Inventory of the caterpillars, their food plants and parasitoids began in 1978 for today's Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 0-2000 m elevation contains at least 10 000 species of non-leaf-mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG-reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co-authors. DNA barcoding -the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species -was added to the taxonomic identification process in 2003.

a b s t r a c tThe Hymenoptera -ants, bees and wasps -represent one of the most successful but least understood insect radiations. We present the first comprehensive molecular study spanning the entire order Hymenoptera. It is based on approximately 7 kb of DNA sequence from 4 gene regions (18S, 28S, COI and EF-1a) for 116 species representing all superfamilies and 23 outgroup taxa from eight orders of Holometabola. Results are drawn from both parsimony and statistical (Bayesian and likelihood) analyses, and from both by-eye and secondary-structure alignments. Our analyses provide the first firm molecular evidence for monophyly of the Vespina (Orussoidea + Apocrita). Within Vespina, our results indicate a sister-group relationship between Ichneumonoidea and Proctotrupomorpha, while the stinging wasps (Aculeata) are monophyletic and nested inside Evaniomorpha. In Proctotrupomorpha, our results provide evidence for a novel core clade of proctotrupoids, and support for the recently proposed Diaprioidea. An unexpected result is the support for monophyly of a clade of wood-boring sawflies (Xiphydrioidea + Siricoidea). As in previous molecular studies, Orussidae remain difficult to place and are either sister group to a monophyletic Apocrita, or the sister group of Stephanidae within Apocrita. Both results support a single origin of parasitism, but the latter would propose a controversial reversal in the evolution of the wasp-waist. Generally our results support earlier hypotheses, primarily based on morphology, for a basal grade of phytophagous families giving rise to a single clade of parasitic Hymenoptera, the Vespina, from which predatory, pollen-feeding, gall-forming and eusocial forms evolved.

The first comprehensive analysis of higher-level phylogeny of the order Hymenoptera is presented. The analysis includes representatives of all extant superfamilies, scored for 392 morphological characters, and sequence data for four loci (18S, 28S, COI and EF-1a). Including three outgroup taxa, 111 terminals were analyzed. Relationships within symphytans (sawflies) and Apocrita are mostly resolved. Well supported relationships include: Xyeloidea is monophyletic, Cephoidea is the sister group of Siricoidea + [Xiphydrioidea + (Orussoidea + Apocrita)]; Anaxyelidae is included in the Siricoidea, and together they are the sister group of Xiphydrioidea + (Orussoidea + Apocrita); Orussoidea is the sister group of Apocrita, Apocrita is monophyletic; Evanioidea is monophyletic; Aculeata is the sister group of Evanioidea; Proctotrupomorpha is monophyletic; Ichneumonoidea is the sister group of Proctotrupomorpha; Platygastroidea is sister group to Cynipoidea, and together they are sister group to the remaining Proctotrupomorpha; Proctotrupoidea s. str. is monophyletic; Mymarommatoidea is the sister group of Chalcidoidea; Mymarommatoidea + Chalcidoidea + Diaprioidea is monophyletic. Weakly supported relationships include: Stephanoidea is the sister group of the remaining Apocrita; Diaprioidea is monophyletic; Ceraphronoidea is the sister group of Megalyroidea, which together form the sister group of [Trigonaloidea (Aculeata + Evanioidea)].

Three new genera are described: Michener (Proteropinae), Bioalfa (Rogadinae), and Hermosomastax (Rogadinae). Keys are given for the New World genera of the following braconid subfamilies: Agathidinae, Braconinae, Cheloninae, Homolobinae, Hormiinae, Ichneutinae, Macrocentrinae, Orgilinae, Proteropinae, Rhysipolinae, and Rogadinae. In these subfamilies 416 species are described or redescribed. Most of the species have been reared and all but 13 are new to science. A consensus sequence of the COI barcodes possessed by each species is employed to diagnose the species, and this approach is justified in the introduction. Most descriptions consist of a lateral or dorsal image of the holotype, a diagnostic COI consensus barcode, the Barcode Index Number (BIN) code with a link to the Barcode of Life Database (BOLD), and the holotype specimen information required by the International Code of Zoological Nomenclature. The following species are treated and those lacking authorship are newly described here with authorship attributable to Sharkey except for the new species of Macrocentrinae which are by Sharkey & van Achterberg: AGATHIDINAE: Aerophilus paulmarshi, Mesocoelus davidsmithi, Neothlipsis bobkulai, Plesiocoelus vanachterbergi, Pneumagathis erythrogastra (Cameron, 1905), Therophilus bobwhartoni, T. donaldquickei, T. gracewoodae, T. maetoi, T. montywoodi, T. penteadodiasae, Zacremnops brianbrowni, Z. coatlicue Sharkey, 1990, Zacremnops cressoni (Cameron, 1887), Z. ekchuah Sharkey, 1990, Z. josefernandezi, Zelomorpha sarahmeierottoae. BRACONINAE: Bracon alejandromarini, B. alejandromasisi, B. alexamasisae, B. andresmarini, B. andrewwalshi, B. anniapicadoae, B. anniemoriceae, B. barryhammeli, B. bernardoespinozai, B. carlossanabriai, B. chanchini, B. christophervallei, B. erasmocoronadoi, B. eugeniephillipsae, B. federicomatarritai, B. frankjoycei, B. gerardovegai, B. germanvegai, B. isidrochaconi, B. jimlewisi, B. josejaramilloi, B. juanjoseoviedoi, B. juliodiazi, B. luzmariaromeroae, B. manuelzumbadoi, B. marialuisariasae, B. mariamartachavarriae, B. mariorivasi, B. melissaespinozae, B. nelsonzamorai, B. nicklaphami, B. ninamasisae, B. oliverwalshi, B. paulamarinae, B. rafamoralesi, B. robertofernandezi, B. rogerblancoi, B. ronaldzunigai, B. sigifredomarini, B. tihisiaboshartae, B. wilberthbrizuelai, Digonogastra montylloydi, D. montywoodi, D. motohasegawai, D. natwheelwrighti, D. nickgrishini. CHELONINAE: Adelius adrianguadamuzi, A. gauldi Shimbori & Shaw, 2019, A. janzeni Shimbori & Shaw, 2019, Ascogaster gloriasihezarae, A. grettelvegae, A. guillermopereirai, A. gustavoecheverrii, A. katyvandusenae, A. luisdiegogomezi, Chelonus alejandrozaldivari, C. gustavogutierrezi, C. gustavoinduni, C. harryramirezi, C. hartmanguidoi, C. hazelcambroneroae, C. iangauldi, C. isidrochaconi, C. janecheverriae, C. jeffmilleri, C. jennyphillipsae, C. jeremydewaardi, C. jessiehillae, C. jesusugaldei, C. jimlewisi, C. jimmilleri, C. jimwhitfieldi, C. johanvalerioi, C. johnburnsi, C. johnnoyesi, C. jorgebaltodanoi, C. jorgehernandezi, C. josealfredohernandezi, C. josefernandeztrianai, C. josehernandezcortesi, C. josemanuelperezi, C. josephinerodriguezae, C. juanmatai, C. junkoshimurae, C. kateperezae, C. luciariosae, C. luzmariaromeroae, C. manuelpereirai, C. manuelzumbadoi, C. marianopereirai, C. maribellealvarezae, C. markmetzi, C. markshawi, C. martajimenezae, C. mayrabonillae, C. meganmiltonae, C. melaniamunozae, C. michaelstroudi, C. michellevanderbankae, C. mingfangi, C. minorcarmonai, C. monikaspringerae, C. moniquegilbertae, C. motohasegawai, C. nataliaivanovae, C. nelsonzamorai, C. normwoodleyi, C. osvaldoespinozai, C. pamelacastilloae, C. paulgoldsteini, C. paulhansoni, C. paulheberti, C. petronariosae, C. ramyamanjunathae, C. randallgarciai, C. rebeccakittelae, C. robertoespinozai, C. robertofernandezi, C. rocioecheverriae, C. rodrigogamezi, C. ronaldzunigai, C. rosibelelizondoae, C. rostermoragai, C. ruthfrancoae, C. scottmilleri, C. scottshawi, C. sergioriosi, C. sigifredomarini, C. stevearonsoni, C. stevestroudi, C. sujeevanratnasinghami, C. sureshnaiki, C. torbjornekremi, C. yeimycedenoae, Leptodrepana alexisae, L. erasmocoronadoi, L. felipechavarriai, L. freddyquesadai, L. gilbertfuentesi, L. manuelriosi, Phanerotoma almasolisae, P. alvaroherrerai, P. anacordobae, P. anamariamongeae, P. andydeansi, P. angelagonzalezae, P. angelsolisi, P. barryhammeli, P. bernardoespinozai, P. calixtomoragai, P. carolinacanoae, P. christerhanssoni, P. christhompsoni, P. davesmithi, P. davidduthiei, P. dirksteinkei, P. donquickei, P. duniagarciae, P. duvalierbricenoi, P. eddysanchezi, P. eldarayae, P. eliethcantillanoae, P. jenopappi, Pseudophanerotoma alanflemingi, Ps. albanjimenezi, Ps. alejandromarini, Ps. alexsmithi, Ps. allisonbrownae, Ps. bobrobbinsi. HOMOLOBINAE: Exasticolus jennyphillipsae, E. randallgarciai, E. robertofernandezi, E. sigifredomarini, E. tomlewinsoni. HORMIINAE: Hormius anamariamongeae, H. angelsolisi, H. anniapicadoae, H. arthurchapmani, H. barryhammeli, H. carmenretanae, H. carloswalkeri, H. cesarsuarezi, H. danbrooksi, H. eddysanchezi, H. erikframstadi, H. georgedavisi, H. grettelvegae, H. gustavoinduni, H. hartmanguidoi, H. hectoraritai, H. hesiquiobenitezi, H. irenecanasae, H. isidrochaconi, H. jaygallegosi, H. jimbeachi, H. jimlewisi, H. joelcracrafti, H. johanvalerioi, H. johnburleyi, H. joncoddingtoni, H. jorgecarvajali, H. juanmatai, H. manuelzumbadoi, H. mercedesfosterae, H. modonnellyae, H. nelsonzamorai, H. pamelacastilloae, H. raycypessi, H. ritacolwellae, H. robcolwelli, H. rogerblancosegurai, H. ronaldzunigai, H. russchapmani, H. virginiaferrisae, H. warrenbrighami, H. willsflowersi. ICHNEUTINAE: Oligoneurus kriskrishtalkai, O. jorgejimenezi, Paroligoneurus elainehoaglandae, P. julianhumphriesi, P. mikeiviei. MACROCENTRINAE: Austrozele jorgecampabadali, A. jorgesoberoni, Dolichozele gravitarsis (Muesebeck, 1938), D. josefernandeztrianai, D. josephinerodriguezae, Hymenochaonia kalevikulli, H. kateperezae, H. katherinebaillieae, H. katherineellisonae, H. katyvandusenae, H. kazumifukunagae, H. keithlangdoni, H. keithwillmotti, H. kenjinishidai, H. kimberleysheldonae, H. krisnorvigae, H. lilianamadrigalae, H. lizlangleyae, Macrocentrus fredsingeri, M. geoffbarnardi, M. gregburtoni, M. gretchendailyae, M. grettelvegae, M. gustavogutierrezi, M. hannahjamesae, M. harisridhari, M. hillaryrosnerae, M. hiroshikidonoi, M. iangauldi, M. jennyphillipsae, M. jesseausubeli, M. jessemaysharkae, M. jimwhitfieldi, M. johnbrowni, M. johnburnsi, M. jonathanfranzeni, M. jonathanrosenbergi, M. jorgebaltodanoi, M. lucianocapelli. ORGILINAE: Orgilus amyrossmanae, O. carrolyoonae, O. christhompsoni, O. christinemcmahonae, O. dianalipscombae, O. ebbenielsoni, O. elizabethpennisiae, O. evertlindquisti, O. genestoermeri, O. jamesriegeri, O. jeanmillerae, O. jeffmilleri, O. jerrypowelli, O. jimtiedjei, O. johnlundbergi, O. johnpipolyi, O. jorgellorentei, O. larryspearsi, O. marlinricei, O. mellissaespinozae, O. mikesmithi, O. normplatnicki, O. peterrauchi, O. richardprimacki, O. sandraberriosae, O. sarahmirandae, O. scottmilleri, O. scottmorii, Stantonia billalleni, S. brookejarvisae, S. donwilsoni, S. erikabjorstromae, S. garywolfi, S. henrikekmani, S. luismirandai, S. miriamzunzae, S. quentinwheeleri, S. robinkazmierae, S. ruthtifferae. PROTEROPINAE: Hebichneutes tricolor Sharkey & Wharton, 1994, Proterops iangauldi, P. vickifunkae, Michener charlesi. RHYSIPOLINAE: Pseudorhysipolis luisfonsecai, P. mailyngonzalezaeRhysipolis julioquirosi. ROGADINAE: Aleiodes adrianaradulovae, A. adrianforsythi, A. agnespeelleae, A. alaneaglei, A. alanflemingi, A. alanhalevii, A. alejandromasisi, A. alessandracallejae, A. alexsmithi, A. alfonsopescadori, A. alisundermieri, A. almasolisae, A. alvarougaldei, A. alvaroumanai, A. angelsolisi, A. annhowdenae, A. bobandersoni, A. carolinagodoyae, A. charlieobrieni, A. davefurthi, A. donwhiteheadi, A. doylemckeyi, A. frankhovorei, A. henryhowdeni, A. inga Shimbori & Shaw, 2020, A. johnchemsaki, A. johnkingsolveri, A. gonodontovorus Shimbori & Shaw, 2020, A. manuelzumbadoi, A. mayrabonillae, A. michelledsouzae, A. mikeiviei, A. normwoodleyi, A. pammitchellae, A. pauljohnsoni, A. rosewarnerae, A. steveashei, A. terryerwini, A. willsflowersi, Bioalfa pedroleoni, B. alvarougaldei, B. rodrigogamezi, Choreborogas andydeansi, C. eladiocastroi, C. felipechavarriai, C. frankjoycei, Clinocentrus andywarreni, Cl. angelsolisi, Cystomastax alexhausmanni, Cy. angelagonzalezae, Cy. ayaigarashiae, Hermosomastax clavifemorus Quicke sp. nov., Heterogamus donstonei, Pseudoyelicones bernsweeneyi, Stiropius bencrairi, S. berndkerni, S. edgargutierrezi, S. edwilsoni, S. ehakernae, Triraphis billfreelandi, T. billmclarneyi, T. billripplei, T. bobandersoni, T. bobrobbinsi, T. bradzlotnicki, T. brianbrowni, T. brianlaueri, T. briannestjacquesae, T. camilocamargoi, T. carlosherrerai, T. carolinepalmerae, T. charlesmorrisi, T. chigiybinellae, T. christerhanssoni, T. christhompsoni, T. conniebarlowae, T. craigsimonsi, T. defectus Valerio, 2015, T. danielhubi, T. davidduthiei, T. davidwahli, T. federicomatarritai, T. ferrisjabri, T. mariobozai, T. martindohrni, T. matssegnestami, T. mehrdadhajibabaei, T. ollieflinti, T. tildalauerae, Yelicones dirksteinkei, Y. markmetzi, Y. monserrathvargasae, Y. tricolor Quicke, 1996. Y. woldai Quicke, 1996. The following new combinations are proposed: Neothlipsis smithi (Ashmead), new combination for Microdus smithi Ashmead, 1894; Neothlipsis pygmaeus (Enderlein), new combination for Microdus pygmaeus Enderlein, 1920; Neothlipsis unicinctus (Ashmead), new combination for Microdus unicinctus Ashmead, 1894; Therophilus anomalus (Bortoni and Penteado-Dias) new combination for Plesiocoelus anomalus Bortoni and Penteado-Dias, 2015; Aerophilus areolatus (Bortoni and Penteado-Dias) new combination for Plesiocoelus areolatus Bortoni and Penteado-Dias, 2015; Pneumagathis erythrogastra (Cameron) new combination for Agathis erythrogastra Cameron, 1905. Dolichozele citreitarsis (Enderlein), new combination for Paniscozele citreitarsis Enderlein, 1920. Dolichozele fuscivertex (Enderlein) new combination for Paniscozele fuscivertex Enderlein, 1920. Finally, Bassus brooksi Sharkey, 1998 is synonymized with Agathis erythrogastra Cameron, 1905; Paniscozele griseipes Enderlein, 1920 is synonymized with Dolichozele koebelei Viereck, 1911; Paniscozele carinifrons Enderlein, 1920 is synonymized with Dolichozele fuscivertex (Enderlein, 1920); and Paniscozele nigricauda Enderlein,1920 is synonymized with Dolichozele quaestor (Fabricius, 1804). (originally described as Ophion quaestor Fabricius, 1804).

Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera), Aleyrodidae (Hemiptera) and Braconidae (Hymenoptera); the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew) was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms.