Doryctopambolus Nunes & Zaldívar-Riverón (Braconidae), a new neotropical doryctine wasp genus with propodeal spines

Abstract The new Neotropical doryctine genus Doryctopambolus gen. n. is erected to contain Doryctopambolus pilcomayensis (van Achterberg & Braet, 2004), comb. n., which was previously placed within Pambolus (Pambolinae), as well as three new species, Doryctopambolus clebschi sp. n., Doryctopambolus dominicanus sp. n. and Doryctopambolus sarochensis sp. n. Members of this new genus are mainly characterised by the presence of at least one pair of conspicuous propodeal apico-lateral projections, which are similar to those present in all members of Pambolinae and in species of three Australasian doryctine genera. We generated DNA barcoding sequences for the three newly described species. We discuss the morphological similarity between species of the Australasian Echinodoryctes Belokobylskij, Iqbal & Austin and Doryctopambolus. A key for the described species of Doryctopambolus is provided.

of this new genus are mainly characterised by the presence of at least one pair of conspicuous propodeal apico-lateral projections, which are similar to those present in all members of Pambolinae and in species of three Australasian doryctine genera. We generated DNA barcoding sequences for the three newly described species. We discuss the morphological similarity between species of the Australasian Echinodoryctes Belokobylskij, Iqbal & Austin and Doryctopambolus. A key for the described species of Doryctopambolus is provided.

Keywords
Doryctinae, Pambolinae, new genus, Hymenoptera introduction The presence of propodeal or scutellar spines is a common feature found in species of a number of hymenopteran parasitoid families, including Eucharitidae, Figitidae, Ichneumonidae and Braconidae, among others. Within the cyclostome group of subfamilies belonging to Braconidae, the presence of acute tubercles or spines on the apical lateral region of propodeum has been considered as the main diagnostic character employed to distinguish members of the cosmopolitan Pambolinae (Quicke andvan Achterberg 1990, van Achterberg 1995). However, species of three Australasian genera (Fijispathius Belokobylskij, Iqbal & Austin, Echinodoryctes Belokobylskij, Iqbal & Austin and Ryukyuspathius Belokobylsij; Belokobylskij et al. 2004, Belokobylskij 2008 and one described species of the Neotropical genus Concurtisella Roman (C. bidens Roman), all belonging to the cyclostome subfamily Doryctinae, also have these propodeal projections, indicating that this feature has actually evolved on separate occasions within Braconidae.
Among the 44 species of Pambolus described to date (Yu et al. 2005, Martínez et al. 2012, two species, P. pilcomayensis van Achterberg & Braet and P. leponcei van Achterberg & Braet, were described from northern Argentina by van Achterberg and Braet (2004). The authors placed these two species within Pambolinae since they have apical-lateral projections on the propodeum. However, a detailed examination of the description digital pictures of the holotype of P. pilcomayensis, as well as additional specimens collected in Brazil and Argentina, have revealed that this species possesses the main diagnostic characters that distinguish members of Doryctinae, viz. fore tibial spines, double nodus and tip of ovipositor strongly sclerotized (Belokobylskij et al. 2004).
In this study we erect the new doryctine genus Doryctopambolus gen. n. to include P. pilcomayensis (van Achterberg & Braet, 2004), comb. n. and three additional Neotropical species described herein. We also molecularly characterise the three newly described species with the DNA barcoding locus (Hebert et al. 2003) and discuss the apparently close relationship between species of this new genus and species of the Australian Echinodoryctes based on their shared external morphological features.

Methods
Specimens belonging to the new genus described in this study were collected in different localities in Argentina, Brazil, Dominican Republic and Venezuela. The Brazilian specimens assigned to P. pilcomayensis were collected in a savannah area at Itumbiara-GO Brazil using Malaise traps. The Argentinian specimens from suburban areas of Buenos Aires, Argentina, were on the other hand collected with pit fall traps associated with riparian vegetation. Specimens assigned to the three newly described species were both collected with yellow pan traps in the Parque Nacional Cerro Saroche, Lara, Venezuela, and in Punta Cana and Parque Nacional Armando Bermudez, in Dominican Republic. All our examined material was compared with other Neotropical doryctine genera using Marsh's (1997Marsh's ( , 2002 keys. Digital colour photographs of all the species included in this work were taken with a Leica® Z16 APO-A stereoscopic microscope and a Leica® DFC295/DFC290 HD camera, and were edited using the Leica Application Suite® program. Digital scanning electron microscope (SEM) photographs of P. pilcomayensis were taken with a FEI QuantaTM 250 SEM in low vacuum mode. The specimens examined in this study are deposited at Universidade Federal de São Carlos -Departamento de Ecologia e Biologia Evolutiva, Brazil (DCBU), Colección Nacional de Insectos, Instituto de Biología, Universidad Nacional Autónoma de México (CNIN-IB UNAM), Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", Buenos Aires, Argentina (MACN), and Museo de La Plata, Argentina (MLP). Terminology for body structure, sculpture and wing venation features follows Marsh (2002).
DNA sequences belonging to the barcoding locus [~650 bp of the cytochrome oxidase I (COI) mitochondrial DNA gene; Hebert et al. 2003] were generated for specimens belonging to the new genus using the DNA extraction and amplification protocols employed in a previous study . Genetic distances were calculated for the sequenced specimens using the K2P distance model with PAUP* version 4.10b (Swofford 2002 cies of Doryctopambolus and C. bidens are the only Neotropical doryctine taxa reported to have these projections, though they mainly differ by their first subdiscal cell (open at apex in Doryctopambolus, closed in C. bidens) and ovipositor length (about the same length as metasoma in Doryctopambolus, longer than body in C. bidens). Doryctopambolus differs from the Australasian Fijispathius and Ryukyuspathius mainly by the fore wing first subdiscal cell open at apex (closed in the later two genera) and the first metasomal segment not petiolate (basal sternal plate at most 0.5 lenght of first tergite in Doryctopambolus, 0.65 to 0.7 in Fijispathius and Ryukyuspathius). Doryctopambolus is morphologically similar to the Australian Echinodoryctes (figs 2A-B). However, species of Doryctopambolus differ from those of Echinodoryctes by having partially reduced to well-developed wings (micropterous in Echinodoryctes), propodeum evenly curved and strongly rugoseareolate (globose and mostly smooth in Echinodoryctes), hind coxa without basoventral tubercle and all femora without dorsal protuberances (both present in Echinodoryctes).
Description. Small size, 2.2-3.6 mm; black to light brown species. Head: head globose; antennal sockets distinctly separated from each other by at least 0.5 times its diameter; frons almost flat, without median carina or furrows; ocelli arranged in equilateral triangle; eye with distinct and sparse setae; gena and temple smooth; malar suture absent; first flagellomere slightly shorter than scape and pedicel combined, slightly longer than second flagellomere; antenna with 16-28 antennomeres; occipital carina meeting hypostomal carina before mandible. Mesosoma: Length of mesosoma about two times its maximum width; neck of pronotum fairly long; pronotal crest conspicuous; mesoscutum declivous anteriorly; mesoscutal lobes smooth and polished medially; notauli complete and strongly impressed; scutellar sulcus deep, with its height 0.8-0.9 times height of scutellar disc; precoxal sulcus complete and scrobiculate, as long as mesopleuron; prepectal carina coarse and complete; propodeum evenly curved and strongly rugose-areolate, with at least one pair of conspicuous apico-lateral projections; propodeal bridge absent. Legs: fore tibia with a row of 7-8 stout spines; middle tibia without spines; femora without dorsal protuberances; hind coxa without basal tubercle. Wings: partially reduced to well-developed wings; fore wing veins r-m and 2RS present; m-cu arising interstitial or slightly antefurcal with vein 2RS, cu-a distinctly postfurcal with vein 1M; first subdiscal cell open at apex; hind wing vein M+CU equal length of vein 1M; cu-a present, m-cu absent; stigma present on male hind wing. Metasoma: length of first metasomal tergum 1.3-1.6 times its apical width, apical width about 2.0-2.3 times basal width; basal sternal plate (acrosternite) about 0.33-0.5 times length of tergum; suture between second and third metasomal tergites absent; second metasomal tergite at least sculptured basally; third metasomal tergite usually smooth, sometimes sculptured basally; remaining metasomal tergites entirely smooth and polished; ovipositor about same length of metasoma.
Distribution. Neotropical. Known from central Argentina to northern Venezuela, and from Dominican Republic in the Caribbean.
Comments. Three new species of Doryctopambolus and D. pilcomayensis comb. nov. are described and redescribed in this study, respectively. Four additional species belonging to this genus, two from Cerro Saroche, Lara, Venezuela, and two from Argentina, were also identified. The two Argentinian species could not be described due to their bad state of preservation. Of the two species from Venezuela, one was represented by a single male and the other one by an incomplete female, and their allospecificity was corroborated with DNA barcoding sequences (DNA voucher nos. DORYC239, 274; GenBank accession numbers JN266989, JN267020). The Parque Nacional Cerro Saroche is a natural reserve of about 32,294 h mainly composed of xeric vegetation with deciduous and semideciduous shrubs (Inparques 1992). The doryctine fauna from this reserve has been previously reported by Briceño et al. (2009), and includes some rarely collected genera such as Verae Marsh, Coiba Marsh and Hecabolus Curtis.
Our morphological observations revealed that the species of Doryctopambolus share various external morphological features with the two described species of the endemic Australian Echinodoryctes (figs 2A-B), including a similar body habitus, at least one pair of apico-lateral propodeal projections and the second metasomal tergite at least partially sculptured. Further morphological and molecular studies will confirm whether or not species of these two genera are congeneric.
Etymology. Combination from the doryctine generic names Doryctes Haliday, 1836 and Pambolus Haliday, 1836, since the type species of this new genus was previously placed within Pambolus. Gender is masculine. Diagnosis. This species distinguishes from D. sarochensis sp. n. by having one pair of propodeal apico-lateral projections (two pairs in D. sarochensis sp. n.) and third metasomal tergite entirely smooth (costate baso-laterally). Doryctopambolus pilcomayensis differs from D. dominicanus sp. n. and D. clebschi sp. n. by having the vertex striate (smooth in D. dominicanus sp. n. and D. clebschi sp. n.), first metasomal tergite not petiolate (at least 0.5 of tergum) and second metasomal tergite entirely sculptured (mostly smooth).
Males. Essentially as females; body length 2.1-3.0 mm; body brown, with antennae, trochanter, trochantellus, tarsi, fore and middle coxae light brown to yellowish; face, frons and vertex slightly sculptured; stigma present in hind wing.  Comments. The presence of D. pilcomayensis near Buenos Aires can be explained by the courses of the Parana and Uruguay basins, which carry downstream many plant and animal species from tropical areas to higher latitudes. n. However, D. clebschi differs from the latter species by having the face smooth medially and slightly rugose laterally (entirely rugose in D. dominicanus sp. n.), mesopleuron mostly smooth (mostly smooth-rugose), first metasomal tergite mostly smooth apically (mostly sculptured apically), and second metasomal tergite mostly smooth, slightly costate baso-laterally (costate basally). These two species distinguish from the remaining two described species of Doryctopambolus, D. pilcomayensis comb. n. and D. sarochensis sp. n., by having the vertex smooth (always sculptured in the latter two species), first metasomal tergite distinctly petiolate (not petiolate), and second metasomal tergite partially smooth (always entirely sculptured).
Etymology. This species is named in honour to our good friend and colleague Hans Clebsch, who collected the specimens assigned to this species. Description. Female. Body length 2.7 mm; fore wing 1.8 mm; ovipositor 1.3 mm. Colour: head light brown, antennae light brown, turning honey yellow to apex, palpi yellow; mesosoma and first and basal two thirds of second metasomal tergites dark brown; wings hyaline; veins and stigma light brown; legs light brown to honey yellow; ovipositor and sheaths honey yellow, apex strongly sclerotised and dark. Head: 16 flagellomeres (one antenna broken, with only 11 flagellomeres; eyes ovoid, small and setose; face rugose, median area not swollen; clypeus slightly rugose; malar space 0.4 times eye height; frons and vertex smooth; temple in dorsal view 2.0 times eye width. Mesosoma: 1.7 times longer its maximum height; pronotum rugose-coriaceous laterally; pronotal groove wide and scrobiculate; propleuron slightly rugose; mesoscutal lobes entirely smooth and polished; notauli wide, deep and scrobiculate, meeting before scutellum in a longitudinally costate area; scutellum smooth, with some setae; scutellar sulcus with five carinae; height of scutellar sulcus 0.9 times height of scutellar disc; subalar sulcus wide, deep and scrobiculate, joining mesopleural sulcus, remaining area of mesopleuron smooth, rugose near precoxal sulcus; venter of mesopleuron smooth; propodeum with one pair of long and sharp apico-lateral projections, slightly shorter than first flagellomere. Legs: hind coxa mostly smooth, slightly rugose ventrally. Wings: fore wing length 3.8 times its maximum width, r:3RSa:3RSb = 1:2:4; 2RS:3RSa:r-m = 2:2:1; m-cu arising antefurcal with vein 2RS; 1cu-a slightly postfurcal with 1M; hind wing vein M + CU about equal length of vein 1M. Metasoma: length of first metasomal tergum 1.6 times its apical width, apical width about 2.1 times basal width, first metasomal tergite costate with slightly rugose microsculpture; basal third of second metasomal tergite costate, apical two thirds smooth and polished; remaining metasomal tergites smooth and polished; basal sternal plate (acrosternite) about 0.5 times length of tergum; ovipositor 0.9 times as long as metasoma. Diagnosis. Doryctopambolus sarochensis sp. n. distinguishes from the remaining species of the genus by having the vertex striate-rugose (striate in D. pilcomayensis comb. n. and smooth in the remaining two species), two pairs of propodeal apico-lateral projections, the basal ones small and blunt and the second pair long and distinctly truncate, and third metasomal tergite costate baso-laterally (entirely smooth in the other species).
Variation Genetic distances. Interspecific variation of the barcoding locus among the examined species of Doryctompambolus, D. clebschi (two specimens), D. dominicanus (one specimen), D. sarochensis (two specimens) and the two undescribed species from Venezuela (one specimen each), was congruent with the interspecific variation observed in other braconid genera, ranging from 4.2 to 14%. The lowest interspecific genetic distance occurred between specimens of the two Dominican species, D. clebschi and D. dominicanus.