Species Diversity in the Parasitoid Genus Asobara (Hymenoptera: Braconidae) from the Native Area of the Fruit Fly Pest Drosophila suzukii (Diptera: Drosophilidae)

Drosophila suzukii (Matsumura), commonly known as Spotted Wing Drosophila (SWD), is a worldwide serious economic threat to the production of berries and stone fruits. The chemical control widely used against this pest is often not able to preventing yield losses because wild flora offers an abundance of fruits to D. suzukii where the pest is able to reproduce and from where it recolonizes neighbouring cultivated fields. Alternatively, within Integrated Pest Management protocols for D. suzukii, biological control could play a key role by reducing its populations particularly in non-cultivated habitats, thus increasing the effectiveness and reducing the side negative effects of other management strategies. Because of the scarcity and of the low efficiency of autochthonous parasitoids in the new invaded territories, in the last few years, a number of surveys started in the native area of D. suzukii to find parasitoid species to be evaluated in quarantine structures and eventually released in the field, following a classical biological control approach. This paper reports the results of these surveys carried out in South Korea and for the first time in China. Among the parasitoids collected, those belonging to the genus Asobara Foerster resulted dominant both by number and species diversity. By combining morphological characters and the mitochondrial COI gene as a molecular marker, we identified seven species of Asobara, of which two associated with D. suzukii, namely A. japonica and A leveri, and five new to science, namely Asobara brevicauda, A. elongata, A mesocauda, A unicolorata, A. triangulata. Our findings offer new opportunity to find effective parasitoids to be introduced in classical biological control programmes in the territories recently invaded by D. suzukii.


Introduction
Drosophila suzukii (Matsumura), commonly known as Spotted Wing Drosophila (SWD), is a worldwide serious economic threat to the production of berries and stone fruits [1,2]. This pest, native of the South-eastern Asia, has been reported simultaneously from Europe and North America in 2008 quickly becoming a key pest of soft and thin skin fruit crops in many locations [3,4,5,6]. More recently new records of the pest include Central and South America [7,8]. The management of SWD in fruit production areas invaded by this pest mainly relays on pesticides [6]. However, this approach is often ineffective in preventing yield losses because wild flora (and fruits) neighbouring the cultivated fields offers a valid alternative for reproduction and refuge sites from where recolonize the crops [9]. Moreover, there is a growing concern about the negative side-effects of pesticides on the environment and for the possible selection of resistant populations. Alternative strategies for controlling this pest are highly desirable and among these, biological control could play a key role particularly in reducing D. suzukii populations in non-cultivated habitats, with positive effects on other management strategies applied in field crops.
To date, field surveys and laboratory tests suggested that only few parasitoid and predator species attacking Drosophilids in Europe and North America adapted to the invasive D. suzukii, although they resulted ineffective inreducing its populations below economic damage thresholds [10][11][12][13]. The absence of endemic effective biocontrol agents in Europe and North America prompted the initiation of surveys in the native range of D. suzukii to find parasitoid species to be evaluated in quarantine and eventually released in the field, following a classical biological control approach. In this work, within the framework of an international project funded by the European Union aiming at enhancing the sustainable control of invasive pests, a survey of parasitoids of D. suzukii has been carried out in China and South Korea, for which no record is available. Among the parasitoids collected, those belonging to the genus Asobara Foerster were dominant both by number and species diversity. By combining a morphological and a molecular approach, we identified seven species of Asobara including five new to science and two associated with D. suzukii. Our findings offer new opportunity to find effective parasitoid/s to be introduced in classical biological control programmes in all countries recently invaded by D. suzukii.

Insect collection
Sampling of D. suzukii and other Drosophilids parasitoids has been carried out in different locations of China (Yunnan Province) and South Korea (see material examined for detailed locations) considered to be part of the native area of D. suzukii. Both unspoiled wild areas and cultivated fields (mainly blueberry but also pear, peach and Chinese bayberry -Myrica rubracultivations) were surveyed. In each location two strategies of collection were adopted. The first one was harvesting soft fruits from trees and shrubs (e.g. blueberries, blackberries and Chinese bayberries) and storing them in aerated boxes in a climatic chamber at 25°C. All boxes were checked daily for parasitoid (and fly) emergence for 3 weeks. The second strategy consisted in positioning fruit-baited traps in the field prepared by cutting a few holes (0.5 mm diameter) on air tight plastic boxes commonly used for preserving food in the fridge (30x15x10cm). Each box was filled with sliced fruits (mainly banana, melon) either uninfested or previously exposed for a few days to hundreds mated females of D. suzukii. A transect of ten fruit-baited traps 100 m away from each other was composed in wild vegetation or in cultivated blueberry fields. Traps were collected one week after set up and parasitoids found crawling in the traps on fruit slices were collected. All traps were kept at 25°C in a climatic chamber (the holes and the lid covered with fine mesh gauze) and checked daily for the emergence of parasitoids (and flies) for 3 weeks. Each parasitoid collected was readily killed in absolute ethanol and preserved at -20°C.
No specific permissions were required for all locations/activities that were conducted with colleagues of local Institutions (see Acknowledgements section). As for private farms we were invited by owners to carry out investigations for reducing the impact of Drosophila suzukii in their propereties. Human participants, specimens or tissue samples, or vertebrate animals, embryos or tissues were not involved in this research. No endangered or protected species were involved in this research.

Morphological analysis
For card mounting, insects stored in absolute ethanol were dried chemically by placing them in a 1:1 absolute ethanol:xylene solution; after 24 hours they were transferred into amyl acetate for 24 hours and then rinsed in amyl acetate until its complete evaporation. They were then mounted on card using a water soluble glue. For slide mounting the protocol reported by Noyes has been followed [14]. In brief, wings were mounted in Canada balsam without processing while the remaining parts were kept for 5 minutes in a 10% KOH solution at 100°C, for 5 minutes in acetic acid and dehydrated in ethanol from 70% to 100%. A drop of clover oil was added to absolute ethanol and after alcohol evaporation the insect was dissected and mounted in Canada balsam. The slides were left for 2 hours on a hotplate to harden the balm before positioning the coverslips. All descriptions and photographs were made using a dissection microscope (LeicaZ16 APO microscope equipped with a Leica DFC 425C digital camera).
Morphological terminology used for species description follows van Achtherberg [15,16]. A new record of distribution is indicated by an asterisk ( Ã ) preceding the country.
The studied material will be deposited in the following depositories:Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS) and Naturalis Biodiversity Center, Leiden, Netherlands (RMNH including collections from former National Museum of Natural History, Leiden, the Entomological Institute, Wageningen and the Zoological Museum, Amsterdam).

Molecular analysis
Before card mounting and after their attribution to the genus Asobara and to a specific morphospecies, whole specimens (see Table 1 for the complete list of samples examined) were subjected to genomic DNA extraction following a non-destructive protocol using a Chelexproteinase K-based without grounding the specimen [17]. Partial sequence of the mitochondrial cytochrome oxidase subunit I gene (COI) was determined for each studied individual and the phylogenetic relationships investigated.
COI was amplified using one of the following primer combinations: LCO and HCO [18], LepF1 and LepR1 [19] or LCOasob and HCO [20]. In a few cases a semi-nested PCR was conducted using one of the above mentioned forward primers and the reverse primer HCOout [21] followed by one of the combinations above cited as second amplification. Reactions were performed in 15 μl volumes containing: 3μl of5 X GoTaq buffer (Promega); 1.5 μl dNTP (2.5 mM each); 0,75 μl of forward and reverse primer (10 μM each); 0.2 μl GoTaq™ DNA Polymerase (5u/ μl) and 1μl template DNA. Amplifications were achieved using a Biorad thermocycler Mycycler (Biorad) programmed for: 1 min at 94°C; followed by 40 cycles of 30 s at 94°C, 90 s at 48°C, and 60 s at 72°C; and a final step of 7 min at 72°C. PCR products were visualized after electrophoresis on 1.5% agarose gel stained with ethidium bromide to confirm the amplification. Fragments obtained were sequenced in both sense and antisense directions by adopting EZ-seq standard  [22] with ClustalW tool and edited manually. COI sequences were verified for protein coding frameshifts and nonsense codons using MEGA6 [23]. COI sequences of Asobara individuals were deposited in GenBank under the accession numbers KT835409-KT835453. Phylogenetic and molecular evolutionary analysis were conducted using MEGA 6 [23]. Phylogeny was constructed using maximum likelihood (ML) and neighbor-joining (NJ) analysis. ML tree was obtained implementing the GTR+G substitution model, selected in MEGA6 as the better to describe the substitution pattern (lowest BIC score), and five discrete gamma categories. Missing data were treated with the pairwise-deletion option and all codon positions were included in the analysis. For tree inference, the nearest-neighbor-interchange (NNI) heuristic method was used and the initial tree selected using the NJ/BioNJ option. ML branch support was based on 1,000 bootstrap replications. NJ tree was constructed using the Tamura 3-parameter method to compute the evolutionary distances, treating missing data with the pairwisedeletion option and running 1,000 bootstrap replications for branch supporting. ML and NJ analyses involved 91 nucleotide sequences and a total of 711 positions in the final dataset. In addition to 44 COI sequences produced in this work, 45 Asobara COI sequences were retrieved from GenBank. These latter included sequences of Asobara citri (Fisher), A. leveri (Nixon), A. japonica Belokobylskij, A. persimilis (Papp), A. pleuralis (Ashmead), A. rossica Belokobylskij, A. rufescens (Foerster), A. tabida (Nees von Esenbeck), and sequences of undescribed Asobara spp. from Japan. Further, COI sequences of Rhygoplitis terminalis (Gahan) and Apanteles anarsiae Ashmead were recovered from GenBank and used as outgroups in phylogenetic analyses. Uncorrected intra-and interspecific p-distances (number of base differences per site) based on COI sequences were also calculated using MEGA6.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:AAF73644-AE86-4546-8D20-AA3A870DCD48. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS [author to insert any additional repositories].

Results
The vast majority of the parasitoids collected from fruit-baited traps and ripe fruits belonged to the genus Asobara Foerster. Morphological analysis allowed the identification of discrete
Diagnosis. Third article of antenna distinctly shorter than fourth article; first subdiscal cell of fore wing widely open because of absence of veins CU1b and 2-1A; maxillary palp with 5-6 articles; veins 2-SR and 1-SR+M of fore wing present; vein cu-a of hind wing of macropterous specimens variable, if absent then lateral carina of mesoscutum absent in front of tegulae and face without a pair of medium-sized vertical grooves above clypeus.
Biology. Rather large genus, contains parasitoids of Drosophilidae and Sepsidae in decaying organic matter, especially fruits and leaves. Species with widened ovipositor sheath have been reared as parasitoids of Tephritidae in fruits.
Distribution. Cosmopolitan [25]. Key to Asobara species from China and South Korea Females. Holotype, length of body (excluding ovipositor) 1.9 mm, length of fore wing 2.1 mm. Head. Width of head 1.4 times its median length ( Fig 1A and 1B), largely glabrous and strongly shiny dorsally; antenna with 23 articles, densely erect bristly setose, length of third article 0.8 times as long as fourth article, slender, length of third, fourth and penultimate articles 4.5, 6.5 and 3.0 times their width, respectively; length of maxillary palp 1.2 times height of head; eye in dorsal view ( Fig 1A) twice as long as temple, sparsely setose; temple in dorsal view subparallel-sided; OOL:diameter of ocellus:POL = 6:2:3 ( Fig 1A); minimum width of face 0.55 times maximum width of head and 0.75 times its height, smooth, with some long and erect setae; clypeus transverse and oval, its surface smooth and flattened; height of clypeus in lateral view equal to width of middle tooth of mandible ( Fig 1B); anterior tentorial pits indistinct ( Fig  1B); length of malar space 0.14 times basal width of mandible; mandible twice as long as wide, largely smooth, with a lamella ventrally and dorsally, with medium-sized carina connected to third tooth ( Fig 1B).
Mesosoma. Length of mesosoma 1.4 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum smooth; epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 2 short crenulae; metapleuron largely smooth, except for a few carinae ventrally; notauli only anteriorly impressed, with strong lateral carina and largely absent on disc; medioposterior depression of mesoscutum small, deep and circular ( Fig 1C); mesoscutum smooth and largely glabrous and its lateral carina interrupted in front of tegulae; scutellar sulcus moderately deep and with one carina, 0.4 times as long as scutellum; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a narrow parallel-sided areola (Fig 1D), its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide.
Wings. Pterostigma narrow triangular ( Fig 1F); vein r about 0.5 times width of pterostigma, issued at apical 0. Legs. Hind coxa smooth and elongate, rounded basally; tarsal claws slender medially; length of femur, tibia and basitarsus of hind leg 4.6, 10.0 and 5.0 times their width, respectively; hind femur, tibia and tarsus with long erect setae. Metasoma. Length of first tergite 2 times its apical width, its surface largely smooth but coarsely rugose medially and laterally lamelliform, its dorsal carinae nearly complete; laterope present; dorsope long and area laterally lamelliform; remainder of metasoma smooth and depressed; length of setose part of ovipositor sheath 0.12 times fore wing and 0.4 times as long as hind tibia and with a few long setae ( Fig 1E); ovipositor sheath more or less widened subapically.
Distribution. South Korea.
Host. Collected in banana traps and in wild berries of Rubus infested by unidentified Drosophilidae.
Diagnostic remarks. The new species is similar to A. orientalis Viereck, 1913, reported from India, Korea and Philippines (Luzon) and to A. pleuralis (Ashmead) from Philippines, Indonesia, Japan and China. It differs by having apical 4-5 antennal articles white (apical 7-8 white in A. orientalis), ovipositor sheath more or less widened subapically (narrow subapically in A. orientalis),first metasomal tergite and mesoscutum dark orange (yellowish brown in A. orientalis); propodeum with rather short lamelliform costulae (without costulae in A. orientalis), third antennal segment of female about 4.5 times as long as wide (about 6.8 times in A. orientalis) and precoxal sulcus complete (absent anteriorly in A. orientalis). The length of ovipositor separates A. brevicauda from A. pleuralis as indicated in the key.
Asobara elongata van Achterberg and Guerrieri, sp. nov. urn:lsid:zoobank.org: act:7B827FFC-4147-4997-AC57-D06E35BB3738 (Fig 2) Material examined. Head. Width of head 1.7 times its median length, largely glabrous and strongly shiny (Fig  2A and 2B); antenna incomplete ( Fig 2C), densely erect bristly setose, length of third article 0.5 times as long as fourth article, slender, length of third and fourth articles 4.5 and 10.0 times their width, respectively; length of maxillary palp 1.2 times height of head; eye in dorsal view 3 times as long as temple, sparsely setose; temple in dorsal view gently curved (Fig 2A); OOL: diameter of ocellus: POL = 9:3:4 (Fig 2A); minimum width of face 0.54 times maximum width of head and 0.7 times its height, smooth, with some long and erect setae; clypeus transverse and rectangular, its surface smooth and flattened; height of clypeus in lateral view equal to width of middle tooth of mandible; anterior tentorial pits indistinct ( Fig 2B); length of malar space 0.4 times basal width of mandible; mandible 2.5 times as long as wide, largely smooth, with a lamella ventrally and dorsally, with medium-sized carina connected to third tooth.
Mesosoma. Length of mesosoma 1.3 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum and epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 3short crenulae; metapleuron largely smooth, except for a few carinae ventrally; notauli only anteriorly impressed, with strong lateral carina and largely absent on disc; medioposterior depression of mesoscutum absent ( Fig 2D); mesoscutum smooth and largely glabrous and its lateral carina almost complete, reaching tegulae; scutellar sulcus moderately deep and with one carina, 0.5 times as long as scutellum; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a parallel-sided areola (Fig 2E), its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide.
Metasoma. Length of first tergite twice its apical width, its surface largely smooth but coarsely rugose medially and laterally lamelliform, its dorsal carinae nearly complete; laterope present; dorsope long and area laterally lamelliform; remainder of metasoma smooth and depressed; setose part of ovipositor sheath0.42 times as long as fore wing and 1.1 times as long as hind tibia and with a few long setae; ovipositor sheath parallel-sided.
Host. Collected in banana traps infested by unidentified Drosophilidae. Diagnostic remarks. The new Oriental species is similar to the East Palaearctic A. japonica. It differs by having the fourth antennal article more elongate (about 10 times as long as wide, about 7 times in A. japonica), the propodeum black (yellowish brown or reddish) andthe head directly narrowed behind eyes (slightly less narrowed behind eye).
Asobara japonica Belokobylskij, 1998. (Fig 3) Material examined. 11♀, 5♂, laboratory rearing on Drosophila suzukii from material collected in SouthKorea, Geochang, Gyeongsangnam-do (35°40'5.43"N 127°52'43.68"E), 11   Head. Width of head 1.5 times its median length, largely glabrous and shiny; antenna with 21 articles and densely erect bristly setose, length of third article 0.6 times as long as fourth article, slender, length of third, fourth and penultimate articles 4.5, 7.0 and 3.0 times their width, respectively; length of maxillary palp 1.4 times height of head; eye in dorsal view 4 times as long as temple, sparsely setose; temple in dorsal view subparallel-sided ( Fig 3A); OOL:diameter of ocellus:POL = 7:3:4 ( Fig 3A); minimum width of face 0.52 times maximum width of head and 1.2 times its height (Fig 3B), evenly smooth, with some long and erect setae; clypeus transverse, oval, its surface smooth and flattened; height of clypeus in lateral view equal to width of middle tooth of mandible; anterior tentorial pits indistinct ( Fig 3B); length of malar space 0.1 times basal width of mandible; mandible 1.9 times as long as wide, largely smooth, with a lamella ventrally and dorsally, medium-sized carina connected to third tooth.
Mesosoma. Length of mesosoma 1.1 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum smooth; epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 1 short crenula; metapleuron largely smooth, except for a few carinae ventrally; notauli ( Fig 3C) only anteriorly impressed, with strong lateral carina and largely absent on disc; mesoscutum smooth and largely glabrous and its lateral carina almost complete, reaching tegulae; scutellar sulcus moderately deep and with one carina, as long as scutellum; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a wide parallel shaped areola (Fig 3D), its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide; propodeal spiracle round, small and in front of apical third of propodeum.
Metasoma. Length of first tergite 2.25 times its apical width, its surface largely smooth but coarsely rugose medially and laterally lamelliform, its dorsal carinae nearly complete; laterope present; dorsope long and area laterally lamelliform; remainder of metasoma smooth and depressed; length of setose part of ovipositor sheath 0.4 times fore wing and 1.1 times as long as hind tibia and with a few long setae; apex of ovipositor sheath parallel-sided medially.
Variation. Not much in the material at hand apart from: antenna of ♂ with 2-3 and of ♀ with 4-5 white apical articles; white apical articles of antenna of ♀ with some more or less dark setae apically; setose part of ovipositor sheath 0.45-0.50 times as long as fore wing and 1.1-1.2 times as long as hind tibia; pterostigma brown to dark brown medially; wing membrane rather infuscate; mesoscutum and metapleuron of ♀ with brown parts appearing black in darker specimens.
Holotype, length of body (excluding ovipositor) 1.9 mm, length of fore wing 2.1 mm. Head. Width of head 1.4 times its median length, largely glabrous and strongly shiny ( Fig  4A and 4B); antenna with 21 articles, densely erect bristly setose, length of third article 0.6 times as long as fourth article, slender, length of third, fourth and penultimate articles 3.5 5.5 and 3.0 times their width, respectively; length of maxillary palp 1.1 times height of head; eye in dorsal view 3 times as long as temple, sparsely setose; temple in dorsal view gently curved ( Fig  4A); OOL:diameter of ocellus:POL = 7:2:3 ( Fig 4A); minimum width of face 0.56 times maximum width of head and 0.8 times its height, smooth, with some long and erect setae; clypeus transverse and oval, its surface smooth and flattened ( Fig 4B); height of clypeus in lateral view equal to width of middle tooth of mandible; anterior tentorial pits indistinct ( Fig 4B); length of malar space 0.1 times basal width of mandible; mandible 1.9 times as long as wide, largely smooth, with a lamella ventrally and dorsally, with medium-sized carina connected to third tooth.
Mesosoma. Length of mesosoma 1.2 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum smooth; epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 1 short crenula; metapleuron largely smooth, except for a few carinae ventrally; notauli only anteriorly impressed, with strong lateral carina and largely absent on disc; medioposterior depression of mesoscutum absent ( Fig 4C); mesoscutum smooth and largely glabrous and its lateral carina almost complete, reaching tegulae; scutellar sulcus moderately deep, 0.5 times as long as scutellum and with one carina; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a narrow parallelsided areola (Fig 4D), its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide.
Metasoma. Length of first tergite 1.6 times its apical width, its surface largely smooth but coarsely rugose medially and laterally lamelliform, its dorsal carinae nearly complete; laterope present; dorsope long and area laterally lamelliform; remainder of metasoma smooth and depressed; length of setose part of ovipositor sheath 0.34 times fore wing and about as long as hind tibia and with a few long setae; ovipositor sheath parallel-sided.
Colour. Body dark brown; with following parts paler: face and clypeus, scapus (and 18 th -21 st articles whitish), apical corner of mesopleuron and all legs; wing membrane slightly infuscate; pterostigma and veins brown.
Variation. Antenna of ♀ with 3-4 white apical articles. Face sometimes bright yellow, apical part of mesopleuron sometimes yellow (heavily contrasting with the remaining dark brown part) Distribution. China (Yunnan).
Host. Collected in banana traps infested by unidentified Drosophilidae Diagnostic remarks. The new Oriental species is similar to the East Palaearctic A. japonica Belokobylskij. It differs by having the face largely or completely brownish yellow, similar to colour of the clypeus (face dark brown, strongly contrasting with yellowish clypeus in A. japonica), first discal cell of the fore wing slightly more elongate (rather robust) and setose part of ovipositor sheath about as long as hind tibia (1.1-1.2 times as long as hind tibia).
Holotype, length of body (excluding ovipositor) 1.9 mm, length of fore wing 2.1 mm. Head. Width of head 1.4 times its median length, largely glabrous and strongly shiny; antenna with 19 articles, densely erect bristly setose, length of third article 0.6 times as long as fourth article, slender, length of third, fourth and penultimate articles 3.5 5.5 and 3.0 times their width, respectively; length of maxillary palp 1.1 times height of head; eye in dorsal view 3 times as long as temple, sparsely setose; temple in dorsal view gently curved; OOL:diameter of ocellus:POL = 7:2:3; minimum width of face 0.56 times maximum width of head and 0.8 times its height, smooth, with some long and erect setae; clypeus transverse and oval, its surface smooth and flattened; height of clypeus in lateral view equal to width of middle tooth of mandible; anterior tentorial pits indistinct; length of malar space 0.1 times basal width of mandible; mandible 1.9 times as long as wide, largely smooth, with a lamella ventrally and dorsally, with medium-sized carina connected to third tooth.
Mesosoma. Length of mesosoma 1.2 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum smooth; epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 1 short crenula; metapleuron largely smooth, except for a few carinae ventrally; notauli only anteriorly impressed, with strong lateral carina and largely absent on disc; medioposterior depression of mesoscutum absent; mesoscutum smooth and largely glabrous and its lateral carina almost complete, reaching tegulae; scutellar sulcus moderately deep, 0.5 times as long as scutellum and with one carina; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a narrow parallel-sided areola, its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide.
Metasoma. Length of first tergite 1.6 times its apical width, its surface largely smooth but coarsely rugose medially and laterally lamelliform, its dorsal carinae nearly complete; laterope present; dorsope long and area laterally lamelliform; remainder of metasoma smooth and depressed; length of setose part of ovipositor sheath 0.34 times fore wing and about as long as hind tibia and with a few long setae; ovipositor sheath parallel-sided.
Colour. Body dark brown; with following parts paler: face and clypeus, scapus (and 15 th -17 th articles whitish), apical corner of mesopleuron and all legs; wing membrane slightly infuscate; pterostigma and veins brown.
Variation. Only one specimen available. Distribution. China (Yunnan), Japan Host. Collected in banana traps infested by unidentified Drosophilidae Diagnostic remarks. The single specimen examined appears almost identical to A. mesocauda if not for the shorter r-m vein in the fore wing and for antenna of 19 segments (21 in A. mesocauda). These differences appear to be supported by molecular evidence (see below) even though it is possible that more morphological differences will emerge when more material will be available.
Head. Width of head 1.3 times its median length, largely glabrous and strongly shiny ( Fig  6A and 6B); antenna with 20 articles, densely erect bristly setose, length of third article 0.7 times as long as fourth article, slender, length of third, fourth and penultimate articles 4.0, 6.0 and 3.5 times their width, respectively; length of maxillary palp 1.1 times height of head; eye in dorsal view 3.5 times as long as temple, sparsely setose; temple in dorsal view subparallel-sided ( Fig 6A); OOL:diameter of ocellus:POL = 6:2:3 ( Fig 6A); minimum width of face 0.55 times maximum width of head and 0.85 times its height, smooth, with some long and erect setae; clypeus transverse, sub-elliptical, its surface smooth and flattened; height of clypeus in lateral view equal to width of middle tooth of mandible; anterior tentorial pits indistinct ( Fig 6B); length of malar space 0.1 times basal width of mandible; mandible 1.9 times as long as wide, largely smooth, with a lamella ventrally and dorsally, with medium-sized carina connected to third tooth.
Mesosoma. Length of mesosoma 1.3 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum smooth; epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 3 short crenulae; metapleuron largely smooth, except for a few carinae ventrally; notauli only anteriorly impressed, with strong lateral carina and largely absent on disc; medioposterior depression of mesoscutum elongated, deep and elliptical ( Fig 6C) and lateral carina absent in front of tegulae; mesoscutum smooth and largely glabrous and its lateral carina almost complete, reaching tegulae; scutellar sulcus moderately deep, as long as scutellum and with one carina; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a wide parallel-sided areola (Fig 6D), its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide.
Wings. Pterostigma narrow triangular ( Fig 6E); vein r 0.6 times width of pterostigma, issued from basal 0.  Fig 7a); minimum width of face 0.5 times maximum width of head and 0.7 times its height, smooth, with some long and erect setae; clypeus transverse and oval, its surface smooth and flattened (Fig 7b); height of clypeus in lateral view equal to width of middle tooth of mandible; anterior tentorial pits indistinct ( Fig 7B); length of malar space 0.1 times basal width of mandible; mandible 1.9 times as long as wide, largely smooth, with a lamella ventrally and dorsally, with medium-sized carina connected to third tooth.
Mesosoma. Length of mesosoma 1.3 times its height; pronotum without pronope; antescutal depression distinct and finely crenulate laterally; side of pronotum smooth; epicnemial area smooth; precoxal sulcus complete; remainder of mesopleuron smooth; episternal scrobe elliptical; pleural sulcus smooth, except for some crenulae medially and ventrally; mesosternal sulcus wide and with 3 short crenulae; metapleuron largely smooth, except for a few carinae ventrally; notauli only anteriorly impressed, with strong lateral carina and largely absent on disc; medioposterior depression of mesoscutum small and nearly round ( Fig 7C); mesoscutum smooth and largely glabrous and its lateral carina absent in front of tegulae; scutellar sulcus moderately deep and with one carina, as long as scutellum; scutellum in lateral view rather flat; metanotum slightly lamelliform protruding dorsally in lateral view; surface of propodeum smooth except for a medium-sized median carina, rather short lamelliform costulae and a narrow barrelshaped areola (Fig 7D), its dorsal corners hardly protruding in lateral view and lateral carina of propodeum wide.
Metasoma. Length of first tergite twice its apical width, its surface largely smooth but coarsely rugose medially and laterally lamelliform, its dorsal carinae nearly complete; laterope present; dorsope long and area laterally lamelliform; remainder of metasoma smooth and depressed; length of setose part of ovipositor sheath 0.37 times fore wing and about as long as hind tibia and with a few long setae; ovipositor sheath parallel-sided.
Variation. Not much in the few specimens at hand. Host. Collected in banana traps infested by unidentified Drosophilidae. Distribution. China (Yunnan), South Korea. Diagnostic remarks. The new species is similar to A. leveri (Nixon). It differs by having the second submarginal cell of fore wing wide basally (narrow basally A. leveri); medio-posterior pit of mesoscutum round or nearly so (short elliptical); yellow scapus slightly or not contrasting with yellowish brown to brown vertex (yellow scapus contrasting with dark brown vertex) and length of setose part of ovipositor sheath 1.0-1.1 times hind tibia and 0.4 times fore wing (1.1-1.2 times and 0.5 times, respectively).

Molecular analysis
Trimmed COI sequences resulted in a fragment of size ranging between 410 and 640 bp. Alignment was straightforward with no frame shifts, nonsense codons, insertions or deletions identified in any sequence. ML and NJ analyses produced trees of almost identical topology, well supporting the distinction between the seven morphospecies collected in China and South Korea in this work, each one appearing as a different lineage on the tree (Fig 8). Overall, at least 15 different species were supported by phylogenetic analysis. The average uncorrected COI pdistance between species ranged from 5.9% and 20.2%. The average uncorrected COI p-distance within species ranged from 0% to 4.7%, except in A. pleuralis (8.7%) (S1 Table. Uncorrected p-distance (number of base differences per site) between COI sequences of Asobara species.; S2 Table. Intraspecific uncorrected p-distances (number of base differences per site) from averaging over all COI sequence pairs within each Asobara species.; S3 Table. Interspecific uncorrected p-distances (number of base differences per site) from averaging over all COI sequence pairs between Asobara species. Standard error estimates are shown above the diagonal.).

Discussion
Asobara is a rather large genus with nearly 40 valid species known worldwide (some of the Neotropical species may not belong in Asobara [24]. Asobara species are mainly larval koinobiont endoparasitoids of flies in the family Drosophilidae and Sepsidae living in decaying organic matter, especially fruits and leaves. Some species with widened ovipositor sheath have been reared as parasitoids of Tephritidae in fruits [25]. The genus is currently revised by the author CvA and colleagues, and many new species are waiting to be described. In this work, using morphological and molecular analyses, we found a considerable diversity of Asobara parasitoids in the Yunnan Province of China and in South Korea. Out of the four species collected in China, all are new to science and are formally described as A. elongata, A. mesocauda, A. unicolorata and A. triangulata. Out of the three species collected in South Korea, only one is new to science and is formally described as A. brevicauda. The other two species, A. japonica and A. leveri, are well known for the Asian continent [27]. However, a further cryptic diversity may be hidden within the new species here described. Because of the high variability of COI sequence observed in some lineages, as for example in A. unicolorata (average uncorrected p-distance 4.7%, range 1.2-8.7%) and A. mesocauda (average uncorrected p-distance 3.3%, range 0-9.7%), it is likely that each of the two morphospecies here described represents a complex of cryptic species whose identity and discriminating characters can be revealed by further investigations and only after the collection of large numbers of individuals necessary to set up a multivariate morphometric analyses [28,29]. Nonetheless, inter and intraspecific genetic distances (as reported in S1 Table. Uncorrected p-distance (number of base differences per site) between COI sequences of Asobara species.; S2 Table. Intraspecific uncorrected p-distances (number of base differences per site) from averaging over all COI sequence pairs within each Asobara species.; S3 Table. Interspecific uncorrected p-distances (number of base differences per site) from averaging over all COI sequence pairs between Asobara species. Standard error estimates are shown above the diagonal) clearly indicate the correct identification of different entities further confirmed by the phylogenetical analysis (Fig 8).
Asobara triangulata shows identical COI sequences to individuals of A. sp. TK1 found in Tokyo but also in the south-western isle of Fukue Japan, not distant from the South Korean coast [27], suggesting a large distribution range of this species in South-eastern Asia. Similarly, COI sequences of A. leveri here collected in South Korea group with sequences of individuals of A. sp. TS1, collected in the small south-western island of Japan, Tsushima and Fukue-jima, very close to South Korean coast [27], strongly supporting conspecificity. Finally, as already reported [27], we found that A. tabida from Japan forms a lineage distantly related to A. tabida from Europe, indicating a status of separate species. Despite the pest status of D. suzukii and the absence of effective natural enemies in the invaded areas of Europe and North America [10][11][12][13] a deep knowledge of parasitoids of D. suzukii in its native area, the Southeastern Asia, is still lacking, limiting the opportunity to implement biological control programmes. Most information is from Japan, where a few parasitoid species were found to parasitize D. suzukii in the field, including Ganaspis xanthopoda (Ashmead) and Leptopilina japonica Novkovic & Kimura (Hymenoptera: Figitidae), Trichopria sp.(Hymenoptera, Diapriidae), and six species of Asobara. The most common resulted A. japonica, while A. rossica, A. rufescens, A. tabida and two unidentified species completed the Asobara complex [30,31,27]. However, the parasitisazion rate observed in the field was very low and in general all this species appeared able to parasitise a wide range of Drosiphilid species, overall resulting of limited interest as biological control agents. Only one species, Asobara sp. TK1, emerged only from D. suzukii [27]. On the basis of our results, this species should be considered within the concept of A. triangulata sp. nov. However, we cannot confirm the specificity to D. suzukii because the single individual here examined was collected in China with banana-baited trap colonized by different species of drosophilids, including D. suzukii. In this work, two Asobara species could be associated with D. suzukii as collected from berries of wild Rubus sp. infested by the fruit fly (A. japonica) or from fruit-baited trap infested with D. suzukii before placement in the field (A. leveri). Our findings, far from indicating a silver bullet in the control of D. suzukii, present new species for quarantine evaluation as biocontrol agents and point out the importance of further collections in the native area of D. suzukii where biodiversity of its parasitoids appears to be extremely wide.
Supporting Information S1 Table. Uncorrected p-distance (number of base differences per site) between COI sequences of Asobara species. (XLS) S2 Table. Intraspecific uncorrected p-distances (number of base differences per site) from averaging over all COI sequence pairs within each Asobara species. (XLS) S3 Table. Interspecific uncorrected p-distances (number of base differences per site) from averaging over all COI sequence pairs between Asobara species. Standard error estimates are shown above the diagonal. (XLS)