ore JHR 64: 191-210 (2018) JOURNAL OF ree soar ice nets (f-) Hymenoptera http://jhr.pensoft.net The Inerrational Society of Hymenoptersts. RESEARCH New internal primers targeting short fragments of the mitochondrial COI region for archival specimens from the subfamily Aphidiinae (Hymenoptera, Braconidae) Milana Mitrovic', Zeljko Tomanovic? | Department of Plant Pests, Institute for Plant Protection and Environment, Banatska 33, 11080 Zemun, Serbia 2 Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia Corresponding author: Milana Mitrovié (milanadesancic@yahoo.co.uk) Academic editor: J. Fernandez-Triana | Received 31 March 2018 | Accepted 11 May 2018 | Published 25 June 2018 http://zoobank. org/OF740AC8-773B-4F69-9437-70FC208 1E526 Citation: Mitrovié M, Tomanovié Z (2018) New internal primers targeting short fragments of the mitochondrial COI region for archival specimens from the subfamily Aphidiinae (Hymenoptera, Braconidae). Journal of Hymenoptera Research 64: 191-210. https://doi.org/10.3897/jhr.64.25399 Abstract Archival specimens are a great resource for molecular research in population biology, taxonomy and con- servation. A primary goal for researchers is to preserve specimens from collections by improving non- invasive methods for DNA extraction and to achieve successful amplification of the short fragments of a target gene in the event of DNA fragmentation. We tested the suitability of a noninvasive method of DNA extraction and amplification of the barcoding region of the mitochondrial gene cytochrome c oxi- dase subunit I from archival specimens of aphid parasitoids belonging to the genera Aphidius, Lysiphlebus and Praon (Aphidiinae, Braconidae, Hymenoptera). Using a commercial kit as a noninvasive method, we successfully extracted DNA from dry 7 to 41 year old samples of 26 different parasitoid species. However, amplification of the barcoding region failed using the standard primer pair LCO1490/HCO2198. In order to reconstruct DNA barcodes we designed internal genus-specific degenerative primers and a new amplification protocol to target the short fragments within the mitochondrial region. Novel primers were designed using as a template the reference sequences from congeners retrieved from the public database. The combination of standard primers with internal primers, in direct and nested amplification reactions, produced short overlapping subsequences, concatenated to recover long barcoding sequences. Additional analyses also confirmed that primers initially designed for Aphidius, Lysiphlebus and Praon can be com- bined in a mixture, and successfully used to obtain short fragments of disintegrated DNA from archival specimens of several other braconid species from the genera Ephedrus and Monoctonus. Copyright Milana Mitrovic, Zeljko Tomanovic. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 192 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) Keywords COL archival specimens, Aphidius, Ephedrus, Lysiphlebus, Monoctonus, Praon, short fragments Introduction The DNA from an archival species is an important source of data in the areas of popu- lation genetics, conservation, taxonomy and phylogeny. In the past researchers were in conflict between the maintenance of specimens undamaged and their use in molecular analyses, which created a strong limitation for studies on museum specimens, in par- ticular studies with rare or extinct species, or those restricted to one or a few individu- als collected many years ago (Gilbert et al. 2007; Mandrioli 2008). However, archival DNA study is now a rapidly developing area of research due to the continual improve- ments of molecular tools with which it is possible to recover DNA information from museum specimens and dry remains, without damaging the material. Insects are a group where these tools have received increasing attention and non- invasive techniques have been developed and used for a variety of orders (Gilbert et al. 2007; Andersen and Mills 2012). Noninvasive methods of DNA extraction from dried specimens are important in order to preserve the quality of museum specimens. Unfortunately, not all specimens contain DNA of suitable quality and in the right amount for conclusive genetic studies. Successful amplification depends on post-mor- tem processes of DNA degradation, which can cause miscoding lesions or physical destruction of the DNA molecule (Rizzi et al. 2012). Degradation of DNA conse- quently produces methodological difficulties in amplification and sequencing of the target region, processes that are limited by the small quantity of template DNA and recovery of short fragments. Besides natural processes of disintegration, another factor that makes archival specimens difficult to work with is the preservation methodology, which can over time result in DNA damage (Dillon et al. 1996; Burrell et al. 2015). In the case of parasitic Hymenoptera, Andersen and Mills (2012) determined that age was a significant factor for successful sequencing, while size and DNA concentration did not influence the amplification of the targeted nuclear and mitochondrial genes. Parasitoid Hymenoptera are a taxonomically challenging group under frequent re- vision, making them a group of great interest for retrieval of genetic information from museum specimens (Andersen and Mills 2012). Among parasitoids that have been in- tensively surveyed by taxonomists and ecologists are aphid endoparasitoids from the sub- family Aphidiinae (Braconidae, Hymenoptera). ‘They are distributed worldwide, closely following the distribution of their aphid hosts (Stary 1988). As solitary endoparasitoids, Aphidiinae are one of the most important natural enemies of aphids and can effectively regulate their populations (Hagvar and Hofsvang 1991). They have been commercially produced and released as classical biological control agents of aphids in many regions and have achieved significant results in diverse agroecosystems. The most important gen- era of aphid parasitoids used in biological control are Aphidius Nees, 1818; Diaeretiella Stary, 1960; Ephedrus Haliday, 1833 and Praon Haliday, 1833 (Boivin et al. 2012). New internal primers targeting short fragments of the mitochondrial COI region... 193 The subfamily Aphidiinae is a diverse group with many cryptic species complexes, and reliable identification is therefore of key importance for their use as biological control agents. This study included aphid parasitoids belonging to the common aphidiine genera Aphidius, Lysiphlebus Forster, 1862 and Praon. Identification based on morphology has often been shown to be inadequate in distinguishing the species of these genera due to the limited number of valid discriminatory morphological characters, as well as their high variation on the intraspecific level (Pungerl 1983; Kavallieratos et al. 2005, 2010; Tomanovi¢ et al. 2003, 2004). Furthermore, several species have confusing taxonomic histories and are in need of revision. In fact, over the last two decades these genera have been constantly rearranged on the basis of new morphological characters and more recently obtained molecular data as well. Mitochondrial barcoding region of the cytochrome oxidase c subunit I (COD) had been used to reconstruct phylogenetic relationships within the genera (Jafari- Ahmadabadi et al. 2011), and examine the phylogenetic affinity and diversity of Aphi- diinae from different geographical regions (Lenin 2015). In addition, it has success- fully detected immature stages of parasitoids inside their aphid hosts, e.g., Lysiphlebus testaceipes Cresson, 1880 inside its host Aphis fabae Scopoli, 1763 (Traugott and Sy- mondson 2008). Either solely or in combination with morphometric methods, the barcoding method was routinely applied in revisiting and resolving the taxonomic status of many species complexes. For example, three species - Aphidius colemani Vier- eck, 1912; A. platensis Bréthes, 1913, and A. transcaspicus Telenga, 1958- were distin- guished within the A. colemani group (Tomanovi¢ et al. 2014); three species - A. rubi Stary, 1962; A. silvaticus Stary, 1962, and A. urticae Haliday, 1834 were re-described within the A. urticae group (Jamhour et al. 2016); two new species - Praon longicaudus Tomanovié & Stary, 2014 and P sambuci Tomanovié & Stary, 2014 - were described within the species complex Praon abjectum Haliday, 1833 (Mitrovski et al. 2013); the species status of P dorsale Haliday, 1833; P longicorne Marshall, 1896; P volucre Hali- day, 1833, and P yomenae Takada, 1968 was confirmed and a new species, viz., Praon staticobii Tomanovic & Petrovic, 2014 was described within the Praon dorsale-yome- nae s. str. group (Mitrovski et al. 2014). Apart from taxonomic revisions, the barcod- ing marker was successfully used to discover new allochthonous species accidentally introduced into new habitats, such as the invasive species Lysiphlebus orientalis Stary & Rakhshani, 2010 (Petrovi¢ et al. 2013) and Aphidius ericaphidis Pike & Stary, 2011 (Petrovié et al. 2017). Considering that these parasitoids are important for fundamental taxonomic and conservation research, as well as being potential biological control agents in aphid man- agement programs, it would be of great value to investigate the possibility of recovering barcoding fragments of COI from museum specimens. Thus, the main objectives of this study were as follows: i) DNA extraction from dry archival specimens belonging to the genera Aphidius, Lysiphlebus and Praon using a noninvasive method; ii) PCR amplification of several short and overlapping fragments within the barcoding region of cytochrome c oxidase subunit I, iii) traditional Sanger sequencing and alignment of 194 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) different short overlapping fragments and concatenation to recover longer target bar- coding region of mitochondrial DNA and iv) testing the suitability of novel primers for targeting barcodes in archival specimens of other braconid species. Material and methods Analyses included species from three different genera of aphid parasitoids, viz., Aphid- ius, Praon and Lysiphlebus. In total 45 specimens were submitted to molecular analy- ses, including 11 species of Aphidius, nine of Lysiphlebus and six of Praon, killed and preserved in dry condition from 7 to 41 years prior to DNA extraction (Table 1). Additionally, in order to test the suitability of these primers in amplification of other parasitiods we chose four species from the genus Monoctonus Haliday, 1833 and four of Ephedrus Haliday, 1833, all dry material up to 31 year old (Table 1). Table |. The list of analyzed species from the genera Aphidius, Lysiphlebus, Praon, Ephedrus, Monoctonus with designated aphid host/plant associations and geographic origin. “} é Specimen Sanple Parasttoud aap year Host plant Aphid host condition code species age of samples pi AF1 ie ee Serbia 2011/7 Metopeurum fuscoviridae F tanacetarius vulgare AF2 Aphidius sussi | Montenegro 2005/13 Delphiniobium junackianum F AF 3 _ | Aphidius sonchi Serbia 2010/8 Sonchus arvensis! Hyperomyzus lactucae FE Aphidius 3, ee : : FF AF4 Fern expats Montenegro 2011/7 Galium sp Linosiphon sp AF5 Aphidius ribis | Montenegro 2011/7 Ribes petreum Cryptomyzus sp. F Aphidius ; : D AD1 Sindbis Serbia 1998/20 Crepis sp Uroleucon sp AD2 a nes Serbia 2001/17 Parte Macrosiphoniella sp. D absinthii vulgaris AD3 | Aphidius sussi Serbia 1998/20 "a Mg Delphiniobium junackianum D toxicum AD4_ | Aphidius ervi Slovenia 2009/9 Sitobion avenae D aestivum AD5 _ | Aphidius eadyi Russia 2007/11 Pisum sativum D AD6 a Pies Serbia 1996/22 Rosa sp. Chaetosiphon sp. D eglanteriae AD7, Aphidius ; AD8 aan Montenegro 2000/18 Salix retusa D ADI | Aphidius sussi Serbia 2000/18 Delphiniobium junackianum D iY AD10 a ga Tran 2010/8 Aphis sargasi D arvensis Aphidius Czech ; 5 ot AD11 7 oe Republic 19999 Pseudobrevicoryne erysimi D AD12 ap Sour Serbia 1998/20 ee Longicaudus trirhodus D eglanteriae elatum AD13 | Aphidius smithi| United States 1977/41 Acyrthosiphon pisum D New internal primers targeting short fragments of the mitochondrial COI region... 195 P Bells & & Sample| Parasitoid Sampling year/ Seems P ; Pans Y x | Host plant Aphid host condition code species age of samples pi AD14 | Aphidius eadyi Tran 1977/41 Medicago sava Acyrthosiphon pisum Aphidius ; AD15 Doitbue: Israel 1979/39 Medicago sativa Acyrthosiphon pisum D PEI Praon volucre Tran 2009/9 Uroleucon sonchi F oleraceus PP2 Praon dorsale Serbia 2010/8 Corylus avelana F PF3 | Praon abjectum Serbia 2011/7 Thallium aquile L. trialeurodes F PD1 gts Montenegro 2009/9 Bake 4 Aphis malvae D longicorne robertianum Filipendula PD2 Praon dorsale | Montenegro 2006/12 be Aberin Macrosiphum cholodkovskyi D Praon : ; PD3 Serbia 2006/12 Rubus sp. Macrosiphum funestum D longicorne PD4 | Praon yomenae | Montenegro 2009/9 D PD5 _ | Praon yomenae Tran 2009/9 Uroleucon sp. 13) repens PD6 eee Cah 2008/10 Rubus sp. Macrosiphum funestum D longicorne Republic PD7 | Praon spinosum Croatia 2005/13 Carex nigra Thripsaphis verrucosa D PD8 _ | Praon spinosum Croatia 2009/9 Thripsaphis verrucosa D keys Pra Czech PD 10; ee is 1998/20 Urtica dioica Microlophium carnosum D longicorne Republic PRI PD12 eae Serbia 2011/7 Medicago sativa Acyrthosiphon pisum 1B barbatum s pee? PD13 | Praon necans Serbia 2005/12 Typha sp. Rhopalosiphum nymphaeae D PD14, Hemerocallis . PD1S Praon yomenae Japan 2002/16 fila Indomegoura indica D LF1 ERS Serbia 2011/7 aa Metopeurum fuscoviridae F hirticornis vulgare Lysiphlebus av ‘Se LF2 Eide Serbia 2010/8 Cirsium arvense| Aphis fabae cirsicanthoides ly Lysiphlebus ; a oe LF3 Serbia 2009/9 Cirsium arvense| Aphis fabae cirsicanthoides F fabarum LD1 pias Serbia 2011/7 deat Metopeurum fuscoviridae D hirticornis vulgare LD2 2 bs ne Serbia 2010/8 Cirsium arvense| Aphis fabae cirsicanthoides D Lysiphlebus a ; wi LD3 Serbia 2009/9 Cirsium arvense| Aphis fabae cirsicanthoides D fabarum Lysiphlebus LD4 : Italy 2006/12 Hedera helix Aphis hederae Is} testaceipes LD5 ue Sesan France 2006/12 Rubus fruticosus Aphis ruborum D testaceipes Lysiphlebus , eg t. LD6o . Costa Rica 2000/18 Eugenia wilsonii Toxoptera aurantii D testaceipes LD7 as ie haga Serbia 2006/12 Vicia cracca Aphis craccae D fritemuelleri LD8 Epsipialelas Tran 2005/13 Verbascum sp. Aphis verbasci D LD9, Lysiphlebus Achillea : LD10 desertorum = so millefolium Pap ESD: 4 196 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) wh é Specimen Parasttond oun: eb” Sanipling yar Host plant Aphid host condition species origin age of samples i Ppupakels Iran 2005/13 rege eae Brachycaudus tragopogonis D fabarum pratensis Lysiphlebus ea Serbia 1996/22 Daucus carota Semiaphis dauci D pis bes Sah 1998/20 Carduus sp. Brachycaudus cardui D melandriicola Republic BIE Os Iran 2005/13 Trego oat Brachycaudus tragopogonis D pratensis Ephedrus ote Serbia 2000/18 Rosa sp. Chaetosiphon sp. D Ephedrus Lonicera ante Montenegro 2004/14 Bite. Hyadaphis sp. D = iat Finland 1987/31 D validus aes Finland 1987/31 D koponeni mS Canada 2005/13 ee Myzus persicae D paulensis annuum Monoctonus 2001/17 Delphinium Nasonovia (Eokakimia) D allisoni galucum wahinkae Monoctonus Ach : ; ieineinenst 1992/26 Triticum sp. Rhopalosiphum padi D Monoctonus Aconitum Ae eh. : MD4 Montenegro 2002/16 Delphiniobium junackianum D leclanthi toxicum * number of years the specimens were kept dry in collections prior to DNA extraction **Specimen condition: (F) fresh refers to specimens kept after collection in 96% ethanol; (D) dry are specimens which were kept dry in collections, pinned or glued to cardboard DNA extraction Dry specimens were carefully removed from the card points so that they could be re- mounted afterwards if the specimens are holotypes. The whole specimens were used for DNA extraction using the QIAGEN Dneasy Blood and Tissue Kit. In the case of parasitoid specimens used as a control, they were preserved in 96% ethanol prior to ex- traction. Whole specimens were placed in 2 ml Eppendorf tubes with proteinase K and ATL buffer. After incubation overnight at 56 °C insect specimens were removed from the buffer, rinsed with 96% ethanol several times, air-dried and put back in the collec- tion. The remaining solution was treated according to the manufacturer's instructions. PCR amplification The first step was an attempt to amplify a barcoding region of mitochondrial gene cytochrome c oxidase subunit I from dry material using the standard primer pair LCO1490/HCO2198 (Folmer et al. 1994). Each PCR reaction was carried out in a volume of 20ul, including: Lpl of extracted DNA, 11.8 ul ELOs 2 ul High Yield Reac- New internal primers targeting short fragments of the mitochondrial COI region... 197 tion Buffer A with 1xMg, 1.8 ul of MgCl, 2.25 mM, 1.2 pl of dNTP 0.6 mM, 1pl LCO1490 0.5 uM, ll HCO2198 0.5 uM, 0.2 ul DNA polymerase 0.05U/ul. The amplification protocol included : i) initial denaturation at 95 °C for 5 min; ii) 35 cycles of 1 min at 94 °C, 1 min at 54 °C and 30 sec at 72 °C and iii) final extension at 72 °C for 7 min. Products were visualized on agarose gel. Due to DNA fragmentation in dry specimens, internal degenerative primers were designed to amplify overlaping short fragments of COI through direct and nested PCR, which could thereafter be aligned to a longer barcoding sequence (Fig. 1). Refer- ence COI sequences of parasitoids retrieved from the GenBank (www.ncbi.nlm.nih. gov/Genbank) were used as a template to design primers for dry material of the genera Aphidius, Praon and Lysiphlebus (Table 2). They were aligned and manually searched for shared conservative regions on which to place the newly designed primers. The initial idea was to divide the barcoding fragment of COI obtained with LCO1490/HCO2198 into three overlapping subsequences, around 260 bp, 270 bp and 280 bp long respectively, and the primers designed for this were marked as for direct PCR. Furthermore, additional internal primers were designed within these three subsequences to amplify even shorter fragments through nested PCR (Fig. 1). The genus-specific degenerative primers were used in combination with standard primers LCO1490 and HCO2198 (Fig. 1). Finally, the position of internal primers al- lowed diverse combinations and targeting of overlapping fragments of different length and position. Due to the shared conservative sites in COI sequences, it was possible for primers initially designed for Aphidius species to be also used in amplification of short fragments in combination with primers specifically designed for Lysiphlebus species (Aph1 Rn, Aph2Fd, Aph3Rn) and for dry Praon specimens as well (Aph2Fn) (Fig. 1). Prior to testing their suitability for amplification of short fragments from dry samples, the designed primers were initially tested on control specimens preserved in 5! 3! wo SAAD eccrine n tL ODD ASOD eee O LADD ene? OOD F Top. FODD cece IBID assesses ‘AphiFn —Aph1 Rn} ‘Aph2Fn Aph3Rn ‘Lys1Fn ‘Lys2Fn Pr3R : PriRn! f ronn PriFn, eae in zp << LCO01490 Aph1Rd HC02198 Lys1Rd PriRd Aph2Fd Aph2Rd Lys2Rd Pr2Fd Pr2Rd Figure |. Position of internal degenerative primers within the barcoding region of COL Aphidius - specific primers: Aph1 Fn, Aph1 Rn, Aph1Rd, Aph2Fd, Aph2Fn, Aph2Rn, Aph2Rd, Aph3Fd, Aph3Fn and Aph3Rn; Lysiphlebus - specific primers: Lys1 Fn, Lys1Rd, Lys2Fn, Lys2Rn, Lys2Rd, Lys3Fd and Lys3Fn; Praon - specific primers: Pr Fn, Pr1Rn, Pr1 Rd, Pr2Fd, Pr2Rn, Pr2Rd, Pr3Fd, Pr3Fn and Pr3Rn. Arrows refer to the direction of the primers, forward or reverse. The exact position of internal primers is designated in comparison to the first nucleotide of the forward LCO1490 primer sequence (5’ GGTCAACAAATCATAAAGATATTGG 3’). 198 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) Table 2. The list of reference Aphidiinae species obtained from GenBank and used in designing the genus-specific primers. Parasitoid species Accession number Aphidius matricariae JN620563 Aphidius urticae JN620590 Aphidius sonchi JN620589 Aphidius rhopalosiphi JN164779 Aphidius ervi JQ723411 Aphidius microlophii JN620566 Aphidius uzbekistanicus JN164751 Aphidius funebris JN620561 Aphidius rosae JN620582 Aphidius eadyi JN620551 Aphidius salicis JN620585 Aphidius ribis JN620579 Aphidius colemani KJ615362 Aphidius transcaspicus KJ615375 Lysiphlebus testaceipes HQ599569 Lysiphlebus orientalis KC237736 Lysiphlebus hirticornis HQ724540 Lysiphlebus fabarum JQ723416 Lysiphlebus cardui JN620640 Lysiphlebus confusus KM408535 Praon barbatum JN620671 Praon yomenae JN620693 Praon gallicum JN620680 Praon abjectum KC128671 Praon dorsale KC128677 Praon exsoletum KJ848478 96% ethanol. In total, five Aphidius species were submitted to initial testing (samples AF1-AF5; Table 1). Three following primer combinations were confirmed successful in direct PCR reactions: i) LCO1490/Aph1Rd, ii) Aph2Fd/Aph2Rd and iii) Aph- 3Fd/HCO2198 (Fig. 2). Three species from the genus Praon were used for test trials (samples PF1- P volucre, PF2- P dorsale and PF3- P abjectum; Table 1). Three indi- vidual analyses were conducted: 1. LCO1490/Pr1Rd; 2. Pr2Fd/Pr2Rd; and 3. Pr3Fd/ HCO2198. All of the products with fresh samples were visualized (Fig. 3). Lysiphlebus hirticornis Mackauer, 1960 (LF1), LZ. cardui Marshall, 1896 (LF2) and L. fabarum Marshall, 1896 (LF3) were included in the initial trials (Table 1). The four follow- ing primer combinations were confirmed suitable: 1) LCO1490/Lys1Rd; 2) Aph2Fd/ Lys2Rd; 3) Pr2Fd/Lys2Rd; and 4) Lys3Fd/ HCO2198 (Fig. 5). After confirmation of their suitability, the new primers were then used in trials with dry specimens. Products of PCR were obtained in 40 ul volumes. In the direct PCR reac- New internal primers targeting short fragments of the mitochondrial COI region... LOD Table 3. The list of primers designed for the genera Aphidius, Lysiphlebus and Praon to amplify short frag- ments of COI barcoding region from dry specimens through direct and nested PCR analyses. Parasitoid group 5’ 3’ primer sequence** Primer direction Aphidius GRGGRAAAGCYATATCAGGAG reverse Aphidius Aph1En TAAGWTTATTAATTCGWATRGA forward Aphidius CAATTWCCAAATCCWCCAATTAT reverse Aphidius ATAATTGGWGGATTTGGWAATTG forward Aphidius GTWCTAATAAAATTAATWGCWCC reverse Aphidius CTCCTGATATRGCTTTYCCYC forward Aphidius GADGAAATHCCTGCTAAATG reverse Aphidius CATTTAGCWGGDATTTCYTC forward Aphidius GGAGCWATTAATTTTATTAGWAC forward Aphidius GTAGTATTTAARTTWCGATC reverse Lysiphlebus GAGGAAAAGCYATATCWGGAG reverse Lysiphlebus TAAGWTTAATTATTCGWATRGA forward Lysiphlebus GTWCTAATAAAATTAATTGCHCC reverse Lysiphlebus CTCCWGATATRGCTTTTCCTC forward Lysiphlebus GAWGAAATACCWGCTAAATG reverse Lysiphlebus CATTTAGCWGGDATTTCWTC forward Lysiphlebus GGDGCAATTAATTTTATTAGWAC forward Praon GAGGRAAAGCTATATCAGGAG reverse Praon AAGWGATCAAATTTAYAATAG forward Praon CAATTWCCAAAYCCWCCAAT TAT reverse Praon ATAATTGGAGGRTTTGGWAATTG forward Praon GTTGWAATAAAATTAATWGCYCC reverse Praon CATTTAGCWGGTATTTCWTC reverse Praon CATTTRGCTGGWATTTCYTC forward Praon GGAGCWATTAATTTTATTWC forward Praon GTWGTATTTAWATTTCGATC reverse *the last letter in the primer’s name refers to PCR reaction: d-direct and n-nested **degenerative base designation/actual base coded: R or - A, or - G; Y or -C or - T; W or -A, or - T. tion, 4 ul of extracted DNA was added into 36 ul of mix, following the recipe described for the LCO1490/HCO2198 primer pair. In nested PCR, 0.25 pl of a product from direct PCR was added into 39.75 ul of mix. The following protocol was developed for direct and nested PCR: i) initial denaturation at 95 °C for 5 min; ii) 37 cycles of 1 min at 95 °C, 1 min at 54 °C, and 30 sec at 72 °C; and iii) final extension at 72 °C for 7 min. Amplified COI fragments were sequenced in both directions using an automated equipment (Macrogen Inc, Seoul, South Korea). Overlapping short fragments of the barcoding region were manually edited in FINCHTV ver.1.4.0 (www.geospiza.com), concatenated to obtain longer sequences and aligned using the CLUSTAL W program integrated in MEGA5 (Tamura et al. 2011). A Maximum likelihood tree was con- structed using the MEGAS software, with 500 bootstrap replicates performed to assess the branch support. The evolutionary distances were computed using the Tamura-Nei 200 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) ‘“AF1 AF2 AF3 AF4 AF5 AF1 AF2 AF3 AF4 AFS AF1 AR2 AF3 AF4 AF5. M a = = ae LCO1490/AphiRd Aph2Fd/Aph2Rd Aph3Fd/HCO2198 Figure 2. Agarose gel visualizing the products of direct PCR in initial trials testing the novel primers with fresh Aphidius samples. Three direct PCR reactions were conducted with the following primer pairs: | LCO1490/Aph1Rd 2 Aph2Fd/Aph2Rd; and 3 Aph3Fd/HCO2198. The species included in trials were: AF 1- A. tanacetarius, AF2- A. sussi, AF3- A. sonchi, AF4- A. linosiphonis and AF5- A. ribis. M — marker. method (Tamura and Nei, 1993). Phylogenetic analyses included the sequenced bar- codes recovered from archival parasitoid specimens combined with the reference COI sequences of Aphidiinae from GenBank. Results Initial trials with dry specimens using standard primer pair for the COI barcoding region LCO1490/HCO2198 failed to give products. Thereafter, 15 dry specimens of 11 different Aphidius species (A. absinthii Marshall, 1896; A. arvensis Stary, 1960; A. avenae Haliday, 1834; A. banksae Kittel, 2016; A. eadyi Subba Rao and Sharma, 1959; A, eglanteriae Haliday, 1834; A. erysimi Stary, 1960; A. funebris Mackauer, 1961; A. ervi Haliday, 1834; A. smithi Subba Rao and Sharma, 1959; A. sussi) were submitted to molecular analyses (Table 1). Insects had been killed and stored dry in collections for 8 to 41 years prior to DNA extraction. The same three combinations of standard and degenerative primers previously confirmed as suitable in the test trials with fresh material were used with dry samples AD1-AD15 as well. Direct PCR produced ampli- cons in all three combinations for samples AD1 to AD6, while in the cases of samples AD7 to AD15 no product was visualized. The products from direct PCR with primer pair LCO1490/Aph1Rd were submitted to two independent nested reactions with primers LCO1490/Aph1Rn and Aph1Fn/Aph1Rd; from direct PCR with primers Aph2Fd/Aph2Rd to nested reactions with Aph2Fd/Aph2Rn and Aph2Fn/Aph2Rd; and products obtained with Aph3Fd/HCO2198 were included in nested trials with the primers Aph3Fd/Aph3Rn and Aph3Fn/HCO2198. In all six individual nested reactions short fragments of the barcoding region were amplified successfully and visualized for all of the tested samples. New internal primers targeting short fragments of the mitochondrial COI region... 201 PF1 ‘PF2:PF3 M PF1PF2PF3° M: PF1 PF2PF3: 5 ae @ = LCO1490/PriRd = Pr2Fd/Pr2Rd Pr3Fd/HCO2198 Figure 3. Agarose gel visualizing the products of direct PCR in initial trials testing the novel primers with fresh Praon samples. Three direct PCR reactions were conducted with primer pairs: 1. LCO1490/Pr1Rd, 2. Pr2Fd/Pr2Rd, 3. Pr3Fd/HCO2198. The species included in trials are PF1- P volucre, PF2- P dorsale, PF3- P abjectum; M — marker. In total 15 specimens of eight Praon species preserved dry for 7 to 20 years prior to DNA extraction were analysed (Table 1). We attempted to retrieve short overlapping frag- ments of COI barcodes from dry samples PD1-PD15 through the same three direct am- plifications as with the fresh material. In analyses with primers targeting the first fragment of the barcoding sequence, all products were obtained. In the second and third reactions short fragments of barcode were amplified in samples PD1-PD11 and PD13, while no product was visualized for samples PD12, PD14 and PD15. The same methodological approach was applied here, namely using the products from direct PCR as a template for secondary nested trials. The amplicons of samples PD12, PD14 and PD15 from the trial with primer pair Pr2Fd/Pr2Rd were processed further in two nested reactions with combi- nations Pr2Fd/Pr2Rn and Aph2Fn/Pr2Rd, while the products of direct PCR with Pr3Fd/ HCO2198 were processed in secondary analyses using the combinations Pr3Fd/Pr3Rn and Pr3Fn/HCO2198. Subsequent analyses successfully targeted short fragments within the subsequences of the barcoding region in all four nested test trials (Fig. 4). The novel primers were tested on Lysiphlebus alpinus Stary, 1971; L. confusus Trem- blay & Eady, 1978; L. desertorum Stary, 1965; L. fabarum; L. fritzmuelleri Mackauer, 1960; L. hirticornis; L. melandriicola Stary, 1961; L. testaceipes), stored dry in collec- tions for 7 to 22 years. Three separate analyses were conducted using the primer com- binations confirmed as suitable with fresh material. Amplicons were visualized in the first direct analysis with the LCO1490/Lys1Rd combination for samples LD1-LD7 and LD10-LD15. No products were visible for samples LD8 and LD9 which were further processed in nested trials with LCO1490/Lys1Rn and Lys1Fn/Lys1Rd. Prod- ucts of the direct PCR conducted with the primer combination Aph2Fd/Lys2Rd were obtained in all samples except LD8, LD9 and LD12 which were thereafter processed in nested analyses with 1. Aph2Fd/Lys2Rn; and 2. Lys2Fn/Lys2Rd. In the third direct 202 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) PD-PD‘PD'M°PD PD PD M |PD PD PD M PD PD’ PD M ee ee ia 12 14-155 ie 4 5 Pr2Fd/Pr2Rn Aph2Fn/Pr2Rd Pr3Fd/Pr3Rn Pr3Fn/HCO2198 Figure 4. Agarose gel visualizing the products of nested trials with products of direct PCR for samples PD12 - P barbatum, PD14 - P yomenae, and PD15 - P yomenae. The products from PCR with Pr2Fd/ Pr2Rd were submitted to secondary nested trials with primer pairs Pr2Fd/Pr2Rn and Aph2Fn/Pr2Rd. Amplicons obtained with Pr3Fd/HCO2198 were used as the template for nested reactions with Pr3Fd/ Pr3Rn and Pr3Fn/HCO2198. LFYLF2 3 MOLI ‘LF2°LF3° M'°LF1 LF2 LF3°M.‘LF1 LF2)LF3) LCO1490/Lys1Rd Aph2Fd/Lys2Rd Pr2Fd/Lys2Rd Lys3Fd/HCO2198 Figure 5. Agarose gel visualizing the products of direct PCR in initial trials testing the novel primers with fresh Lysiphlebus samples. Tested combinations of primers were: 1) LCO1490/Lys1Rd; 2) Aph2Fd/ Lys2Rd; 3) Pr2Fd/Lys2Rd; and 4) Lys3Fd/HCO2198. The species included in trials were: LF1 - L. hirti- cornis; LF2 - L. cardui; and LF3 - L. fabarum; M — marker. PCR trial, amplicons were visualized in all analyzed specimens besides LD8, LD9 and LD13 which were further submitted to analyses with primers 1. Lys3Fd/Aph3Rn; and 2. Lys3Fn/HCO2198. We obtained products in all nested trials (Fig. 6). Our research covers different taxonomically challenging Aphidiinae, for which reason we tested suitability of the newly designed primers on several other archival specimens from the genera Monoctonus and Ephedrus. In order to preserve the limited amount of DNA obtained from dry specimens, we avoided blind PCR trials as well as testing of all possible combinations by doing initial alignment of barcode sequences of fresh material (unpublished data) and degenerative primers (Table 4). According to the alignment we chose the primers best suited to target the species of interest. New internal primers targeting short fragments of the mitochondrial COI region... 203 Table 4. Comparison of barcode fragments of COI for Monoctonus and Ephedrus with degenerative primers sequences. Difference in base pair substitutions (bp) Ucecnerste puma Monoctonus sp. Ephedrus sp. Aph1Rd 0-2 bp 4-6 bp AphFln 2-5 bp 0-3 bp Aph1Rn 0-4 bp 0-4 bp Aph2Fd 0-4 bp 2-3 bp Aph2Rd 0-2 bp 2-5 bp Aph2Fn 0-2 bp 4-7 bp Aph2Rn 1-3 bp 2-5 bp Aph3Fd 0-3 bp 1-4 bp Aph3Fn 0-2 bp 4-7 bp Aph3Rn 0-1 bp 1=4 bp Lys1Rd 0-2 bp 4—5 bp Lys1Fn 0-4 bp 0-4 bp Lys2Rd 0-1 bp 1-2 bp Lys2Fn 0-2 bp 5~7 bp Lys2Rn 1-4 bp 0-5 bp Lys3Fd 0-3 bp 1-3 bp Lys3Fn 0-1 bp 5-7 bp Pr1Rd 0-3 bp 3—5 bp PrlFn 1-4 bp 4-6 bp Pr1Rn 0-4 bp 1-3 bp Pr2Fd 0—4 bp 0-2 bp Pr2Rd 1-2 bp 0-1 bp Pr2Rn 1-4 bp 0-4 bp Pr3Fd 0-4 bp 0-4 bp Pr3Fn 1-3 bp 4-7 bp Pr3Rn 0-2 bp 0-3 bp LD‘LD'M ‘LD ‘LD FLD'LD‘LD’°M ‘LD LD LD | (D tO LD M LWLD LD BS. 8 9 Fee 8 120s Oe Ore 5: dee S43 LCO1490/ LysiFn/ Aph2Fd/ Lys2Fn/ Lys3Fd/ _Lys3Fn/ LysiRn LysiRd Lys2Rn Lys2Rd Aph3Rn HCO2198 Figure 6. Agarose gel visualizing the products of nested trials with products of direct PCR for samples LD8 — L. confusus, LD9 — L. desertorum; LD12 — L. fabarum; and LD13 — L. alpinus. The products of LD8 and LD9 from PCR with LCO1490/Lys1 Rd were submitted to secondary reactions combining two primer pairs, viz., 1. LCO1490/Lys1Rn; and 2. Lys1Fn/Lys1Rd. Amplicons of LD8, LD9 and LD 12 ob- tained with Aph2Fd/Lys2Rd were submitted to secondary nested trials with primer pairs Aph2Fd/Lys2Rn and Lys2Fn/Lys2Rd. Products from direct PCR with Lys3Fd/HCO2198 were used as the template for nested reactions with Lys3Fd/Aph3Rn and Lys3Fn/HCO2198. 204 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) In the case of Ephedrus species, we chose two combinations for direct PCR, i.e., 1. LCO1490/Pr2Rd, and 2. Aph3Fd/HCO2198. Four species preserved in dry con- dition for 14 to 31 years in collections were included in the test trials, viz., E. plagia- tor Nees, 1811 (ED1); E. Laevicollis Thomson, 1895 (ED2); E. validus Haliday, 1833 (ED3); and E. koponeni Halme, 1992 (ED4) (Table 1). Amplicons of both targeted fragments were visualized on gel for specimens ED1, ED3, and ED4, while in the case of the ED2 sample a PCR product was visible only with primer pair Aph3Fd/ HCO2198. Products of the ED2 were subjected to separate nested reactions with primer pair LCO1490/Pr1Rd and Pr2Fd/Pr2Rd. Both short fragments of the barcode were successfully amplified and concatenated with the third subsequence obtained in direct PCR to retrieve a longer barcode fragment of COI. Dry specimens of the following four Monoctonus species preserved for 13 to 26 years were subjected to PCR analyses: MZ. paulensis (Ashmead) (MD1); M. allisoni Pike and Stary, 2003 (MD2); M. washingtonensis Pike and Stary, 1995 (MD3); and M. leclanthi Tomanovi¢ and Stary, 2002 (MD4). The same approach was repeated as with Ephedrus, ie., barcoding sequences of fresh material were aligned and analysed for primers suit- ability prior to molecular analyses (Table 4). The final choice fell on three combinations in direct PCR to retrieve three overlapping short fragments within the barcoding COI fragment: 1. LCO1490/Aph1Rd; 2. Pr2Fd/Lys2Rd; and 3. Pr3Fd/HCO2198. The fi- nal results show that the tested combinations of standard and degenerative primers suc- cessfully amplified all three short subsequences in all tested Monoctonus species. The overall results of combining different primers in direct and secondary nested reactions are summarized in Fig. 7. Short fragments of the COI barcodes obtained from direct and nested PCR analyses of the following samples were deposited in the GenBank: AD4 - A. ervi (MG991997), AD7 - A. avenae (MG991998), AD 10 - A. arvensis (MG991999), LD1 - L. hirticornis (MG992000), LD4 - L. testaceipes (MG992001), LD7 - L. fritemuelleri (MG992002), PD2 - P dorsale (MG992003), PD5 - P yomenae (MG992004), ED2 - E. plagiator (MG991993), ED4 - E. koponeni (MG991992), MD1 - M. paulensis (MG991996), MD2 - ™. allisoni (MG991995), MD3 - M. washingtonensis (MG991994). Several reference COI sequences from different Aphidiinae species were obtained from the public database and used with the archival material for tree construction. A total of 31 barcoding sequences were aligned, trimmed to the same length and submitted to phy- logenetic analysis. A Maximum likelihood tree shows evident clustering of congeneric species in separate lineages with substantial bootstrap support (Fig. 8), confirming the quality of COI barcoding sequences retrieved from archival parasitoids specimens by targeting the short overlapping fragments with newly designed primers. Discussion and conclusion The barcoding method has shown to be a useful tool in discriminating parasitoid species from the five Aphidiinae genera studied, enabling further research on their biodiversity New internal primers targeting short fragments of the mitochondrial COI region... 205 AD1, AD2, AD3, AD4, ADS, AD6 3 LCO1490/AphiRd Aph2Fd/Aph2Rd Aph3Fd/HCO2198 = AD7, AD8, AD9, AD10, AD11, ADi2, AD13, AD14, AD1i5 t . tt : a a ~ pp4, PD2, PD3, PD4, PDS, PD6, PD7, PD8, PD9, PD10, PD11, PD13 S LCO1490/PriRd Pr2Fd/Pr2Rd aati Pr3Fd/HCO2198 = PD12, PD14, PD15 a wd ~~" D1, LD2, LD3, LD4, LDS, LD6, LD7, LD10, LD11, LD14, LD15 LCO1490/Lys1Rd Aph2Fd/Lys2Rd iit Lys3Fd/HCO2198 wy LD8, LD9 = i S 2 1p12 Ww g nam Kar LDi3 ‘J ED1i, ED3, ED4 = LCO1490/Pr2Rd Aph3Fd/HCO2198 vy = _&D2 iy a es aac " MD1, MD2, MD3, MD4 — —— : Monoctonus LCO1490/Aphi1Rd Pr2Fd/Lys2Rd Pr3Fd/HCO2198 Figure 7. Scheme with overview of PCR attempts to recover the barcoding region of cytochrome c oxidase subunit I with novel primers from archival specimens from the genera Aphidius, Praon, Lysiphlebus, Ephedrus and Monoctonus. Primer pairs coloured red were used in direct PCR; black coloured primers were used in secondary nested reactions. Positions where short fragments within the subsequences overlap are marked with a pattern. and phylogeny. ‘The results presented here indicate the possibility of testing many other different combinations of primers in future research on archival specimens with the ex- pectation of achieving success in retrieving the targeted subsequences. The position of the newly designed primers was evidently well chosen, targeting sites conservative enough to permit their multiple uses on a much wider spectrum of museum material than initially planned. Similar to the results obtained by Andersen and Mills (2012), in our study age was apparently a limiting factor for successful amplification with the newly designed internal primers. On the other hand, the starting point in this study was awareness that museum specimens are not always available, or that the type material is sometimes re- stricted to a single specimen, etc., and thus cannot be manipulated in numerous trials. For this reason, blank PCR products were always further processed through secondary analyses with additional internal primers. This assumption was confirmed to be the basis of a good methodological approach with substantial success. The results presented above refer only to combination of primers randomly select- ed to test their suitability in retrieving the barcoding region from Ephedrus and Monoc- tonus species. Without the need for further expenditure of limited DNA sources, the 206 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) iog | Praon dorsale KC1268 7? 61) ' PD? Praon dorsale 79 /L Praon volucre JNB20690 98 Praon exsoletum KJe46476 Praon yormenae JNB20693 100'} PDS Praon yomenae ED4 Ephedrus koponeni ED? Ephedrus plagiator Ephedrus plagiator JNB20625 99 |Ephedrus plagiatar MF1I54179 MDS Monactonus washingtonensis fis) 100 = MD1 Monactonus paulensis MD Monoctonus allisoni a Monoctonus crepidis JNb20bb2 a, ; Lysiphlebus testaceipes HOIS99569 23 |°LDOY Lysiphlebus fritzmuelleri LD4 Lysiphlebus testaceipes io | L¥Siphlebus hirticoris HO 24540 LDO1 Lysiphlebus hirticornis 93 | - Lysiphlebus fabarum JO?235416 93 4 Lysiphlebus cardui JN620640 Aphidius ribis JN6205/79 70 Aphidius coleman KJ61S5362 AD? Aphidius avenae 100 Aphidius avenae KYoor926 Aphidius uzbekistanicus JNTb4751 ag) Aphidius erv JOV2541 1 ADd Aphidius ervi Aphidius eadyi JNB20551 ADO Aphidius arvensis a3 Binodoxy¥s communis FI?9o201 0.1 Figure 8. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood is shown. There were a total of 568 positions in the final dataset. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The percentage of replicate trees >50% in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. New internal primers targeting short fragments of the mitochondrial COI region... 207 here presented overview of nucleotide differences between the barcodes of parasitoids and information about primers clearly indicate that quite a few other combinations can be tested with the expectation of successfully retrieving short fragments. Many benefits of using novel primers in conservation genetics and phylogeny stud- ies are recognized, above all, the possibility of analyzing archival material of Aphidiinae parasitoids with unresolved taxonomic status. To date there have been many phylogenetic studies with different hypotheses about the origin and classification of certain taxa. Many examples in the literature show the importance of an integrative approach combining mo- lecular and morphological data in taxonomic, phylogenetic and conservation studies, but even when using such an approach, researchers are quite often left with open questions. In view of the many confronting opinions held by different groups of authors, we can assume that the involvement of archival remains of Aphidiinae in molecular analyses will prove to be of great usefulness by yielding results enabling us to resolve the problems of phylogenetic relationships and the taxonomic recognition of different parasitoid groups. It can be predicted that the herein described method of retrieving the barcoding region in parasitoids will take on increasing importance by making it possible to in- clude not only extinct species preserved in museums, but also endemic or rare species under threat of extinction as well. Good examples of parasitoid species with potential risk of extinction are various associations of aphid hosts/parasitoids whose distribution are restricted to habitats under constant anthropogenic pressure of degradation such as the wetlands (Tomanovi¢ et al. 2012). Modern genomic research opened complex questions exceeding the capacity of traditional DNA sequencing technologies. The Next-generation sequencing has revo- lutionized the biological sciences allowing us to study biological systems at higher level. In the light of an ongoing rapid progress in the field of modern sequencing technolo- gies, newly designed primers could meet the demands in terms of depth of informa- tion in studying genomics of different Aphidiinae by delivering an insight into DNA variation of the target mitochondrial region. Acknowledgments This research has been funded by the Ministry of Education, Science and Techno- logical Development from Serbia, through the grant No. III43001. We would like to thank PhD students Aiman Jamhour, Miljana Jakovljevi¢ and Korana Koci¢ for assis- tance in molecular analyses. References Andersen JC, Mills NJ (2012) DNA Extraction from Museum Specimens of Parasitic Hyme- noptera. PLoS ONE 7(10): e45549. https://doi.org/ 10.137 1/journal.pone.0045549 208 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) Boivin G, Hance T, Brodeur J (2012) Aphid parasitoids in biological control. Canadian Journal of Plant Science 92: 1-12. https://doi.org/10.4141/cjps2011-045 Burrell AS, Disotell TR, Bergey CM (2015) The use of museum specimens with high-through- put DNA sequencers. Journal of Human Evolution 79: 35-44. https://doi.org/10.1016/j. jhevol.2014.10.015 Dillon N, Austin AD, Bartowsky E (1996) Comparison of preservation techniques for DNA extraction from hymenopterous insects. Insect Molecular Biology 5: 21-24. https://doi.org/10.1111/j.1365-2583.1996.tb00036.x Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294-299. https://pdfs.semanticscholar.org/943d/38b9 d96f8222e883604822b cafb7930 ca6da.pdf Gilbert MTP, Moore W, Melchior L, Worobey M (2007) DNA Extraction from Dry Museum Beetles without Conferring External Morphological Damage. PLoS ONE 2(3): e272. https:// doi.org/10.1371/journal.pone.0000272 Hagvar EB, Hofsvang T (1991) Aphid parasitoids (Hymenoptera, Aphidiidae): biology, host selection and use in biological control. Biocontrol News and Information 12: 13-42. https://www.cabdirect.org/cabdirect/abstract/ 19911154713 Hebert PDN, Cywinska A, Ball SL, Dewaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society London (B) 270: 313-321. https://doi.org/10.1098/ rspb.2002.2218 Jafari-Ahmadabadi NJ, Karimi J, Modarres M, Rakhshani E (2011). Morphological and molecular methods in identification of Aphidius transcaspicus Telenga (Hym: Braconidae: Aphidiinae) parasitoid of Hyalopterus spp. (Hom: Aphididae) with additional data on Aphidiinae phylog- eny. Journal of the Entomological Research Society 13: 91-103. http://www.entomol.org/ journal/index.php?journal=JERS &page=article&cop=viewArticle&path%5B%5D=303 Jamhour A, Mitrovi¢ M, Petrovic A, Stary P, Tomanovié Z, (2016) Re-visiting the Aphidius urticae s. str. group: re-description of Aphidius rubi Stary and A. silvaticus Stary (Hyme- noptera: Braconidae: Aphidiinae). Zootaxa 4178: 278-288. http://doi.org/10.11646/ zootaxa.4178.2.6 Kambhampati S, Volkl W, Mackauer M (2000) Phylogenetic relationships among genera of Aphidii- nae (Hymenoptera: Braconidae) based on DNA sequence of the mitochondrial 16S rDNA gene. Systematic Entomology 25: 437-445. https://doi.org/10.1046/j.1365-3113.2000.00129.x Kavallieratos NG, Tomanovié Z, Athanassiou CG, Stary P, Zikié V, Sarlis GP, Fasseas C (2005) Aphid parasitoids infesting cotton, citrus, tobacco, and cereal crops in southeast- ern Europe: aphid-plant associations and keys. Canadian Entomologist 137: 516-531. https://doi.org/10.4039/N05-020 Kavallieratos NG, Tomanovié Z, Stary P, Zikié V, Petrovi¢-Obradovié O (2010) Parasitoids (Hyme- noptera: Braconidae: Aphidiinae) attacking aphids feeding on Solanaceae and Cucurbitaceae crops in southeastern Europe: aphidiine-aphid-plant associations and key. Annals of the Ento- mological Society of America 103: 153-164. https://doi.org/10.1603/AN09004 Lenin EA (2015) Intergeneric affinity of sixty two Aphidiine parasitoids (Hymenoptera: Braconidae: Aphidiinae) from different geographical regions. Journal of Entomol- New internal primers targeting short fragments of the mitochondrial COI region... 209 ogy and Zoology Studies 3: 405-410. http://www.entomoljournal.com/archives/2015/ vol3issue6/PartF/3-5-93.pdf Mandrioli M (2008) Insect collections and DNA analyses: how to manage collections? Museum Managementand Curatorship 23: 193-199. https://doi.org/10.1080/09647770802012375 Mitrovski-Bogdanovi¢é A, Petrovié A, Mitrovié M, Ivanovié A, Ziki¢ V, Stary P, Vorburger C, Tomanovié Z (2013) Identification of two cryptic species within the Praon abjectum group (Hymenoptera: Braconidae: Aphidiinae) using molecular markers and geomet- ric morphometrics. Annals of the Entomological Society of America 106: 170-180. https://doi.org/10.1603/AN12100 Mitrovski-Bogdanovié A, Tomanovi¢ Z, Mitrovié M, Petrovié A, Ivanovic A, Ziki¢ V, Stary P, Vorburger C (2014) The Praon dorsale-yomenae s.str. complex (Hymenoptera, Braconidae, Aphidiinae): Species discrimination using geometric morphometrics and molecular mark- ers with description of a new species. Zoologischer Anzeiger 253: 270-282. https://doi. org/10.1016/j.jcz.2014.02.001 Petrovic A, Mitrovié M, Stary P, Petrovi¢-Obradovié O, Ziki¢ V, Tomanovi¢ Z, Vorburger C (2013) Lysiphlebus orientalis (Hymenoptera, Braconidae), a new invasive aphid parasitoid in Europe — evidence from molecular markers. Bulletin of Entomological Research 103: 451-457. https://doi.org/10.1017/S000748 5313000035 Petrovié A, Ckrki¢ J, Jamhour A, Obradovi¢ OP, Mitrovi¢ M, Stary PB, Nedstam B, Tomanovi¢ Z (2017) First record of Aphidius ericaphidis (Hymenoptera, Braconidae) in Europe: North American hitchhiker or overlooked Holarctic citizen? Journal of Hymenopteran Research 57: 143-153. https://doi.org/10.3897/jhr.57.12517 Pungerl NB (1983) Variability in characters commonly used to distinguish Aphidius spe- cies (Hymenoptera: Aphidiidae). Systematic Entomology 8: 425-430. https://doi. org/10.1111/j.1365-3113.1983.tb00493.x Rizzi E, Lari M, Gigli E, De Bellis G, Caramelli D (2012) Ancient DNA studies: new perspec- tives on old samples. Genetics Selection Evolution 44: 21. https://doi.org/10.1186/1297- 9686-44-21 Stary P (1988) Aphidiidae. In: Minks AK, Harrewijn P (Eds) Aphids: Their Biology, Natural Enemies and Control. Volume B. Elsevier, Amsterdam, 171-184. Tamura K, Nei M (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolu- tion 10: 512-526. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGAS: mo- lecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739. https://doi.org/10.1093/molbev/msr121 Tomanovié Z, Kavallieratos NG, Stary PB, Athanassiou CG, Ziki¢ V, Petrovi¢-Obradovié O, Sarlis GP (2003) Aphidius Nees (Hymenoptera: Braconidae: Aphidiinae) in Serbia and Montenegro: tritrophic associations and keys. Acta Entomologica Serbica 8: 15 —39. Tomanovié Z, Kavallieratos NG, Athanassiou CG, Petrovié O, Stanisavljevié LJ (2004) A new Aphidius species (Hymenoptera: Braconidae: Aphidiinae) from high-montane areas of south- eastern Europe. Phytoparasitica 32: 221-225. https://doi.org/10.1007/BF02979816 210 M. Mitrovié & Z. Tomanovié / Journal of Hymenoptera Research 64: 191-210 (2018) Tomanovié Z, Stary P, Kavallieratos NG, Gagi¢ V, Ple¢as M, Jankovié M, Rakhshani E, Cetkovié A, Petrovic A (2012) Aphid parasitoids (Hymenoptera: Braconidae: Aphidiinae) in wetland habitats in western Palaearctic: key and associated aphid parasitoid guilds. Annales- Societe Entomologique de France 48: 189-198. https://doi.org/10.1080/0037927 1.2012.10697763 Tomanovié Z, Petrovi¢ A, Mitrovi¢ M, Kavallieratos NG, Stary P, Rakhshani E, Rakhshanipour M, Popovi¢é A, Shukshuk AH, Ivanovi¢é A (2014) Molecular and morphological variability within the Aphidius colemani group with redescription of Aphidius platensis Brethes (Hy- menoptera: Braconidae: Aphidiinae). Bulletin of Entomological Research 104: 552-565. https://doi.org/10.1017/S000748 5314000327 Traugott M, Symondson WOC (2008) Molecular analysis of predation on parasitized hosts. Bulle- tin of Entomological Research 98: 223-231. https://doi.org/10.1017/S0007485308005968