CONSERVATION BIOLOGY AND BIODIVERSITY
DNA Barcodes and Targeted Sampling Methods Identify a New Species and Cryptic Patterns of Host Specialization Among North American Coptera (Hymenoptera: Diapriidae) ANDREW A. FORBES,1,2 SERDAR SATAR,3,4 GABRIELA HAMERLINCK,1 AMANDA E. NELSON,1 3 AND JAMES J. SMITH
Ann. Entomol. Soc. Am. 105(4): 608Ð612 (2012); DOI: http://dx.doi.org/10.1603/AN12012
ABSTRACT Fewer than half of the 80 Ð100 North American species in parasitoid genus Coptera Say (Hymenoptera: Diapriidae) have been described. Hosts are known for just nine of these. The taxonomy of Coptera has been complicated by its cryptic morphology and a life history that includes parasitism of pupae beneath the surface of soils. Here, we describe collections targeting the host genus with which Coptera have most frequently been associated: ßies in genus Rhagoletis (Loew) (Diptera: Tephritidae). DNA barcodes, morphology, and ecology (host associations) were used to understand species limits for Coptera collected from Rhagoletis. Four species of Coptera were recovered from Þve species of Rhagoletis, including a new species: Coptera n. sp. 1. Two of the associations with particular species of Rhagoletis were previously unknown, and no two species of Coptera were found to be attacking the same host, suggesting these four Coptera are specialist parasites. As several of the 25 North American species of Rhagoletis are agricultural pests, a better understanding of their natural associations with Coptera may prove valuable to biological control efforts. KEY WORDS Rhagoletis, parasitoid, genetic barcode, biological control, cryptic species
The parasitic wasp genus Coptera Say (Hymenoptera: Diapriidae) is species-rich and common in North America. However, most species remain undescribed, while host-associations and other relevant biology are unknown for all but a handful of species (Muesebeck 1980, Masner and Garcia 2002). Coptera has great potential as an economically important genus. A native Mexican species, Coptera haywardi (Ogloblin), is cultured for control of the Caribbean fruit ßy, Anastrepha suspensa (Loew) (Diptera: Tephritidae) (Sivinski et al. 1998), and African Coptera have been bred and released in Hawaii for control of the Mediterranean fruit ßy Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (Silvestri 1914, Yoder and Wharton 2002). Nevertheless, just 29 of the estimated 80 Ð100 native North American species of Coptera have been described, and host associations are known for only nine (Muesebeck 1980). Our poor understanding of the genus Coptera may be a symptom of a “perfect storm” of taxonomic impediments. First, many of the most commonly applied character suites are uninformative for Coptera. Coptera always have uniformly black bodies, and their 1 Department of Biology, The University of Iowa, 434A Biology Building, Iowa City, IA 52242. 2 Corresponding author, e-mail:
[email protected]. 3 Department of Entomology and Lyman Briggs College, Michigan State University, 238 Farm Lane, East Lansing, MI 48824. 4 Department of Plant Protection, Faculty of Agriculture, Cukurova University, Balcali, 01330 Adana, Turkey.
sclerites rarely bear surface sculpturing (Yoder and Wharton 2002). Characters used in distinguishing species are often quantitative and continuous, such as the relative lengths and shapes of body parts, which can become cumbersome if many species are under consideration (Muesebeck 1980). Second, Coptera are sexually dimorphic, making the association of males with females in sympatry problematic and necessitating the creation of separate keys for each sex (Muesebeck 1980, Masner and Garcia 2002). A third complication is that Coptera have a life history that results in their host associations being mostly unknown or overlooked. Adult Coptera walk along fruit ßy larval trails as they search for pupal hosts buried in the soil (Buckingham 1975, Granchietti et al. 2012). Females dig into the soil, unearth concealed hosts, and oviposit a single egg (Buckingham 1975). Because hosts are attacked in soil, deÞnitive host associations for Coptera are only known from cases when insect pupae were ßoated from soil samples and both host and parasitoid were allowed to eclose (Cameron and Morrison 1974, Maier 1981). As soil-based collecting is far less common (and much more difÞcult) than other trapping methods, Coptera are almost exclusively collected as adults in low-to-the-ground traps, resulting in their host-associations being mostly anonymous. It is unclear whether most Coptera are specialist or generalist parasitoids. Muesebeck (1980) reports multiple pupal hosts for some species, including some
0013-8746/12/0608Ð0612$04.00/0 䉷 2012 Entomological Society of America
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FORBES ET AL.: REVEALING CRYPTIC PARASITOID DIVERSITY IN Coptera
from different Dipteran families. Few, if any, of these have been conÞrmed in the time since that revision of the genus. Several were reared solely from lab cultures, indicating that these may not be “natural” hosts. Other spurious associations are because of errors in host ßy identiÞcation, such as the reference to C. occidentalis Muesebeck having been reared from Rhagoletis cingulata (Loew) (Diptera: Tephritidae) in Oregon (Hagen et al. 1980, Muesebeck 1980). R. cingulata is an Eastern species that does not occur in Oregon (Foote et al. 1993). Studies of host acceptance in lab settings have indicated that Coptera may have the ability to develop on several different host species, but nevertheless have a more limited host range than other pupal parasitoids (Sivinski et al. 1998). Additionally, behavioral experiments indicate a preference both for ancestral hosts and for volatile odors associated with fruit habitats (Sivinski et al. 1998, Granchietti et al. 2012), suggesting specialized host-searching behavior. One method that can be used to determine the diversity and understand host speciÞcity for North American Coptera is to conduct directed sampling efforts from their most frequently reported hosts, which are ßies in genus Rhagoletis Loew (Diptera: Tephritidae). Of the nine host records for North American Coptera, Þve are with Rhagoletis. Rhagoletis are specialist frugivores with nonoverlapping host ranges whose third instar larvae emerge from fruit hosts and pupate in soil. The genus has 25 named North American species, each with well characterized host associations and geographic ranges (Bush 1966, Foote et al. 1993). Further, egg and larval stage parasitoids of each Rhagoletis species are well known for many taxa, but pupal parasitoids are not (Forbes et al. 2010). The standing store of knowledge about this genus of ßies provides an opportunity for targeted sampling of their pupal parasitoids, which will in turn reveal more about Coptera. Here, we report the collection of Coptera from six species of Rhagoletis associated with eight different host plant species using two different sampling techniques. We used DNA barcodes (mtDNA COI) as a manner by which to identify preliminary species limits (Hebert et al. 2003). SpeciÞcally, we asked 1) whether new Coptera–Rhagoletis associations would be revealed by targeting Rhagoletis for which no such association has been previously published, and 2) whether Coptera associated with Rhagoletis appear to be specialist or generalist. Materials and Methods Six different species of Rhagoletis were identiÞed as targets for our collections, three of which had been previously associated with Coptera taxa, and three that had not. R. pomonella Walsh infests both hawthorns (Crataegus spp.) and apples (Malus domestica Borkhausen), and it is a documented host of C. pomonellae Muesebeck (Maier 1981). R. cingulata infests black cherries (Prunus serotina Ehrhart) and is host to C. cingulatae Muesebeck (Muesebeck 1980). R. suavis
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(Loew) infests husks of walnuts (Juglans spp.) and is another host of C. pomonellae (Muesebeck 1980). R. juniperina Marcovitch, R. mendax Curran and R. zephyria Snow infest Eastern redcedar (Juniperus virginiana L.), blueberries (Vaccinium corymbosum L.), and snowberries (Symphoricarpos spp.), respectively. No Coptera associations have been reported from any of these potential hosts. All six Rhagoletis species included here are found sympatrically in the Midwestern United States, which is where this study was conducted. Coptera collections were performed in the summer of 2011. The primary trapping method used was the deployment of 4 cm deep, 18 cm diameter yellow plastic pans (Solo Cup Co., Lake Forest, IL) half-Þlled with a dilute water/detergent mixture, for 24 h under Rhagoletis host fruit plants. Only fruiting plants known by the authors to be infested with Rhagoletis ßies in previous years were used for this study. Pans were checked daily for Coptera, then emptied and reÞlled with new liquid. Different numbers of pans were deployed, and for varying numbers of total days, as dictated by time and space constraints. Pans under Eastern redcedar and hawthorns in East Lansing, MI, and under black cherries in Iowa City, IA, were maintained for the longest duration (see Table 1 for deployment details). At three of the same sites where yellow pan traps were used (hawthorn and juniper in East Lansing, MI, and walnut in Riverside, IA), Coptera were also collected by excavating soil samples, which were then air dried for 2 d and sifted through a #10 soil sieve (Hubbard ScientiÞc Co., Chippewa Falls, WI) to isolate Rhagoletis puparia. Rhagoletis pupae were held at room temperature (⬇23⬚C) until all ßies and parasitoids eclosed. The number of Rhagoletis ßies, Coptera wasps, and other parasitoid taxa emerging from each pupal collection was recorded, and the identities of Rhagoletis hosts were conÞrmed using Foote et al. (1993). Live adult Coptera were also collected from beneath walnuts in Riverside, IA. R. suavis-infested walnut fruits were moved into small piles to attract hostsearching Coptera. After 1 h, Coptera found searching on and around fruits were collected using an aspirator. These, and all Coptera collected in this study, were frozen at ⫺80⬚C. For a subset of the yellow pan-trapped Coptera, and for all Coptera collected from soil under juniper and hawthorn in East Lansing, MI, whole genomic DNA was extracted using DNeasy Blood & Tissue Kits (Qiagen Sciences, Germantown, MD). A 648 bp segment of the mitochondrial COI gene was polymerase chain reaction (PCR)-ampliÞed using the primers LepF1 and LepR1 (Smith et al. 2007) and using the following cycling parameters: 94⬚C for 3 min, followed by 35 cycles of 94⬚C for 30 s, 46⬚C for 1 min, and 72⬚C for 2 min, with a Þnal extension of 72⬚C for 4 min. Reactions were cleaned using Shrimp Alkaline Phosphatase (USB, Swampscott, MA) and Exonuclease I (New England Biolabs, Ipswich, MD), and cycle sequencing was performed in both forward and reverse directions
610 Table 1.
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Coptera collected in yellow pan traps under fruit trees known to be hosts to one of six species of Rhagoletis fly
Host ßy
Fruit habitat
City, state
No. pans
No. days
Coptera species
No. Coptera collected
R. pomonella
Hawthorn Hawthorn Apple Apple Apple Apple Walnut Walnut Walnut Eastern Red Cedar Black Cherry Black Cherry Blueberry Snowberry
East Lansing, MI Fennville, MI East Lansing, MI Fennville, MI Homestead, IA Iowa City, IA Iowa City, IA Riverside, IA South Bend, IN East Lansing, MI Rose Lake, MI Iowa City, IA East Lansing, MI East Lansing, MI
7 18 9 20 30 10 5 3 10 3 7 39 12 9
23 2 49 2 5 7 20 7 1 83 9 40 7 3
C. pomonellae and Coptera. n. sp. 1 C. pomonellae C. pomonellae and Coptera. n. sp. 1 C. pomonellae None Coptera. n. sp. 1 C. pomonellae C. pomonellae C. pomonellae C. pomonellae and Coptera. n. sp. 1 C. cingulatae C. cingulatae C. pilosa None
368 45 30 17 0 4 17 3 21 208 24 63 3 0
R. suavis R. juniperina R. cingulata R. mendax R. zephyria
For sites where more than one species of Coptera were collected, the total no. in the right-hand column refers to the total Coptera collected of both species because mtDNA COI was not sequenced for all individuals.
on an ABI 3730 DNA Analyzer using BigDye 3.1 (Applied Biosystems, Foster City, CA) sequencing chemistry. Forward and reverse sequences were used to create consensi for each individual, which were then aligned by hand in BioEdit (Hall 1999). Bayesian phylogenetic analysis was performed using MrBayes 3.1.2 (Ronquist et al. 2012) (5,000,000 generations, 1,250 burnin, GTR⫹G model). The best-Þt substitution model was determined to be GTR⫹G (general time reversible with gamma rates) using the Phylogenetic Inference with Automatic Likelihood model Selectors (PALM) web server, which integrates PhyML and MODELTEST to select a model via maximum likelihood methods (Chen et al. 2009). An individual wasp from genus Aneurhynchus (Hymenoptera: Diapriidae) was also sequenced at COI and used as an outgroup. Sequences were deposited into GenBank (Accession Nos.: JQ889990-JQ890048). All individuals from which DNA was extracted were pinned and labeled for future taxonomic work.
Results In total, 831 individual Coptera were collected in this study. Yellow pan collections yielded 803 Coptera across all sites (Table 1). Coptera were collected in yellow pans associated with all hosts except for snowberry, the host plant of R. zephyria. Rhagoletis pupae sifted from soil collections yielded 25 individual Coptera, and three host-searching Coptera were aspirated from the surface of R. suavis-infested walnuts. Bayesian analysis of COI sequences resolved four different clades of Coptera among our collections (Fig. 1). Each clade Þts the deÞnition of a Ôbarcode speciesÕ based on interclade sequence divergence; pairwise divergence between clades ranged from 9.7Ð13.7%, far exceeding the conservative 2% divergence limit typically used in barcoding studies (Smith et al. 2007, 2011). Intraclade sequence divergence was also low, ranging between 0 and 0.59%. Pairwise comparisons also therefore satisfy the “10⫻ rule” threshold for barcode species, wherein inter-
Fig. 1. Bayesian tree (5,000,000 generations, 1,250 burn in, and a JC model of substitution) of four species of Coptera reared from or collected under the host plants of Þve species of Rhagoletis ßy. Tree is based on an alignment of 648 bp near the 5⬘ end of mitochondrial COI. Fly hosts are denoted by their host plant: HAW ⫽ hawthorn (R. pomonella); BLB ⫽ blueberry (R. mendax); ERC ⫽ Eastern redcedar (R. juniperina); BLCH ⫽ black cherry (R. cingulata). Coptera associated with hawthorns and Eastern redcedar were reared from ßy host pupae. All others were collected under host trees in yellow pan traps. C. pomonellae and Coptera. n. sp. 1 were both pan trapped under hawthorns and junipers, but C. pomonellae was reared only from R. pomonella pupae, while Coptera. n. sp. 1 was reared only from R. juniperina pupae.
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Table 2. COI haplotypes of a subset of the yellow pan trapped Coptera vs those from soil collections under both hawthorns and junipers in East Lansing, MI (sites are ⬇0.8 km apart) Haplotype class C. pomonellae Coptera. n. sp. 1
Soil collections (reared)
Pan traps Hawthorn
Juniper
Hawthorn
Juniper
14 1
15 4
12 0
0 11
speciÞc variation must exceed intraspeciÞc variation by a factor of ten (Hebert et al. 2004), though sample sizes of genotyped individuals within each clade were often small (n ⫽ 2Ð41). The Þrst species cluster, which keyed out as C. pomonellae in the key by Muesebeck (1980), was pantrapped under fruit hosts of R. pomonella, R. suavis, and R. juniperina. A second cluster, keyed to C. cingulatae, was pan trapped only under black cherries (P. serotina), the fruit host of R. cingulata. A third cluster, keyed to C. pilosa (Ashmead), was pan-trapped under highbush blueberries, the fruit host of R. mendax. Finally, the fourth cluster also keyed morphologically to C. pomonellae based on MuesebeckÕs (1980) key. However, based on the extent of divergence of the COI sequence from all other clusters (ⱖ9.7%), and the Þdelity of the host association with J. virginiana, we putatively identify individuals in this fourth cluster as a new species. This species (from here on ÔCoptera. n. sp. 1Õ) was trapped in yellow pans under hawthorns and junipers in Michigan, and under apple trees in both Michigan and Iowa. Soil-collections, however, showed that only Coptera. n. sp. 1 was reared from R. juniperina, while only C. pomonellae was reared from R. pomonella (Table 2). We were not able to determine diagnostic characters to distinguish Coptera. n. sp. 1 from C. pomonellae morphologically. Some variation in setal distribution across the dorsal mesosoma was observed, but these traits were not uniformly different. Discussion Our sampling effort targeting Rhagoletis ßy host pupal environments was effective in uncovering new diversity and host records for Coptera. We collected one new species of Coptera (Coptera n. sp. 1), morphologically. This new species was cryptic with C. pomonellae, but differed both in haplotype and host use. Further, C. pilosa, a species described Þrst by Ashmead (1893), revised by Muesebeck (1980), and unmentioned in any subsequent peer reviewed literature was collected under blueberries, the host plant of the blueberry maggot, R. mendax. This is the Þrst suggestion of a host association for C. pilosa, though we hesitate to Þrmly associate it with R. mendax until it has been reared from pupae of this ßy. We also conÞrmed published host associations for C. cingulatae (R. cingulata) and C. pomonellae (R. pomonella) (Muesebeck 1980, Maier 1981). C. pomonellae was also aspirated from above walnuts, but Coptera reared from R. suavis pupae were not of sufÞcient quality for ge-
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netic work, and so we consider the association of C. pomonellae with R. suavis unconÞrmed. This study also revealed new information about host specialization. Coptera. n. sp. 1 was collected in pan traps beneath plant hosts of both R. pomonella and R. juniperina, but reared exclusively from pupae of the latter (Table 2). Conversely, soil-collected R. pomonella pupae yielded only C. pomonellae. These results indicate that Coptera may search for hosts over a wide area (the two sites in question were both in East Lansing, MI, ⬇0.8 km apart), but only attack certain hosts. Alternatively, Coptera may attack many hosts, but fail to develop in all but a subset. In either case, pan trapping beneath black cherry trees and blueberries in the same city each produced just a single species of Coptera, strongly suggesting that different species of Coptera specialize on one or a few hosts. Many larval parasitoids of Rhagoletis ßies show evidence of specialization on just one or a few ßy species (Forbes et al. 2009, 2010), but all of these also interact directly with the fruit host. Here, we demonstrate the Þrst evidence that direct interaction with the plant environment may not be necessary for specialization among Rhagoletis parasitoids. We note that without mtDNA sequences, Coptera. n. sp. 1 would not have been distinguished from C. pomonellae because of their extreme morphological similarity. Parasitoids as a whole tend to be morphologically cryptic, especially to nonexperts, and genetic barcodes have shown great value in revealing hidden diversity, even in temperate regions (Smith et al. 2011). Critics of the use of barcode sequences to deÞne species boundaries argue that incomplete lineage sorting or introgression of mitochondrial genomes across species boundaries may result in a misrepresentation of true species limits (Funk and Omland 2003). Here, however, the extremely divergent haplotypes (⬎10% in all pairwise comparisons), as well as the 100% host Þdelity between mtDNA haplotypes and the Rhagoletis pupae from which these Coptera were reared via soil collections (Table 2), strongly support Coptera. n. sp. 1 as a new species, parasitizing only R. juniperina, and being different from C. pomonellae. We do not formally describe Coptera n. sp. 1 here, as we anticipate its inclusion in a future formal circumscription of North American Coptera. This and future efforts to identify Coptera–Rhagoletis associations may prove informative to biological control of Rhagoletis ßies. For instance, C. pilosa, which we can now tentatively associate with pupae of the blueberry maggot, R. mendax, might be a completely overlooked, yet potentially beneÞcial control organism for that commercially important pest species. Additionally, several other economically relevant Rhagoletis ßies have not yet been assessed for potential Coptera associations, including R. fausta and R. indifferens, which both infest cultivated cherries, R. ribicola (currants) and R. striatella (tomatillos). Attempts have been made to use Coptera as biological control agents, but the results to date have been mixed. An African Coptera species, C. silvestri, was released in Hawaii in the early 1900s to control the Mediterranean fruit ßy (Silvestri 1914), but its success has not been
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measured, and it is known to (counterproductively) hyperparasitize other introduced larval and egg parasitoids. More promisingly, C. haywardii is being cultured in Mexico for control of its apparently ancestral hosts in the genus Anastrepha (Tephritidae) in citrus fruits (Sivinski et al. 1998, Guillen et al. 2002). Another potential success has been the use of C. occidentalis to control its natural host, R. completa, which has invaded walnuts in Europe (Granchietti et al. 2012). These latter two examples highlight the promise for control of pest species using ancestrally associated Coptera. Acknowledgments The authors thank those who helped collect Coptera: Eric Anderson collected R. suavis pupae from soil in Riverside, IA; William Armstrong and Megan Frayer pan-trapped Coptera and sifted pupae from soil in East Lansing, MI; and Glen Hood pan trapped Coptera in Fennville, MI, and South Bend, IN. Matt Yoder provided our outgroup species, and conversations with Matt were instrumental in understanding the current state of Coptera taxonomy. The authors also thank Gery Hehman and the Carver Center for Genomics at the University of Iowa for assistance with sequencing.
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Granchietti, A., P. Sacchetti, M. Cristiana Rosi, and A. Belcari. 2012. Fruit ßy larval trail acts as a cue in the host location process of the pupal parasitoid Coptera occidentalis. Biol. Control 61: 7Ð14. Gullien, L., M. Aluja, M. Equihua, and J. Sivinski. 2002. Performance of two fruit ßy (Diptera: Tephritidae) pupal parasitoids (Coptera haywardi [Hymenoptera: Diapriidae] and Pachycrepoideus vindemiae [Hymenoptera: Pteromalidae]) under different environmental soil conditions. Biol. Control 23: 219 Ð227. Hagen, K. S., R. L. Tassan, and M. Fong. 1980. Biological control of the walnut husk ßy. University of California Walnut Research Board Report. (www.walnutresearch. ucdavis.edu/1980/1980_9.pdf). Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41: 95Ð98. Hebert, P.D.N., S. Ratnasingham, and J. R. deWaard. 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. Royal Soc. B. 270: S96 ÐS99. Hebert, P.D.N., M. Y. Stoeckle, T. S. Zemlak, and C. M. Francis. 2004. IdentiÞcation of birds through DNA barcodes. PLOS Biol. 2: e312. Maier, C. T. 1981. Parasitoids emerging from puparia of Rhagoletis pomonella (Diptera: Tephritidae) infesting hawthorn and apple in Connecticut. Can. Entomol. 113: 867Ð 870. Masner, L., and J. L. Garcia. 2002. The genera of Diapriinae (Hymenoptera: Diapriidae) in the New World. Bull. Am. Mus. Nat. Hist. 268: 1Ð138. Muesebeck, C.F.W. 1980. The Nearctic parasitic wasps of the genera Psilus Panzer and Coptera Say (Hymenoptera, Proctotrupoidea, Diapriidae). U.S. Dept. Agric. Tech. Bull. 1617: 1Ð71. Ronquist, F., M. Teslenko, P. van der Mark, D. Ayres, A. Darling, S. Ho¨ hna, B. Larget, L. Liu, M. A. Suchard, and J. P. Huelsenbeck. 2012. MrBayes 3.2: efÞcient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61: 539 Ð542. Silvestri, F. 1914. Report of an expedition to Africa in search of the natural enemies of fruit ßies (Trypaneidae) with descriptions, observations and biological notes. Bulletin of the Division of Entomology, Board of Commissioners of Agriculture and Forestry, Hawaii 3: 1Ð176. Sivinski, J., K. Vulinec, E. Menezes, and M. Aluja. 1998. The bionomics of Coptera haywardi (Ogloblin) and other pupal parasitoids of Tephritid fruit ßies. Biol. Control 11: 193Ð202. Smith, M. A., D. M. Wood, D. H. Janzen, W. Hallwachs, and P.D.N. Hebert. 2007. DNA barcodes afÞrm that 16 species of apparently generalist tropical parasitoid ßies (Diptera, Tachinidae) are not all generalists. Proc. Nat. Acad. Sci. U.S.A. 104: 4967Ð 4972. Smith, M. A., E. S. Eveleigh, K. S. McCann, M. T. Merilo, P.C. McCarthy, and K. I. Van Rooyen. 2011. Barcoding a quantiÞed food web: crypsis, concepts, ecology and hypotheses. PLoS ONE 6: e14424. (doi: 10.1371/journal. pone.0014424). Yoder, M. J., and R. A. Wharton. 2002. Nomenclature of African Psilini (Hymenoptera: Diapriidae) and status of Coptera robustior, a parasitoid of Mediterranean fruit ßy (Diptera: Tephritidae). Can. Entomol. 134: 561Ð576. Received 24 January 2012; accepted 9 April 2012.
