James B. Whitfield

Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections

We DNA barcoded 2,597 parasitoid wasps belonging to 6 microgastrine braconid genera reared from parapatric tropical dry forest, cloud forest, and rain forest in Area de Conservación Guanacaste (ACG) in northwestern Costa Rica and combined these data with records of caterpillar hosts and morphological analyses. We asked whether barcoding and morphology discover the same provisional species and whether the biological entities revealed by our analysis are congruent with wasp host specificity. Morphological analysis revealed 171 provisional species, but barcoding exposed an additional 142 provisional species; 95% of the total is likely to be undescribed. These 313 provisional species are extraordinarily host specific; more than 90% attack only 1 or 2 species of caterpillars out of more than 3,500 species sampled. The most extreme case of overlooked diversity is the morphospecies Apanteles leucostigmus. This minute black wasp with a distinctive white wing stigma was thought to parasitize 32 species of ACG hesperiid caterpillars, but barcoding revealed 36 provisional species, each attacking one or a very few closely related species of caterpillars. When host records and/or within-ACG distributions suggested that DNA barcoding had missed a species-pair, or when provisional species were separated only by slight differences in their barcodes, we examined nuclear sequences to test hypotheses of presumptive species boundaries and to further probe host specificity. Our iterative process of combining morphological analysis, ecology, and DNA barcoding and reiteratively using specimens maintained in permanent collections has resulted in a much more fine-scaled understanding of parasitoid diversity and host specificity than any one of these elements could have produced on its own.

Estimating the age of the polydnavirus/braconid wasp symbiosis

Polydnaviruses are essential components mediating host–parasitoid relationships between some braconid wasps and their caterpillar hosts largely by suppressing or misdirecting the host immune systems. The polydnavirus–wasp relationship is an unusual apparent mutualism between viruses and eukaryotes and remarkably has evolved to the stage where the two entities no longer can be considered separate. Estimations of the age of the polydnavirus-bearing clade of braconid wasps based on separate calculations from the mitochondrial 16S rRNA and COI genes and the nuclear 28S rRNA gene, calibrated using fossil data, converge to indicate a date of origin of ≈73.7 ± 10 million years ago. This range provides an upper bound on the time during which these wasps and viruses have been functionally associated.

Virus or not? Phylogenetics of polydnaviruses and their wasp carriers.

Our current, still limited, understanding of the comparative biology and evolution of polydnaviruses (PDVs) is reviewed, especially in the context of the possible origins of these parasitoid viruses and of their coevolution with carrier wasps. A hypothetical scenario of evolution of PDVs from ascovirus (or ascovirus-like) ancestors is presented, with examples of apparent extant transitional forms. PDVs appear, in the case of bracoviruses, to show phylogenetic relationships that mirror those of their wasp carriers: with ichnoviruses, the picture is less clear. Ongoing sequencing studies of entire PDV genomes from diverse wasp species are likely to greatly contribute to our understanding of PDV evolution.

Networks: expanding evolutionary thinking

Networks allow the investigation of evolutionary relationships that do not fit a tree model. They are becoming a leading tool for describing the evolutionary relationships between organisms, given the comparative complexities among genomes.

Wolbachia and DNA barcoding insects: Patterns, potential, and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein – wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor – for which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

Genomic and Morphological Features of a Banchine Polydnavirus: Comparison with Bracoviruses and Ichnoviruses

ABSTRACT Many ichneumonid and braconid endoparasitoids inject a polydnavirus (PDV) into their caterpillar hosts during oviposition. The viral entities carried by wasps of these families are referred to as “ichnoviruses” (IVs) and “bracoviruses” (BVs), respectively. All IV genomes characterized to date are found in wasps of the subfamily Campopleginae; consequently, little is known about PDVs found in wasps of the subfamily Banchinae, the only other ichneumonid taxon thus far shown to carry these viruses. Here we report on the genome sequence and virion morphology of a PDV carried by the banchine parasitoid Glypta fumiferanae. With an aggregate genome size of ∼290 kb and 105 genome segments, this virus displays a degree of genome segmentation far greater than that reported for BVs or IVs. The size range of its genome segments is also lower than those in the latter two groups. As reported for other PDVs, the predicted open reading frames of this virus cluster into gene families, including the protein tyrosine phosphatase (PTP) and viral ankyrin (ank) families, but phylogenetic analysis indicates that ank genes of the G. fumiferanae virus are not embedded within the IV lineage, while its PTPs and those of BVs form distinct clusters. The banchine PDV genome also encodes a novel family of NTPase-like proteins displaying a pox-D5 domain. The unique genomic features of the first banchine virus examined, along with the morphological singularities of its virions (IV-like nucleocapsids, but enveloped in groups like some of the BVs), suggest that they could have an origin distinct from those of IVs and BVs.

DNA barcoding and the taxonomy of Microgastrinae wasps (Hymenoptera, Braconidae): Impacts after 8 years and nearly 20 000 sequences

Microgastrine wasps are among the most species‐rich and numerous parasitoids of caterpillars (Lepidoptera). They are often host‐specific and thus are extensively used in biological control efforts and figure prominently in trophic webs. However, their extraordinary diversity coupled with the occurrence of many cryptic species produces a significant taxonomic impediment. We present and release the results of 8 years (2004–2011) of DNA barcoding microgastrine wasps. Currently they are the best represented group of parasitoid Hymenoptera in the Barcode of Life Data System (BOLD), a massive barcode storage and analysis data management site for the International Barcoding of Life (iBOL) program. There are records from more than 20 000 specimens from 75 countries, including 50 genera (90% of the known total) and more than 1700 species (as indicated by Barcode Index Numbers and 2% MOTU). We briefly discuss the importance of this DNA data set and its collateral information for future research in: (1) discovery of cryptic species and description of new taxa; (2) estimating species numbers in biodiversity inventories; (3) clarification of generic boundaries; (4) biological control programmes; (5) molecular studies of host‐parasitoid biology and ecology; (6) evaluation of shifts in species distribution and phenology; and (7) fostering collaboration at national, regional and world levels. The integration of DNA barcoding with traditional morphology‐based taxonomy, host records, and other data has substantially improved the accuracy of microgastrine wasp identifications and will significantly accelerate further studies on this group of parasitoids.

Preliminary evolutionary relationships within the parasitoid wasp genus Cotesia (Hymenoptera: Braconidae: Microgastrinae): combined analysis of four genes

Abstract.  The braconid wasp genus Cotesia Cameron (Braconidae: Microgastrinae) is one of the largest genera of parasitoid wasps, and its species are employed frequently as biological control agents against pest insects. Several Cotesia species are also used as model organisms in physiology, ecology and population genetics studies. The genus thus has considerable importance from both applied and basic science perspectives. We investigated the phylogenetic relationships of twenty‐five species of Cotesia commonly used in field and laboratory research, using the genes 16S, ND1, 28S and LW opsin and employing a range of phylogeny estimation methods including maximum parsimony, maximum likelihood, minimum evolution and Bayesian inference. Phylogenetic estimates differed little among methods, especially for the combined data analysis. The combined analysis of four genes identified four well‐supported clades within Cotesia: the melanoscela group (containing C. melanoscela, the C. flavipes species complex and probably also C. ruficrus), the kariyai group (containing C. kariyai, C. kazak, C. cyaniridis, C. flaviconchae and probably also C. anisotae and C. griffini), the rubecula group (containing C. congregata, C.electrae, C.euchaetis, C. marginiventris, C. obsuricornis and C. schizurae), and the glomerata group (consisting of C. glomerata, C. melitaearum and C. plutellae), plus a basal unresolved complex including C. hyphantriae, C. diacrisiae and C. empretiae. These groups correspond poorly with previous broad subgroups of Cotesia defined by Papp based on morphology. The current work constitutes the first real framework for comparative studies in systematics, ecology, physiology and population genetics of Cotesia. A preliminary analysis of the evolution of gregarious development from solitary is presented, in which it is apparent that solitary development is ancestral, and gregariousness has arisen several times independently within separate groups.

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.

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

Abstract 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.