and species-groups
Idarnes is the sister group of Sycophaga, a diversified Old World genus mostly associated with Ficus subgenus Sycomorus, but two species are associated with F. subg. Urostigma. Idarnes carme species-group is sister to a clade grouping I. flavicollis species-group and I. incertus species-group (Cruaud et al., 2011a;Cruaud et al., 2011b).
Sycophaga and all Idarnes species-groups can be identified using the following key:   The I. incertus species-group shares similarities with Anidarnes Bouček, 1993-another neotropical Sycophaginae genus-but can be distinguished by the following characters: (1)  antennae usually inserted closer to the clypeal margin than to the median ocellus, or at most at the same distance, whereas in Anidarnes they are inserted closer to the median ocellus; (2) ovipositor without the median constriction apomorphic of Anidarnes (to the exception of A. dissidens Farache & Rasplus 2013); (3) metascutellum at least 3× as wide as long in incertus species-group whereas at most 2× as wide as long, or trapezoidal, in Anidarnes (sometimes the metascutellum is inconspicuous in both groups). Keys to the genera of neotropical fig wasps are provided by Bouček (1993) and Rasplus & Soldati (2005).

Idarnes incertus species-group
1993 Bouček, Z., Journal of Natural History 27: 200-203-species-group treatment for Idarnes. Description Females. Body length 1.3-2.8 mm. Ovipositor length 0.4-1.6 mm. Body colour yellow to black, metallic tinge mostly absent. Wings hyaline. Head transverse (1.2-1.4× as wide as high). Face sculpture reticulate. Malar sulcus absent. Maxillary and labial palpi composed at most of two or three segments, the last one frequently reduced and setae-like. Clypeal margin bilobed. Frontal depression (scrobal cavity) shallow, rarely including median ocellus. Supraclypeal area delimited laterally by subantennal grooves. Antennae inserted closer to clypeal margin than to median ocellus (sometimes nearly equidistant from them). Toruli separated by one torulus diameter or less, but never closer than 0.5× torulus diameter. Antenna with 12-13 antennomeres (11-12 flagellomeres; one or two anelli) and a very small terminal protuberance. Clava not well delimited. Vertex slightly concave. POL 2.2-3.4× OOL. Mesosoma slightly curved dorsally. Pronotum 0.7-1.0× as long as mesoscutum. Notaulus complete and usually crenulated. Mesoscutellum 1.1-1.5× as long as wide near transscutal articulation. Metascutellum transverse, at most as long as frenum, sometimes inconspicuous. Postmarginal vein very short, at most 0.5× as long as stigmal vein. Ovipositor sheaths shorter than body length, and 0.8-3× as long as hind tibia. Males. Similar to females, sometimes showing variation in tinge/colour and body proportions (see Supplemental Information 4). Flagellum shorter than in females. Wings medially infuscated, with dense pilosity, especially at the costal and apical margin, and near marginal vein. Remarks. Idarnes is treated here as masculine as explained in Farache et al. (2013).
Supraclypeal area wider than torulus (Fig. 4E) (1906), but we could not find any female specimens at NMW.

Host plant. Ficus gomelleira Kunth & Bouché
Remarks: There is only one male specimen collected by Mayr at NMW, minuten-mounted and decapitated. Despite the absence of head, the following characters ascertain its position within the Idarnes incertus species-group: (1) winged male, (2) body colour, (3) postmarginal vein compared to stigmal vein, (4) shape of mesoscutellum and (4) striated axillula. Etymology. The specific name refers to the predominant body colour. Diagnosis (♀). Body colour predominantly dark brown. Pronotum laterally yellow. Legs yellow, femora and coxae yellow brown. Supraclypeal area as wide as torulus, or slightly narrower. Subantennal groove as long as torulus. Distance from torulus to median ocellus 0.9× distance from torulus to oral margin. Metascutellum nearly 0.5× as long as frenum to inconspicuous in dorsal view. Ovipositor sheaths 1.5× as long as hind tibia.

Female.
Size and colour. Body length 1.3-1.6 mm. Ovipositor length 0.5 mm. Predominantly dark brown. Scape yellow. Pedicel and flagellum yellow brown. Lower face yellow. Pronotum laterally yellow. Legs yellow, femora and coxae yellow brown. Head. Supraclypeal area as wide as torulus. Subantennal groove as long as torulus. Distance from torulus to median ocellus 0.9× distance from torulus to oral margin. POL 2.9× OOL. Scape 2.1× as long as pedicel. Antenna with two anelli (character sometimes inconspicuous). First funicular segment 0.7-0.8× as long as wide, with 5-9 multiporous plate sensilla.  Etymology. The name refers to the sexual colour dimorphism observed in this species. Diagnosis (♀). Head and mesosoma predominantly yellow orange. Metasoma black. Predominant colour of males brown black. Supraclypeal area as wide as torulus. Subantennal groove longer than torulus. Distance from torulus to median ocellus 1-1.1× the distance from torulus to oral margin. Metascutellum nearly 0.3-1.0× as long as frenum in dorsal view. Anterior margin of propodeum angulose medially. Ovipositor sheaths 0.9-1× as long as hind tibia.

Female.
Size and colour. Body length 1.8 mm. Ovipositor length 0.7-0.8 mm. Head yellow orange. Scape and pedicel yellow. Flagellum yellow brown. Mesosoma bown black. Pronotum laterally yellow. Legs yellow. Metasoma brown black Head. Supraclypeal area narrower than torulus. Subantennal groove shorter than torulus. Distance from torulus to median ocellus 1-1.2× distance from torulus to oral margin. POL 2.9× OOL. Scape 2.3× as long as pedicel. Antenna with two anelli. First funicular segment 0.8-0.9× as long as wide, with approximately 6 multiporous plate sensilla. Etymology. The specific name refers to the coloration of the metasoma, especially in ventral view. Diagnosis (♀). Body predominantly yellow orange. Margin of metasomal tergites brown. Subantennal groove shorter than torulus. Distance from torulus to median ocellus 1.3× distance from torulus to oral margin. Metascutellum inconspicuous to approximately 0.5× as long as frenum in dorsal view. Anterior margin of propodeum angulose medially. Propodeal median line present, traceable at least in the anterior half of propodeum. Male. Similar to female. Distance from torulus to median ocellus 1.7× distance from torulus to oral margin. POL 2.0× OOL. Antennal flagellum shorter than scape plus pedicel (longer in female). Host plant. Ficus aurea Nuttal, form isophlebia (Standley) Berg. Ficus isophlebia was synonymized with F. aurea, however the differences observed between entities within the F. aurea species complex led C. C. Berg to recognize four informal entities within the species (Berg, 2007). Taking into account the morphological differences observed in the host-plants (Ficus aurea form isophlebia and Ficus aurea form aurea), the fact that these forms are sympatrically pollinated by different pollinator species and that non-pollinating communities associated with these forms are composed of different species (J-Y Rasplus, 2016, unpublished data), including different species of the Idarnes incertus species-group, we suspect that these forms of Ficus aurea may be in fact different but closely related Ficus species. Etymology. The specific name refers to the mesoscutum, which is particularly curved in lateral view. Diagnosis (♀). Body predominantly black. Supraclypeal area narrower than torulus. Subantennal groove longer than torulus. Distance from torulus to median ocellus 0.8× distance from torulus to oral margin. Mesoscutum and scutellar-axillar complex strongly curved in lateral view. Metascutellum nearly as long as, or longer than frenum in dorsal view. Ovipositor sheaths 2× as long as hind tibia.
The trees reconstructed using ML and Bayesian methods showed the same topology (Fig. 16). Idarnes incertus species-group was recovered monophyletic (PP = 1; ML BP = 100%) and divided in two main clades (clade 1 and clade 2; Fig. 16). The first clade is well resolved and composed by five species; I. brunneus was recovered sister to I. comptoni (PP = 1; ML BP = 100%), and I. amazonicus was sister to I. gordhi plus I. ramirezi (PP = 1; ML BP = 100%). The deeper nodes within the second clade were not well resolved, yet we could retrieve a well supported clade formed by I. pseudoflavus, I. brevis, and I. dimorphicus (PP = 1; ML BP = 100%) but the relationships among these tree species were uncertain. Also, I. incertus was retrieved as sister to I. flaviventris + I. nigriventris (PP = 1; ML BP = 100%). The relationships of I. maximus and I. gibberosus were not well established (Fig. 16). Idarnes maximus was recovered sister to I. brevis + I. dimorphicus + I. pseudoflavus with relatively high Bayesian posterior probability support (PP = 0.97) but low maximum likelihood booststrap support (ML BP 52%), while I. gibberosus was recovered as sister to the clade I. flaviventris + I. incertus + I. nigriventris with Bayesian posterior probability support (PP = 0.9) yet low ML BP support (ML BP = 63%)

DISCUSSION
The Idarnes incertus species-group is clearly distinct from the remaining Idarnes. Indeed, they rarely exhibit metallic tinge and their ovipositor sheaths are always shorter than the body length, whereas the remaining Idarnes exhibit metallic colour and the ovipositor is always longer than body. Species belonging to the I. incertus species-group are globally similar, and the main differences between species concern the body coloration and the relative length of the ovipositor. According to recent phylogenetic analyses, the I. incertus species-group appears to be a recent radiation within Sycophaginae (Cruaud et al., 2011a;Cruaud et al., 2011b) and the morphological similarity of the species may be partly linked to their recent divergence (∼20-10 Ma, during the Miocene; Cruaud et al., 2011a). Species of the Idarnes incertus species-group are usually species-specific with the exception of I. americanae, that was found associated with F. americana and F. costaricana (both species occurring in Costa Rica), and I. incertus that is associated with F. aurea and F. citrifolia in Florida.
Several These patterns strongly suggest that the diversification of the I. incertus species-group within Ficus do not follow a ''one-to-one rule'' of diversification as discussed for pollinators (Rasplus, 1996). Our results clearly show that host shifts between Ficus species and diversification on the same Ficus host are frequent. Our phylogenetic analyses show one case of diversification within the same host species. Indeed, species associated with the Ficus aurea complex (I. flaviventris + I. incertus + I. nigriventris) formed a strongly supported monophyletic clade and were morphologically closely related. On the other hand, species associated with the F. americana complex (I. amazonicus, I. gibberosus, and I. ramirezi) belong to different clades, which strongly suggests that host shifts happened. Patterns of diversification within host species were also observed in Anidarnes for which sister species occurred on the same host complexes, i.e., F. aurea and F. americana complexes (Farache et al., 2013). This suggests the existence of different diversification patterns among genera even when they show similar life histories and belong to a same subfamily.
Here we recognized three previously described species belonging to Idarnes incertus species-group. Additionally, 17 species new to science are recognized and described. Most species were collected in Costa Rica (11 species), Brazil (4 species) and Colombia (three species). Sampling efforts in Brazil and Costa Rica are comparable, and therefore this shows that the group is probably more diversified in lower latitudes. The high number of new species found in this study highlights the lack of taxonomic information on the Neotropical fig wasps. Despite an increasing number of phylogenetic studies including these wasps (Cruaud et al., 2011a;Cruaud et al., 2010;Cruaud et al., 2011b;Cruaud et al., 2012;Heraty et al., 2013;Munro et al., 2011) just a few recent (i.e., 20th century onwards) taxonomic papers are available on non-pollinating and pollinating wasps (Bouček, 1993;Farache et al., 2013;Jansen-Gonzalez & Sarmiento, 2008;Schiffler, Azevedo & Kawada, 2002;Wiebes, 1995).
This study yields taxonomic and phylogenetic frameworks for a group of Idarnes, which represents an important part of the Sycophaginae diversity (ca. 33% of the species). This contribution is an important step to a well-established taxonomic foundation for Agaonidae, and we hope it will subsidize further investigations addressing taxonomy, evolution, and host specificity in fig wasps.