Redescription of Mononeuron duguetiae Fischer ( Braconidae , Doryctinae ) , a gall associated species on Duguetia furfuracea ( St . Hil . ) ( Annonaceae )

We redescribe the poorly known, gall associated doryctine wasp Mononeuron duguetiae Fischer (Braconidae) based on museum and recently collected material. This species shares various morphological features with the members of the also gall associated Allorhogas Gahan, suggesting their close relationship. A Bayesian phylogenetic analysis using mitochondrial (COI) and nuclear (28S) markers recovered M. duguetiae within a clade with another five gall associated doryctine genera + Heterospilus Haliday. Surprisingly, M. duguetiae was recovered, though with low support, as the sister taxon of a Monitoriella Hedqvist + Labania Hedqvist clade, and not closely related to Allorhogas. Information is provided about the galls on Duguetia furfuracea (St. Hil.) (Annonaceae) where the specimens of M. duguetiae were reared.


Introduction
The braconid subfamily Doryctinae is a cosmopolitan, highly diverse group of parasitoid wasps containing more than 1,300 described species (Yu et al. 2005), although this probably only represents half of its actual species diversity (Jones et al. 2009).
Species of this subfamily are grouped into about 200 recognized genera, some of which are considerably species rich with wide geographical distributions (e.g.Heterospilus Haliday, Spathius Nees, Notiospathius Matthews and Marsh, Allorhogas Gahan).The doryctine genus Mononeuron Fischer was erected by Fischer (1981) to contain a single species, M. duguetiae Fischer, reared from galls on Duguetia furfuracea (St.Hil.) (Annonaceae) at a locality in the state of São Paulo, Brazil.In this work, Fischer distinguished Mononeuron from other doryctine genera by its reduced hind wing vein cu-a, first metasomal tergite without wing-like projections, and remaining metasomal tergites without grooves.These features, however, are also present in other doryctine genera.
Mononeuron duguetiae instead appears to be morphologically very similar to the species of the mainly New World Allorhogas Gahan, which are also known to be gall associated.
Here, we redescribe M. duguetiae based on museum (including the holotype specimen) and recently collected material, and provide information about the galls where the species was reared.We also employed one mitochondrial (mt) [cytochrome oxidase I (COI)] and one nuclear [28S ribosomal (r) RNA gene] marker to elucidate the phylogenetic affinities of this taxon.

Methods
All the specimens examined in this study were collected in the type locality of the species, the vegetation of which consists of the remains of Brazilian savannah ('cerrado') forest owned by the Universidade Federal de São Carlos, in São Carlos, São Paulo, Brazil.Some of the specimens were reared from galls on D. furfuracea in February of 2011.The specimens were preserved in 100% ethanol and kept at -20°C until they were mounted or processed for DNA sequencing.The examined material is deposited in the Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos, São Carlos, SP, Brazil (DCBU), the Colección Nacional de Insectos, Instituto de Biología, Universidad Nacional Atónoma de México (CNIN IB-UNAM), and the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", Buenos Aires, Argentina (MACN).
The terminology employed follows Sharkey and Wharton (1997), except for the surface sculpture, which follows Harris (1979).Colour digital photographs were taken and edited with a Leica® Z16 APO-A stereoscopic microscope, a Leica® DFC295/ DFC290 HD camera, and the Leica Application Suite® program.Digital SEM photographs were taken with a FEI QuantaTM 250 SEM in low vacuum mode.

Phylogenetic affinities of Mononeuron based on DNA sequence data
The phylogenetic placement of Mononeuron was assessed using DNA sequences of one fast evolving (629 bp of the COI mtDNA gene) and one slow evolving [~650 bp of the second and third domain regions of the 28S rRNA gene] gene marker.Two COI and one 28S sequence were generated from specimens of M. duguetiae (COI: Gen-Bank accession nos JQ320372-3; 28S: GenBank accession no.JQ320371).Protocols for genomic DNA extraction of the samples (using a non-destructive technique) and amplification of the PCR products were the same as those employed by Ceccarelli et al. (2012).Amplification of the COI and 28S products employed the LepF1/LepRI (Hebert et al. 2004) and the 28Sfwd/28Srev primers (Belshaw and Quicke 1997;Mardulyn and Whitfield 1999), respectively.PCR products were sent directly to the High-Throughput Genomics Unit at the University of Washington (http://www.htseq.org/index.html) for sequencing.All sequences were edited using Sequencher 4. 1.4 (Gene Codes Corp.).
COI genetic distances between the two sequenced specimens of M. duguetiae and among these and previously published sequences of species belonging to Heterospathius, Heterospilus and to the gall associated doryctine genera Allorhogas Gahan, Labania Hedqvist, Monitoriella Hedqvist, Percnobracon Kieffer & Jörgensen and Psenobolus Reinhard were calculated using the K2P distance model (Kimura 1980) with PAUP version 4.0b10 (Swofford 2002).GenBank accession numbers for the above taxa are found in Zaldívar-Riverón et al. (2008).Heterospilus was recovered in previous studies as the sister group of a clade with the species of the above gall associated doryctine genera (Zaldívar-Riverón et al. 2007, 2008).The phylogenetic affinities of M. duguetiae were assessed by including the 28S and COI sequences of one specimen of M. duguetiae in a previously published COI+28S matrix (Zaldivar-Riverón et al. 2008) composed of 94 species belonging to 64 different doryctine genera.This matrix excluded the regions of ambiguous alignment detected for the 28S marker from the analyses performed.
A Bayesian MCMC partitioned analysis was run with MrBayes version 3.1.2(Ronquist and Huelsenbeck 2003) through the University of Oslo bioportal (http:// www.bioportal.uio.no/).The analysis employed two simultaneous runs of 10 million generations each, using four chains, default priors, and four partitions, one for 28S and three for COI according to its first, second and third codon positions.The evolutionary model employed for each partition was obtained with MrModeltest version 2.3 (Nylander 2004) and PAUP version 4.0b10 (Swofford 2002) using the Akaike information criterion.The first five million sampled trees of each run were deleted and the remaining trees were pooled to build a majority rule consensus tree, considering posterior probability (PP) of clades ≥ 0.95 as significantly supported (Huelsenbeck and Ronquist 2001).
We used a Bayesian approach (Buckley et al. 2002;Reeder 2003) to test for an alternative topology not present in the 50% majority rule consensus trees derived from the two simultaneous analyses.This alternative topology places the three included specimens of Allorhogas and M. duguetiae as monophyletic.Details about this approach are found in Zaldívar-Riverón et al. (2008).

Phylogenetic relationships
Corrected COI genetic distances between the two specimens of M. dugetiae and among the latter specimens and the species belonging to other gall associated genera were of 0.5% (3 bp) and of 13.5 to 16.5% (71 to 86 bp), respectively.Corrected COI distances among species of the above gall associated taxa and species of Heterospilus varied from 14.4 to 21.1 % (79 to 111 bp).Our Bayesian phylogram recovered the same major doryctine clades obtained in Zaldívar-Riverón's ( 2008) phylogenetic study: a South American, an Australian, a Holcobraconini, and an African-Holarctic-Madagascan clade.Within the South American clade, M. duguetiae is nested within a significantly supported clade (PP= 1.0) composed of members belonging to the five gall associated doryctine genera and the included species of Heterospilus Haliday (Fig. 1).This latter clade is composed of two subclades, one (PP = 0.92) with Perconobracon at the base of a monophyletic Allorhogas (PP = 0.96) that are sister to a cluster containing M. duguetiae and Monitoriella + Labania (PP = 0.94), and a second one with a Heterospilus + Heterospathius cluster that is sister to Psenobolus (PP = 0.95).The Bayesian test showed that the alternative topology with the three included specimens of Allorhogas and M. duguetiae as monophyletic is present in some of our 95% credible set of trees sampled from Bayesian analysis.We therefore cannot statistically reject the paraphyly of Allorhogas with respect to M. duguetiae.
Mononueuron is morphologically similar to Allorhogas, but it can be distinguished from members of this genus by having the vertex at least partially striate (Fig. 2B) (coriaceous in all described species of Allorhogas); hind wing vein SC+R absent (present in all described species of Allorhogas except A. ingavera Marsh); hind wing vein cu-a absent or spectral (sclerotized or nebulose in most described species of Allorhogas, only spectral in A. argentinus Brèthes and A. shawi Marsh); hind wing vein 1A incomplete (always reaching vein cu-a in other described species of Allorhogas); ovipositor long, about 1.5 times length of metasoma (Fig. 3B) (generally shorter than metasoma in most of the described species of Allorhogas, longer than metasoma in A. gallicola Gahan).
Male.Slightly smaller than female.Body length 2.0-2.5 mm; hind femur swollen, length 2.35 times its maximum width.
Biology.We collected 20 leaves of D. furfuracea infested with galls at the type locality of M. duguetiae in February of 2011.All leaves were placed in a plastic bag and maintained at room temperature.Each of the collected leaves had between three to 10 spheroidal galls with a maximum diameter of approximately 1 cm (Fig. 3A).Twenty five specimens of M. duguetiae emerged from the galls a few days after being collected, and two or more individuals were observed to emerge from each gall.Moreover, two specimens of an unidentified cecydomiid species and the following wasp taxa emerged after most of the specimens of M. duguetiae had emerged: Torymidae (six specimens belonging to an unidentified species), Eurytomidae (13 specimens of an unidentified species assigned to Sycophila Walker, and eight specimens of an unidentified species), Eulophidae (21 individuals of an unidentified species), and Diapriidae (one unidentified specimen).Members of some of these wasp families are usually parasitoids (Torymidae, Eulophidae), whereas others are generally phytophagous (e.g.Eurytomidae).We could not confirm that M. duguetiae is the species that induces the above galls, though its early emergence suggests that it might be cecidogenic.
Comments.The genus Mononeuron has been mainly characterised by the absence of hind wing vein cu-a.This feature was employed to distinguish the genus in the key to New World doryctine genera provided by Marsh (1997).We found that some of the examined specimens of M. duguetiae have hind wing vein cu-a spectral (sensu Mason 1986) but present.Some described species of Allorhogas also have a nebulous or even spectral hind wing vein cu-a (e.g. A. argentinus, A. shawi;Marsh 2002).
In our Bayesian analysis, M. duguetiae was more closely related to Monitoriella and Labania than to the species of Allorhogas, although with marginally non-significant support.This contrasts with our morphological examinations, which show that there are various external morphological features in M. duguetiae shared with species of Allorhogas.The latter genus is a mainly Neotropical and Nearctic group with 33 described and an undetermined number of undescribed species.Among the features shared by M. duguetiae and members of Allorhogas are an excavated frons, hind wing vein r-m present, pronotal collar very short or absent, propodeum usually with semicircular or subtriangular shaped basal median areas, basal sternal plate of first metasomal tergum less than 0.25 length of tergum; metasoma sculptured on first three terga, hind coxa with small but distinct basal tubercle, fore wing vein r-m present, first subdiscal cell open at apex, and hind wing vein m-cu usually curved toward wing apex, often straight to wing margin but never angled toward wing base.Further molecular phylogenetic studies employing additional markers and more taxa, especially for the species of Allorhogas, will confirm the relationships of M. duguetiae with respect to other gall associated doryctine genera.
We also examined various specimens assigned to M. duguetiae deposited in the DCBU collection that show some morphological differences, and which might represent an undescribed species.These specimens were collected in the type locality of M. duguetiae and all of them were reared from the same type of galls on D. furfuracea.They differ from our concept of M. duguetiae by having the vertex, mesopleuron and basal areas of propodeum mostly smooth, and the first metasomal median tergite smooth and with brown colour.Further molecular data will allow us to confirm whether this represents an undescribed species of Mononeuron or intraspecific variation within M. duguetiae.

Figure 1 .
Figure 1.Part of the Bayesian phylogram obtained in this study showing the phylogenetic affinities of M. duguetiae Fischer with respect to other gall associated doryctine genera (see results for details of the complete reconstructed topology).Numbers near clades represent posterior probabilities.