A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (π), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

A revision of the parasitoid wasp genus Alphomelon Mason with the description of 30 new species (Hymenoptera, Braconidae).

The parasitoid wasp genus Alphomelon Mason, 1981 is revised, based on a combination of basic morphology (dichotomous key and brief diagnostic descriptions), DNA barcoding, biology (host data and wasp cocoons), and distribution data. A total of 49 species is considered; the genus is almost entirely Neotropical (48 species recorded from that region), but three species reach the Nearctic, with one of them extending as far north as 45° N in Canada. Alphomelon parasitizes exclusively Hesperiinae caterpillars (Lepidoptera: Hesperiidae), mostly feeding on monocots in the families Arecaceae, Bromeliaceae, Cannaceae, Commelinaceae, Heliconiaceae, and Poaceae. Most wasp species parasitize either on one or very few (2-4) host species, usually within one or two hesperiine genera; but some species can parasitize several hosts from up to nine different hesperiine genera. Among species with available data for their cocoons, roughly half weave solitary cocoons (16) and half are gregarious (17); cocoons tend to be surrounded by a rather distinctive, coarse silk (especially in solitary species, but also distinguishable in some gregarious species). Neither morphology nor DNA barcoding alone was sufficient on its own to delimit all species properly; by integrating all available evidence (even if incomplete, as available data for every species is different) a foundation is provided for future studies incorporating more specimens, especially from South America. The following 30 new species are described: cruzi, itatiaiensis, and palomae, authored by Shimbori & Fernandez-Triana; and adrianguadamuzi, amazonas, andydeansi, calixtomoragai, carolinacanoae, christerhanssoni, diniamartinezae, duvalierbricenoi, eldaarayae, eliethcantillanoae, gloriasihezarae, guillermopereirai, hazelcambroneroae, josecortesi, keineraragoni, luciarosae, manuelriosi, mikesharkeyi, osvaldoespinozai, paramelanoscelis, paranigriceps, petronariosae, ricardocaleroi, rigoi, rostermoragai, sergioriosi, and yanayacu, authored by Fernandez-Triana & Shimbori.

Checking the checkered taxonomy of Plötz's checkered skippers (Hesperiidae: Pyrgini).

We present an analysis of the names proposed by Carl Plötz in 1884 for the New World species in the genus Pyrgus Hübner, [1819] facilitated by the genomic sequencing of extant primary type specimens comparatively with a larger sample of more recently collected specimens of these species and their relatives. The changes to nomenclature suggested here are only caused by the identity of primary type specimens as revealed by their phenotypes or through genomic sequencing. All neotypes are designated to stabilize nomenclature in agreement with the current usage of these names, which in unison agrees best with the information available about them. Lectotypes are designated for the following 5 taxa: Pyrgus (Scelothrix [sic]) bellatrix Plötz, 1884 (type locality Argentina: Buenos Aires), Pyrgus (Pyrgus) willi Plötz, 1884 (type locality in Brazil: Minas Gerais), Pyrgus (Pyrgus) albescens Plötz, 1884 (type locality in Mexico), Pyrgus (Syrichthus [sic]) lycurgus Plötz, 1884 (type locality in "Central America", likely southern Mexico), and Pyrgus occidentalis Skinner, 1906 (type locality USA: Texas, San Antonio). Neotypes are designated for the following 4 taxa: Pyrgus (Pyrgus) adepta Plötz, 1884 (Herrich-Schäffer in litt.) (type locality Colombia: Bogota), Pyrgus (Scelothrix [sic]) dion Plötz, 1884 (type locality Colombia: Bogota), Pyrgus (Scelothrix [sic]) adjutrix Plötz, 1884 (Herrich-Schäffer in litt.) (type locality in Mexico: Nuevo Leon), Pyrgus (Pyrgus) insolatrix Plötz, 1884 (Herrich-Schäffer in litt.) (type locality in "Central America", likely southern Mexico). As a result, P. lycurgus and P. insolatrix are objective synonyms. The following are junior subjective synonyms: P. dion of Burnsius adepta (Plötz, 1884), Pyrgus (Syrichthus [sic]) varus Plötz, 1884 of Burnsius orcus (Stoll, 1780) and P. adjutrix of Burnsius oileus (Linnaeus, 1767). Heliopetes (Heliopyrgus) willi (Plötz, 1884) is a species-level taxon and not a subspecies of Heliopetes (Heliopyrgus) domicella (Erichson, [1849])). Genomic analysis of the lectotypes of P. albescens, P. lycurgus, and P. occidentalis establishes them as conspecific with Burnsius communis (Grote, 1872), thus depriving a distinct species currently identified as Burnsius albescens from its name, that becomes a name for Burnsius communis albescens (Plötz, 1884) in accord with its lectotype identity; P. lycurgus and P. insolatrix are its junior subjective synonyms, but P. occidentalis is a junior subjective synonym of B. communis communis. A new name Burnsius albezens Grishin sp. n. (type locality USA: Arizona, Cochise Co., Portal) is proposed for the species misidentified as B. albescens. Furthermore, genomic comparisons reveal two other new species and one new subspecies of Burnsius Grishin, 2019: B. burnsi Grishin sp. n. (type locality Mexico: Veracruz, Huatusco), B. adepta inepta Grishin ssp. n. (type locality Ecuador: Pichincha, Tandapi), and B. orcynus Grishin sp. n. (type locality Curaçao: Hato Field) that are cryptic and can be confidently identified only by their genotype.

Minimalist revision and description of 403 new species in 11 subfamilies of Costa Rican braconid parasitoid wasps, including host records for 219 species.

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: Aerophiluspaulmarshi, Mesocoelusdavidsmithi, Neothlipsisbobkulai, Plesiocoelusvanachterbergi, Pneumagathiserythrogastra (Cameron, 1905), Therophilusbobwhartoni, T.donaldquickei, T.gracewoodae, T.maetoi, T.montywoodi, T.penteadodiasae, Zacremnopsbrianbrowni, Z.coatlicue Sharkey, 1990, Zacremnopscressoni (Cameron, 1887), Z.ekchuah Sharkey, 1990, Z.josefernandezi, Zelomorphasarahmeierottoae. BRACONINAE: Braconalejandromarini, 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, Digonogastramontylloydi, D.montywoodi, D.motohasegawai, D.natwheelwrighti, D.nickgrishini. CHELONINAE: Adeliusadrianguadamuzi, A.gauldi Shimbori & Shaw, 2019, A.janzeni Shimbori & Shaw, 2019, Ascogastergloriasihezarae, A.grettelvegae, A.guillermopereirai, A.gustavoecheverrii, A.katyvandusenae, A.luisdiegogomezi, Chelonusalejandrozaldivari, 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, Leptodrepanaalexisae, L.erasmocoronadoi, L.felipechavarriai, L.freddyquesadai, L.gilbertfuentesi, L.manuelriosi, Phanerotomaalmasolisae, 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, Pseudophanerotomaalanflemingi, Ps.albanjimenezi, Ps.alejandromarini, Ps.alexsmithi, Ps.allisonbrownae, Ps.bobrobbinsi. HOMOLOBINAE: Exasticolusjennyphillipsae, E.randallgarciai, E.robertofernandezi, E.sigifredomarini, E.tomlewinsoni. HORMIINAE: Hormiusanamariamongeae, 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: Oligoneuruskriskrishtalkai, O.jorgejimenezi, Paroligoneuruselainehoaglandae, P.julianhumphriesi, P.mikeiviei. MACROCENTRINAE: Austrozelejorgecampabadali, A.jorgesoberoni, Dolichozelegravitarsis (Muesebeck, 1938), D.josefernandeztrianai, D.josephinerodriguezae, Hymenochaoniakalevikulli, H.kateperezae, H.katherinebaillieae, H.katherineellisonae, H.katyvandusenae, H.kazumifukunagae, H.keithlangdoni, H.keithwillmotti, H.kenjinishidai, H.kimberleysheldonae, H.krisnorvigae, H.lilianamadrigalae, H.lizlangleyae, Macrocentrusfredsingeri, 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: Orgilusamyrossmanae, 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, Stantoniabillalleni, S.brookejarvisae, S.donwilsoni, S.erikabjorstromae, S.garywolfi, S.henrikekmani, S.luismirandai, S.miriamzunzae, S.quentinwheeleri, S.robinkazmierae, S.ruthtifferae. PROTEROPINAE: Hebichneutestricolor Sharkey & Wharton, 1994, Proteropsiangauldi, P.vickifunkae, Michenercharlesi. RHYSIPOLINAE: Pseudorhysipolisluisfonsecai, P. mailyngonzalezaeRhysipolisjulioquirosi. ROGADINAE: Aleiodesadrianaradulovae, 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, Bioalfapedroleoni, B.alvarougaldei, B.rodrigogamezi, Choreborogasandydeansi, C.eladiocastroi, C.felipechavarriai, C.frankjoycei, Clinocentrusandywarreni, Cl.angelsolisi, Cystomastaxalexhausmanni, Cy.angelagonzalezae, Cy.ayaigarashiae, Hermosomastaxclavifemorus Quicke sp. nov., Heterogamusdonstonei, Pseudoyeliconesbernsweeneyi, Stiropiusbencrairi, S.berndkerni, S.edgargutierrezi, S.edwilsoni, S.ehakernae, Triraphisbillfreelandi, 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, Yeliconesdirksteinkei, Y.markmetzi, Y.monserrathvargasae, Y.tricolor Quicke, 1996. Y.woldai Quicke, 1996. The following new combinations are proposed: Neothlipsissmithi (Ashmead), new combination for Microdussmithi Ashmead, 1894; Neothlipsispygmaeus (Enderlein), new combination for Microduspygmaeus Enderlein, 1920; Neothlipsisunicinctus (Ashmead), new combination for Microdusunicinctus Ashmead, 1894; Therophilusanomalus (Bortoni and Penteado-Dias) new combination for Plesiocoelusanomalus Bortoni and Penteado-Dias, 2015; Aerophilusareolatus (Bortoni and Penteado-Dias) new combination for Plesiocoelusareolatus Bortoni and Penteado-Dias, 2015; Pneumagathiserythrogastra (Cameron) new combination for Agathiserythrogastra Cameron, 1905. Dolichozelecitreitarsis (Enderlein), new combination for Paniscozelecitreitarsis Enderlein, 1920. Dolichozelefuscivertex (Enderlein) new combination for Paniscozelefuscivertex Enderlein, 1920. Finally, Bassusbrooksi Sharkey, 1998 is synonymized with Agathiserythrogastra Cameron, 1905; Paniscozelegriseipes Enderlein, 1920 issynonymized with Dolichozelekoebelei Viereck, 1911; Paniscozelecarinifrons Enderlein, 1920 is synonymized with Dolichozelefuscivertex (Enderlein, 1920); and Paniscozelenigricauda Enderlein,1920 is synonymized with Dolichozelequaestor (Fabricius, 1804). (originally described as Ophionquaestor Fabricius, 1804).

A DNA barcode library for the butterflies of North America.

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.

Nuclear genomes distinguish cryptic species suggested by their DNA barcodes and ecology.

DNA sequencing brings another dimension to exploration of biodiversity, and large-scale mitochondrial DNA cytochrome oxidase I barcoding has exposed many potential new cryptic species. Here, we add complete nuclear genome sequencing to DNA barcoding, ecological distribution, natural history, and subtleties of adult color pattern and size to show that a widespread neotropical skipper butterfly known as Udranomia kikkawai (Weeks) comprises three different species in Costa Rica. Full-length barcodes obtained from all three century-old Venezuelan syntypes of U. kikkawai show that it is a rainforest species occurring from Costa Rica to Brazil. The two new species are Udranomia sallydaleyae Burns, a dry forest denizen occurring from Costa Rica to Mexico, and Udranomia tomdaleyi Burns, which occupies the junction between the rainforest and dry forest and currently is known only from Costa Rica. Whereas the three species are cryptic, differing but slightly in appearance, their complete nuclear genomes totaling 15 million aligned positions reveal significant differences consistent with their 0.00065-Mbp (million base pair) mitochondrial barcodes and their ecological diversification. DNA barcoding of tropical insects reared by a massive inventory suggests that the presence of cryptic species is a widespread phenomenon and that further studies will substantially increase current estimates of insect species richness.

Varying and unchanging whiteness on the wings of dusk-active and shade-inhabiting Carystoides escalantei butterflies.

Whiteness, although frequently apparent on the wings, legs, antennae, or bodies of many species of moths and butterflies, along with other colors and shades, has often escaped our attention. Here, we investigate the nanostructure and microstructure of white spots on the wings of Carystoides escalantei, a dusk-active and shade-inhabiting Costa Rican rain forest butterfly (Hesperiidae). On both males and females, two types of whiteness occur: angle dependent (dull or bright) and angle independent, which differ in the microstructure, orientation, and associated properties of their scales. Some spots on the male wings are absent from the female wings. Whether the angle-dependent whiteness is bright or dull depends on the observation directions. The angle-dependent scales also show enhanced retro-reflection. We speculate that the biological functions and evolution of Carystoides spot patterns, scale structures, and their varying whiteness are adaptations to butterfly's low light habitat and to airflow experienced on the wing base vs. wing tip.

Mitochondrial and nuclear phylogenetic analysis with Sanger and next-generation sequencing shows that, in Área de Conservación Guanacaste, northwestern Costa Rica, the skipper butterfly named Urbanus belli (family Hesperiidae) comprises three morphologically cryptic species.

BACKGROUND: Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. However, a combination of DNA barcodes, morphology, and natural history data has revealed several cryptic species complexes within them. Here, we investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica. RESULTS: Although no morphological traits appear to distinguish among the three, congruent nuclear and mitochondrial lineage patterns show that "Urbanus belli" in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic compensatory base pair changes were discovered in the secondary structure of ITS2. CONCLUSION: These findings corroborate the existence of three cryptic species. Our confirmation of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues for using a multi-marker approach coupled with next-generation sequencing for exploration of other suspected species complexes.

Review of Apanteles sensu stricto (Hymenoptera, Braconidae, Microgastrinae) from Area de Conservación Guanacaste, northwestern Costa Rica, with keys to all described species from Mesoamerica.

More than half a million specimens of wild-caught Lepidoptera caterpillars have been reared for their parasitoids, identified, and DNA barcoded over a period of 34 years (and ongoing) from Area de Conservación de Guanacaste (ACG), northwestern Costa Rica. This provides the world's best location-based dataset for studying the taxonomy and host relationships of caterpillar parasitoids. Among Hymenoptera, Microgastrinae (Braconidae) is the most diverse and commonly encountered parasitoid subfamily, with many hundreds of species delineated to date, almost all undescribed. Here, we reassess the limits of the genus Apanteles sensu stricto, describe 186 new species from 3,200+ parasitized caterpillars of hundreds of ACG Lepidoptera species, and provide keys to all 205 described Apanteles from Mesoamerica - including 19 previously described species in addition to the new species. The Mesoamerican Apanteles are assigned to 32 species-groups, all but two of which are newly defined. Taxonomic keys are presented in two formats: traditional dichotomous print versions and links to electronic interactive versions (software Lucid 3.5). Numerous illustrations, computer-generated descriptions, distributional information, wasp biology, and DNA barcodes (where available) are presented for every species. All morphological terms are detailed and linked to the Hymenoptera Anatomy Ontology website. DNA barcodes (a standard fragment of the cytochrome c oxidase I (COI) mitochondrial gene), information on wasp biology (host records, solitary/gregariousness of wasp larvae), ratios of morphological features, and wasp microecological distributions were used to help clarify boundaries between morphologically cryptic species within species-complexes. Because of the high accuracy of host identification for about 80% of the wasp species studied, it was possible to analyze host relationships at a regional level. The ACG species of Apanteles attack mainly species of Hesperiidae, Elachistidae and Crambidae (Lepidoptera). About 90% of the wasp species with known host records seem to be monophagous or oligophagous at some level, parasitizing just one host family and commonly, just one species of caterpillar. Only 15 species (9%) parasitize species in more than one family, and some of these cases are likely to be found to be species complexes. We have used several information sources and techniques (traditional taxonomy, molecular, software-based, biology, and geography) to accelerate the process of finding and describing these new species in a hyperdiverse group such as Apanteles. The following new taxonomic and nomenclatural acts are proposed. Four species previously considered to be Apanteles are transferred to other microgastrine genera: Dolichogenidea hedyleptae (Muesebeck, 1958), comb. n., Dolichogenidea politiventris (Muesebeck, 1958), comb. n., Rhygoplitis sanctivincenti (Ashmead, 1900), comb. n., and Illidops scutellaris (Muesebeck, 1921), comb. rev. One European species that is a secondary homonym to a Mesoamerican species is removed from Apanteles and transferred to another genus: Iconella albinervis (Tobias, 1964), stat. rev. The name Apanteles albinervican Shenefelt, 1972, is an invalid replacement name for Apanteles albinervis (Cameron, 1904), stat. rev., and thus the later name is reinstated as valid. The following 186 species, all in Apanteles and all authored by Fernández-Triana, are described as species nova: adelinamoralesae, adrianachavarriae, adrianaguilarae, adrianguadamuzi, aichagirardae, aidalopezae, albanjimenezi, alejandromasisi, alejandromorai, minorcarmonai, alvarougaldei, federicomatarritai, anabellecordobae, rostermoragai, anamarencoae, anamartinesae, anapiedrae, anariasae, andreacalvoae, angelsolisi, arielopezi, bernardoespinozai, bernyapui, bettymarchenae, bienvenidachavarriae, calixtomoragai, carloscastilloi, carlosguadamuzi, eliethcantillanoae, carlosrodriguezi, carlosviquezi, carloszunigai, carolinacanoae, christianzunigai, cinthiabarrantesae, ciriloumanai, cristianalemani, cynthiacorderoae, deifiliadavilae, dickyui, didiguadamuzi, diegoalpizari, diegotorresi, diniamartinezae, duniagarciae, duvalierbricenoi, edgarjimenezi, edithlopezae, eduardoramirezi, edwinapui, eldarayae, erickduartei, esthercentenoae, eugeniaphilipsae, eulogiosequeira, felipechavarriai, felixcarmonai, fernandochavarriai, flormoralesae, franciscopizarroi, franciscoramirezi, freddyquesadai, freddysalazari, gabrielagutierrezae, garygibsoni, gerardobandoi, gerardosandovali, gladysrojasae, glenriverai, gloriasihezarae, guadaluperodriguezae, guillermopereirai, juanmatai, harryramirezi, hectorsolisi, humbertolopezi, inesolisae, irenecarrilloae, isaacbermudezi, isidrochaconi, isidrovillegasi, ivonnetranae, jairomoyai, javiercontrerasi, javierobandoi, javiersihezari, jesusbrenesi, jesusugaldei, jimmychevezi, johanvargasi, jorgecortesi, jorgehernandezi, josecalvoi, josecortesi, josediazi, josejaramilloi, josemonteroi, joseperezi, joserasi, juanapui, juancarrilloi, juangazoi, juanhernandezi, juanlopezi, juanvictori, juliodiazi, juniorlopezi, keineraragoni, laurahuberae, laurenmoralesae, leninguadamuzi, leonelgarayi, lilliammenae, lisabearssae, luciariosae, luisbrizuelai, luiscanalesi, luiscantillanoi, luisgarciai, luisgaritai, luishernandezi, luislopezi, luisvargasi, manuelarayai, manuelpereirai, manuelriosi, manuelzumbadoi, marcobustosi, marcogonzalezi, marcovenicioi, mariachavarriae mariaguevarae, marialuisariasae, mariamendezae, marianopereirai, mariatorrentesae, sigifredomarini, marisolarroyoae, marisolnavarroae, marvinmendozai, mauriciogurdiani, milenagutierrezae, monicachavarriae, oscarchavesi, osvaldoespinozai, pablotranai, pabloumanai, pablovasquezi, paulaixcamparijae, luzmariaromeroae, petronariosae, randallgarciai, randallmartinezi, raulacevedoi, raulsolorsanoi, wadyobandoi, ricardocaleroi, robertmontanoi, robertoespinozai, robertovargasi, rodrigogamezi, rogerblancoi, rolandoramosi, rolandovegai, ronaldcastroi, ronaldgutierrezi, ronaldmurilloi, ronaldnavarroi, ronaldquirosi, ronaldzunigai, rosibelelizondoae, ruthfrancoae, sergiocascantei, sergioriosi, tiboshartae, vannesabrenesae, minornavarroi, victorbarrantesi, waldymedinai, wilbertharayai, williamcamposi, yeissonchavesi, yilbertalvaradoi, yolandarojasae, hazelcambroneroae, zeneidabolanosae.

Reading the complex skipper butterfly fauna of one tropical place.

BACKGROUND: An intense, 30-year, ongoing biodiversity inventory of Lepidoptera, together with their food plants and parasitoids, is centered on the rearing of wild-caught caterpillars in the 120,000 terrestrial hectares of dry, rain, and cloud forest of Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. Since 2003, DNA barcoding of all species has aided their identification and discovery. We summarize the process and results for a large set of the species of two speciose subfamilies of ACG skipper butterflies (Hesperiidae) and emphasize the effectiveness of barcoding these species (which are often difficult and time-consuming to identify). METHODOLOGY/PRINCIPAL FINDINGS: Adults are DNA barcoded by the Biodiversity Institute of Ontario, Guelph, Canada; and they are identified by correlating the resulting COI barcode information with more traditional information such as food plant, facies, genitalia, microlocation within ACG, caterpillar traits, etc. This process has found about 303 morphologically defined species of eudamine and pyrgine Hesperiidae breeding in ACG (about 25% of the ACG butterfly fauna) and another 44 units indicated by distinct barcodes (n = 9,094), which may be additional species and therefore may represent as much as a 13% increase. All but the members of one complex can be identified by their DNA barcodes. CONCLUSIONS/SIGNIFICANCE: Addition of DNA barcoding to the methodology greatly improved the inventory, both through faster (hence cheaper) accurate identification of the species that are distinguishable without barcoding, as well as those that require it, and through the revelation of species "hidden" within what have long been viewed as single species. Barcoding increased the recognition of species-level specialization. It would be no more appropriate to ignore barcode data in a species inventory than it would be to ignore adult genitalia variation or caterpillar ecology.

A tropical horde of counterfeit predator eyes.

We propose that the many different, but essentially similar, eye-like and face-like color patterns displayed by hundreds of species of tropical caterpillars and pupae-26 examples of which are displayed here from the dry, cloud, and rain forests of Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica-constitute a huge and pervasive mimicry complex that is evolutionarily generated and sustained by the survival behavior of a large and multispecific array of potential predators: the insect-eating birds. We propose that these predators are variously and innately programmed to flee when abruptly confronted, at close range, with what appears to be an eye of one of their predators. Such a mimetic complex differs from various classical Batesian and Müllerian mimicry complexes of adult butterflies in that (i) the predators sustain it for the most part by innate traits rather than by avoidance behavior learned through disagreeable experiences, (ii) the more or less harmless, sessile, and largely edible mimics vastly outnumber the models, and (iii) there is no particular selection for the eye-like color pattern to closely mimic the eye or face of any particular predator of the insect-eating birds or that of any other member of this mimicry complex. Indeed, selection may not favor exact resemblance among these mimics at all. Such convergence through selection could create a superabundance of one particular false eyespot or face pattern, thereby increasing the likelihood of a bird species or guild learning to associate that pattern with harmless prey.

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity.

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. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to 'variation' or thought to be insignificant for species-level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.

DNA barcodes and cryptic species of skipper butterflies in the genus Perichares in Area de Conservacion Guanacaste, Costa Rica.

DNA barcodes can be used to identify cryptic species of skipper butterflies previously detected by classic taxonomic methods and to provide first clues to the existence of yet other cryptic species. A striking case is the common geographically and ecologically widespread neotropical skipper butterfly Perichares philetes (Lepidoptera, Hesperiidae), described in 1775, which barcoding splits into a complex of four species in Area de Conservación Guanacaste (ACG) in northwestern Costa Rica. Three of the species are new, and all four are described. Caterpillars, pupae, and foodplants offer better distinguishing characters than do adults, whose differences are mostly average, subtle, and blurred by intraspecific variation. The caterpillars of two species are generalist grass-eaters; of the other two, specialist palm-eaters, each of which feeds on different genera. But all of these cryptic species are more specialized in their diet than was the morphospecies that held them. The four ACG taxa discovered to date belong to a panneotropical complex of at least eight species. This complex likely includes still more species, whose exposure may require barcoding. Barcoding ACG hesperiid morphospecies has increased their number by nearly 10%, an unexpectedly high figure for such relatively well known insects.

DNA barcodes distinguish species of tropical Lepidoptera.

Although central to much biological research, the identification of species is often difficult. The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. However, the effectiveness of DNA barcoding for identifying specimens in species-rich tropical biotas is unknown. Here we show that cytochrome c oxidase I DNA barcodes effectively discriminate among species in three Lepidoptera families from Area de Conservación Guanacaste in northwestern Costa Rica. We found that 97.9% of the 521 species recognized by prior taxonomic work possess distinctive cytochrome c oxidase I barcodes and that the few instances of interspecific sequence overlap involve very similar species. We also found two or more barcode clusters within each of 13 supposedly single species. Covariation between these clusters and morphological and/or ecological traits indicates overlooked species complexes. If these results are general, DNA barcoding will significantly aid species identification and discovery in tropical settings.

Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding.

By facilitating bioliteracy, DNA barcoding has the potential to improve the way the world relates to wild biodiversity. Here we describe the early stages of the use of cox1 barcoding to supplement and strengthen the taxonomic platform underpinning the inventory of thousands of sympatric species of caterpillars in tropical dry forest, cloud forest and rain forest in northwestern Costa Rica. The results show that barcoding a biologically complex biota unambiguously distinguishes among 97% of more than 1000 species of reared Lepidoptera. Those few species whose barcodes overlap are closely related and not confused with other species. Barcoding also has revealed a substantial number of cryptic species among morphologically defined species, associated sexes, and reinforced identification of species that are difficult to distinguish morphologically. For barcoding to achieve its full potential, (i) ability to rapidly and cheaply barcode older museum specimens is urgent, (ii) museums need to address the opportunity and responsibility for housing large numbers of barcode voucher specimens, (iii) substantial resources need be mustered to support the taxonomic side of the partnership with barcoding, and (iv) hand-held field-friendly barcorder must emerge as a mutualism with the taxasphere and the barcoding initiative, in a manner such that its use generates a resource base for the taxonomic process as well as a tool for the user.

Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator.

Astraptes fulgerator, first described in 1775, is a common and widely distributed neotropical skipper butterfly (Lepidoptera: Hesperiidae). We combine 25 years of natural history observations in northwestern Costa Rica with morphological study and DNA barcoding of museum specimens to show that A. fulgerator is a complex of at least 10 species in this region. Largely sympatric, these taxa have mostly different caterpillar food plants, mostly distinctive caterpillars, and somewhat different ecosystem preferences but only subtly differing adults with no genitalic divergence. Our results add to the evidence that cryptic species are prevalent in tropical regions, a critical issue in efforts to document global species richness. They also illustrate the value of DNA barcoding, especially when coupled with traditional taxonomic tools, in disclosing hidden diversity.