Download - 2014 Preliminary phylogeny of the genus Copidosoma (Hymenoptera, Encyrtidae), polyembryonic parasitoids of Lepidoptera

Systematic Entomology (2014), DOI: 10.1111/syen.12057

Preliminary phylogeny of the genus Copidosoma(Hymenoptera, Encyrtidae), polyembryonic parasitoidsof LepidopteraF A N G Y U1,2, F U - Q I A N G C H E N1, S H E N - H O R N Y E N3, L I - H O N GT U2, C H A O - D O N G Z H U1, E M I L I O G U E R R I E R I 4,5 and Y A N - Z H O UZ H A N G 1

1Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China,2School of Life Sciences, Capital Normal University, Beijing, China, 3Department of Biological Sciences, National Sun Yat-SenUniversity, Kaohsiung, Taiwan, 4Istituto per la Protezione delle Piante, CNR, Portici (Napoli), Italy and 5Department of LifeSciences, The Natural History Museum, London, U.K.

Abstract. The genus Copidosoma (Hymenoptera: Chalcidoidea: Encyrtidae) is adiverse group of polyembryonic parasitoids of Lepidoptera, including species thathave the potential to control agricultural and forestry pests. Moreover, some species ofCopidosoma display polyembryony. Despite their economic and scientific importance,little is known about the phylogeny of Copidosoma and its relations to other groupsof Encyrtidae. Here we infer the phylogenetic relationships of this genus fromnucleotide sequences of two nuclear (18S and 28S ) and one mitochondrial (COI )genes. Forty-four species of Copidosoma and three species of Copidosomopsis plustwo outgroup species are included in Maximum Parsimony and Bayesian analyses.Copidosomopsis syn. n. is proposed as a junior synonym of Copidosoma basedon phylogenetic analysis results. Each of nine identical clades, resulting from bothanalyses, is proposed as informal species group: cervius group (cervius , chalconotumand serricorne), recovered as the basal lineage of Copidosoma; nacoleiae group(nacoleaie, meridionalis and an undescribed species, formerly belonging to the genusCopidosomopsis); boucheanum group (boucheanum , terebrator , peticus , phaloniae,ancharus , tibiale and sosares); noyesi group (noyesi and probably undescribed relatedspecies); albipes group (albipes and coimbatorense); varicorne group (includingvaricorne and subalbicorne in one subclade, and aretas and fuscisquama in theother); thebe group (thebe and probably undescribed related species); exiguum group(exiguum and probably undescribed related species); floridanum group (floridanum ,primulum , transversum , truncatellum and agrotis). Host associations of the genusand host specificity of recognized groups are discussed. The current work offers afoundation for a comprehensive phylogeny of Copidosoma and the possibility toreconstruct cophylogeny between Copidosoma and their lepidopteran hosts.

Correspondence: Yan-Zhou Zhang, Institute of Zoology, ChineseAcademy of Sciences, 1 Beichen West Road, Chaoyang District,100101, Beijing, China. E-mail: [email protected]; Emilio Guerrieri,Istituto per la Protezione delle Piante, Consiglio Nazionale delleRicerche, Sez. Portici, Via Universita’ 133, 80055 Portici (NA), Italy.E-mail: [email protected]

Introduction

The genus Copidosoma was erected by Ratzeburg (1844) forthe species Copidosoma boucheanum (Hymenoptera: Chalci-doidea: Encyrtidae). It is a diverse and cosmopolitan groupcurrently with about 190 described species, developing asprimary parasitoids of lepidopteran caterpillars (Noyes, 2012).Several species of Copidosoma play a key role in the control of

© 2014 The Royal Entomological Society 1

2 F. Yu et al.

agricultural and forestry pests (Hain & Wallner, 1973; Guerrieri& Noyes, 2005). For example, Copidosoma floridanum (Ash-mead) was introduced into Hawaii in 1898 for the control ofChrysodeixis chalcites (Esper) (Noctuidae) (Swezey, 1931);Copidosoma koehleri Blanchard has been widely used forthe control of the potato tuber moth Phthorimaea poerculella(Zeller) (Gelechiidae) (e.g. Whiteside, 1980; Horne, 1990;Guerrieri, 1995). Copidosoma primulum (Mercet) (misidenti-fied as Copidosoma heliothis Liao) has been released in Chinaagainst Helicoverpa armigera (Hubner) (Noctuidae) in wheatfields (Li et al., 1996). Apart from potential biocontrol agents,species of Copidosoma are of great biological interest becauseof polyembryony (Grbic et al., 1992; Harvey et al., 2000).These polyembryonic parasitoids have evolved a caste systemin which embryos from the same egg develop into either repro-ductive wasps or soldier caste larvae (Donnell et al., 2004;Giron et al., 2004). Despite obligate polyembryony found inseveral animal groups, little is known about its origin and evo-lution in parasitoid wasps. Although not all host associationsof Copidosoma are reliable or even known, a total of 13 fam-ilies distributed among seven superfamilies of the Lepidopteraare reported so far (Guerrieri & Noyes, 2005). Nonetheless,some species of Copidosoma appear to exhibit quite strict hostspecificity.

In the most recent revision of Copidosoma (Guerrieri &Noyes, 2005), a detailed history of synonymies is reported.The most significant ones include Litomastix (Noyes & Hayat,1984; Hayat, 1986; Trjapitzin, 1989; Guerrieri & Noyes,2005; Zhang & Huang, 2007) and Paralitomastix (Kazmi &Hayat, 1998; Guerrieri & Noyes, 2005). A potential synonymywith Copidosomopsis Girault remains debatable. Besides thedifferent number of funicular segments (five in Copidosomop-sis , six in Copidosoma), Kazmi & Hayat (1998) suggestedthat females of the two genera also could be separated bythe shape of hypopygium. Guerrieri & Noyes (2005) notedthat this difference did not apply for at least two Europeanspecies of Copidosoma but retained Copidosomopsis as validgenus on the basis of the elongated and pear-shaped propodealspiracle found in some New World species. In his Ph.D. thesis,Zolnerowich (1995) proposed the synonymy of RaffaelliaGirault and Apsilophrys De Santis with Copidosoma , butthese two cannot be considered until formal publication.

Several regional revisions of Copidosoma were finishedover the last 20 years (Zolnerowich, 1995, North Americanspecies; Kazmi & Hayat, 1998, Indian species; Guerrieri &Noyes, 2005, European species; Zhang & Huang, 2007, Chi-nese species). Nevertheless, there is need of comprehensivephylogenetic analysis of this genus: so far, only one phylo-genetic study of the tribe Copidosomatini has been conductedbased on morphological characters (Zolnerowich, 1995) andanother on four Copidosoma species associated with Noctuidaewas carried out using 28S rDNA (Zhang et al., 2008).

In this work, in order to understand the evolutionaryrelationships of the species in the genus Copidosoma , weinfer a molecular phylogeny using 18S rDNA (18S ), the 28SrDNA (28S ) and the mitochondrial cytochrome c oxidase I(COI ) gene. These three genes have been frequently used

for phylogenetic reconstruction ranging from the superfamilyto the generic level in Chalcidoidea (Campbell et al., 2000;Gauthier et al., 2000; Heraty et al., 2004; Owen et al., 2007;Schmidt & Polaszek, 2007; Cruaud et al., 2010; Burks et al.,2011; Munro et al., 2011; Heraty et al., 2013). The aim ofthis preliminary phylogeny is to provide a framework fora more comprehensive phylogenetic analysis of the speciesof Copidosoma . The pattern of host use and host specificityrelated to the Lepidoptera is also discussed.

Materials and methods

Specimen handling

Specimens were obtained from laboratory rearing and fieldcollection (sweeping, yellow pan or Malaise trapping) (Noyes,1982). All samples were preserved in 95–100% ethanoland kept at −20◦C until DNA extraction. Identification wasperformed by the author Z.Y.Z. at species/genus level withthe aid of available keys and type material. The sequencedspecimens were deposited as voucher specimens in the Instituteof Zoology, Chinese Academy of Sciences, Beijing, China. Onthe basis of biology and currently accepted tribal classificationof Encyrtidae (Trjapitzin, 1973), the genus Ageniaspis waschosen as outgroup. A total of 58 taxa, including 56 ingrouptaxa and two outgroup taxa (Ageniaspis citricola and A. sp.),were used for phylogenetic analysis. Details of the sequencedspecimens and voucher information are listed in Table S1.

DNA extraction, PCR and sequencing

Parasitoids were removed from 95–100% ethanol and driedin open Eppendorf tubes prior to extraction. Genomic DNAwas extracted using the DNeasy Blood & Tissue Kit (Qia-gen GmbH, Hilden, Germany) following the manufacturer’sprotocols.

Primer sequences for PCR amplification of 18S , 28S andCOI are listed in Table S2. The 28S sequences (D2 expansionsegment) were generated using the primer pairs D2-3549and D2-4068 (Campbell et al., 1993), or D2-3566 (Gillespieet al., 2005a) and D2-4057 (Heraty et al., 2004). Partial18S sequences were amplified using the primer combinations18S_H17-35F and 18S_H17-35R (Heraty et al., 2004), or Saiand Sbi (Whiting et al., 1997). The PCR program for bothribosomal DNA genes was as follows: 3 min at 94◦C; 30 cyclesof 45 s at 94◦C, 45 s at 56◦C, 1 min at 72◦C; followed by afinal extension at 72◦C for 10 min. The COI gene fragmentwas amplified using the universal DNA barcoding primersLCO1490 and HCO2198 (Folmer et al., 1994). In some taxa,the primer FWPTF1 (Li et al., 2010) paired with Lep-R1(Hebert et al., 2004) was used to generate an approximately500-bp internal sequence. The PCR cycle program for COIfollowed Hebert et al. (2003).

Polymerase chain reactions (PCR) were carried out in50-μL reaction volumes using TaKaRa ExTaq Polymerasekits (TaKaRa, Dalian, China). Final volumes contained 5 μL

© 2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057

Phylogeny of Copidosoma 3

ten × Buffer, 25 mm MgCl2, 2.5 mm dNTP mixture, 10 pmolof each primer, 1 U of ExTaq and 5 μL genomic DNA. AllPCRs were performed on an Eppendorf Mastercycler gradient(Hamburg, Germany). Each PCR product was electrophoresedthrough 1% agarose gel and sequencing was performeddirectly from positive products on both directions usingBigDye v3.1 on an ABI 3730xl DNA Analyzer (AppliedBiosystems, Carlsbad, CA, USA). All sequences have beendeposited in GenBank (see Table S1 for accession numbers).

Sequence alignment and phylogenetic analysis

All nucleotide sequences were verified as Encyr-tidae using BLAST searches of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences of COI gene were alignedusing ClustalW implemented in Bioedit v7.1.3.0 (Hall, 1999)and translated into amino acid sequences using MEGA v4.0(Tamura et al., 2007) to test the presence of stop codons. Theribosomal DNA sequences were aligned manually using sec-ondary structure models following Gillespie et al. (2005a,b).Phylogenetic trees were reconstructed using the combineddataset of 28S , 18S and COI .

The parsimony analysis of the combined dataset wasconducted in TNT v1.1 (Goloboff et al., 2008) under NewTechnology Search. Gaps were coded as missing. Equallyweighted heuristic searches were performed employing 1000random addition sequence replicates with default sectorial,ratchet, drift and tree-fusing parameters. Nodal supports wereevaluated with 1000 standard bootstrap replicates.

For Bayesian analysis, the dataset included five partitions:28S , 18S , COI first codon positon, COI second codonposition, COI third codon position. The best-fitting model ofnucleotide substitution was selected for each partition usingjModelTest v2.1.3 (Darriba et al., 2012) based on the correctedAkaike information criterion (AICc). Bayesian analyses wereconducted using MrBayes v3.2.1 (Huelsenbeck & Ronquist,2001) consisting of two Markov chain Monte Carlo (MCMC)analyses run for 4 000 000 generations sampling trees every100 generations and using four chains and default priors.Convergence between the two runs was assessed using theaverage standard deviation of split frequencies (below 0.01).The two runs were combined after the removal of the first1 000 000 generations from each run as burn-in.

Results

Alignments

The final alignment of the combined dataset, including28S sequences of Copidosoma floridanum (AY599319) andC . truncatellum (AY599320) from GenBank, was 2158 bpin length with 864 parsimony-informative characters. Thenumbers of taxa and characters of each gene (total andparsimony informative), and the best-fitting model of eachpartition are summarized in Table S3.

Phylogenetic analysis

Parsimony analysis of the combined data resulted ina well-resolved phylogeny with several strongly supportedclades. The parsimony analysis yielded two most parsimonioustrees with a tree length of 4707 steps (Fig. 1).

Bayesian analysis of the combined dataset resulted in a well-resolved and strongly supported phylogenetic tree, regardlessof a few weak posterior probability (< 0.70) nodes and somecollapse in the more apical groups Clade IV, Clade V andClade VI (Fig. 2).

In both Bayesian and MP analysis, Copidosoma was notmonophyletic, with the examined species of Copidosomopsismonophyletic and nested within Copidosoma . Although therelationships between some deeper nodes were problematicand poorly supported, the following nine clades (Figs 1, 2)(= groups) are recognized:

Clade I: C. cervius , C . chalconotum , C . serricorne andC . sp. near notatum . This clade was strongly supportedas the sister group to Copidosomopsis + the remainingCopidosoma (Figs 1, 2).Clade II: all sampled species of the genus Copidosomop-sis , Co. nacoleiae, Co. meridionalis , and Co. sp. The sistergroup relationship between Copidosomopsis + the remain-ing Copidosoma had high support.Clade III: C . boucheanum , C . terebrator , C . peticus ,C . phaloniae, C . ancharus , C . tibiale, C . sosares andspecies near C . peticus . The similar internal relationshipswere retrieved in both analyses (Figs 1, 2).Clade IV: C . noyesi and two species near C. noyesi . Despitesister group to the Clade III and Clades V–IX in the MPanalysis (Fig. 1), this group was unresolved in the Bayesiananalysis (Fig. 2).Clade V: C. albipes , C. coimbatorense and C . sp. nearcoimbatorense. This clade is strongly supported as sistergroup to Clade VI in the Bayesian analysis (Fig. 2).Clade VI: C . varicorne, C . subalbicorne (both speciesformerly included in the genus Paralitomastix ),C . fuscisquama , C . aretas and five species nearC . subalbicorne. This clade was sister group to CladesVII–IX in the MP analysis (Fig. 1), but sister to Clade Vin the Bayesian analysis (Fig. 2).Clade VII: C . thebe, C . sp1 near thebe and C . sp2 nearthebe. This group was sister to the remaining clades andC . lucidum in both analyses.Clade VIII: C . exiguum , C . sp1 near exiguum and C . sp2near exiguum . This clade was supported as sister group toClade IX in the Bayesian analysis (Fig. 2), but sister toC . lucidum in the MP analysis.Clade IX: C . floridanum , C . primulum , C . transversum ,C . truncatellum , C . agrotis and three species nearC . agrotis . This group is the most apical clade in MPanalysis, but unsupported.

In both analyses, Clade I was the sister group to Copido-somopsis and the remaining Copidosoma , with strong support


4 F. Yu et al.

Fig. 1. Strict consensus of two most-parsimonious trees from the combined molecular dataset with equally weighted characters. Values abovethe branches indicate clade bootstrap support (> 50) using 1000 replicates. In taxon names, A. = Ageniaspis (out group), C. = Copidosoma ,Co. = Copidosomopsis . Grey bars indicate the monophyletic clades from I to IX.



Fig. 2. Bayesian tree of the partitioned dataset using a mixed model (4 million generations; burn-in = 1 million generations). Values above thebranches indicate posterior probabilities (≥ 0.50). Grey bars indicate the monophyletic clades from I to IX.


6 F. Yu et al.

(BS = 100, PP = 1.00). Copidosomopsis (Clade II) wasmonophyletic and strongly supported (BS = 92, PP = 1.00) assister group to the remaining Copidosoma . The topology ofthe remaining clades was inconsistent, depending on analyticalmethods. In MP analysis, the relationship between clades wasresolved but unsupported. In the Bayesian result Clade IIIwas strongly supported as sister to the remaining clades in thegenus (PP = 0.99), whereas Clade IV was sister group to CladeIII and Clades V–IX in the MP tree. Clade IV was unresolvedbecause of collapse in the Bayesian analysis, and Clade Vwas sister group to Clade VI with strong support (PP = 0.95).A sister relationship between Clade VII and remaining cladeswas consistent in both trees, but with moderate support onlyin the Bayesian analysis (PP = 0.80). Clade IX was sistergroup to Clade VIII in the Bayesian analysis (PP = 0.76), butsister to Clade VIII + C. lucidum in the MP result (Fig. 1).Additionally, the position of C . lucidum was unstable. On theone hand, C . lucidum was found sister to Clade VIII + CladeIX with poor support (PP = 0.61) in the Bayesian analysis,but on the other hand, C . lucidum appeared sister to CladeVIII without support in MP analysis.

Discussion

Phylogenetic relationships, taxonomic inferences and hostranges

Our phylogenetic analysis confirmed the synonymy of Lit-omastix and Paralitomastix with Copidosoma as suggestedin the previous revisions based on morphological characters(Kazmi & Hayat, 1998; Guerrieri & Noyes, 2005). Copidoso-mopsis was nested with strong support within Copidosoma inboth MP and Bayesian analyses. Given some considerations onthis controversial genus (Guerrieri & Noyes, 2005), we proposeCopidosomopsis syn.n. as a new synonymy of Copidosomaon the basis of our reconstructed phylogenetic relationships.We further propose the recognition of the following species-groups: cervius group (Clade I), nacoleiae group (Clade II),boucheanum group (Clade III), noyesi group, (Clade IV),albipes group (Clade V), varicorne group (Clade VI), thebegroup (Clade VII), exiguum group (Clade VIII) and floridanumgroup (Clade IX).

cervius group. The cervius group (Clade I) was recoveredas the most basal clade of Copidosoma in both analyses (Figs1, 2). Morphologically, this group has a number of distinctivefeatures, including forewing venation with postmarginal veinas long as stigmal vein, long antennal segments with clavatransversally truncated at the apex, similar thoracic sculptureconsisting of small rounded cells that are moderately deepon the mesoscutum and very superficial on scutellum, andmale genitalia with long parameres and digiti (Guerrieri& Noyes, 2005). Trjapitzin (1971, 1977) considered anelongate postmarginal vein as plesiomorphic in Encyrtidae;as the only species group showing this feature, the cerviusgroup confirmed this character assumption. Where known, the

biology of the species of this group also appears homogeneous,with species associated with Geometridae.

Within the cervius group, C . cervius was the sister groupof C. chalconotum + C. serricorne (BS = 99, PP = 1.00). Thegrouping of C . cervius , C. chalconotum + C. serricorne wasfirst proposed based on morphology by Guerrieri & Noyes(2005). Females of C . cervius can be readily separated fromthe other two species by the shorter length of the funicularsegments. Similarly, males of C . cervius can be separated bythe relatively longer digiti and the gently curved inner marginsof the parameres (sinuous in serricorne and chalconotum)(Guerrieri & Noyes, 2005). The sister species C . serricorneand C . chalconotum can be separated from each other by onlyslight but consistent differences in the shape of the apex of theovipositor sheaths (gonostyli) and in the relative length of theclava (Guerrieri & Noyes, 2005).

nacoleiae group. The examined species of Copidosomop-sis were monophyletic and nested within Copidosoma in bothanalyses, with robust support values. Copidosomopsis is verysimilar to Copidosoma (Noyes & Hayat, 1984; Kazmi &Hayat, 1998; Guerrieri & Noyes, 2005) and here we proposethis genus as a junior synonym of Copidosoma . Kazmi &Hayat (1998) separated Copidosomopsis from Copidosoma onthe basis of the shape of hypopygium in females and male gen-italia with sclerotized digiti without denticles and parameresreduced or absent. Subsequently, Guerrieri & Noyes (2005)noted that some of these characters were shared by Europeanspecies of Copidosoma; in fact, they found that Copidoso-mopsis could be kept separated from Copidosoma only forNew World species, showing a distinctive propodeal spira-cle elongate and pear-shaped. A molecular characterizationof these species would be of great help in assessing theircorrect taxonomic position because our analysis and biolog-ical features strongly support a formal synonymy of Copido-somopsis with Copidosoma . C. nacoleiae, C. plethorica andC. tanytmema , are indeed polyembryonic endoparasitoids ofLepidoptera, mainly Pyralidae and Tortricidae (Noyes & Hayat,1984; Yu et al., 2010).

boucheanum group. The boucheanum group was recoveredas sister group to Clades IV–IX (see Fig. 2) with strongsupport (PP = 0.99) in the Bayesian analysis. Overall, themales of this group share similar genitalia with phallobasenarrowing towards its base and parameres no longer than digiti,aedeagus long and slender, bilaterally concave and pointed atthe apex. Biologically, species of this group were associatedwith different lepidopteran families; including Gelechiidae,Depressariidae, Coleophoridae, Blastodacnidae and Tortricidae(see Fig. 3 and Appendix S1).

This group divided into two subclades in both anal-yses (BS = 100, PP = 1.00), including C. boucheanum ,C. terebrator , C . phaloniae, C. peticus and species nearC. peticus in one and C. ancharus , C. tibiale and C. sosaresin the other. In the first subclade, two species, C . ancharusand C . tibiale were recovered as sister species with strongsupport (BS = 100, PP = 1.00). The morphological characters



Fig. 3. Bayesian cladogram with each species group labelled in square brackets and host associations marked in family level by colour bars.For the ingroup, coloured branches represent species with known hosts and black branches stand for species of unknown host. In taxon names,A. = Ageniaspis (out group), C. = Copidosoma , Co. = Copidosomopsis .

are coherent with this result: females of ancharus and tibialeshare a similar antennal structure and a very superficialsculpture on scutellum, and can be separated on the relativelength of F1, gonostyli and exerted part of the ovipositor(Guerrieri and Noyes, 2005). In both analyses, the two specieswere found close to C . sosares (BS = 82, PP = 1.00).

In the second subclade, the species relationships were rel-atively stable in both MP and Bayesian analyses. Copido-soma phaloniae was close to C . peticus and species nearC . peticus . C. boucheanum was sister to C. terebrator , withstrong support (PP = 0.99) in Bayesian analysis, confirminga morphological closeness (Guerrieri & Noyes, 2005) in


8 F. Yu et al.

general habitus, thoracic sculpture and strongly exerted ovipos-itor. On the basis of morphological characters, we believe thatthe recently described species C. longicaudata Japoshvili &Guerrieri (Japoshvili et al., 2013) could reasonably fall withinthis subgroup.

noyesi group. In the Bayesian analysis, the noyesi group wasunresolved. However, the sister relationship between this groupand remaining ones was recovered in the MP analysis, althoughthis was not well supported (Fig. 1). Females of this groupare quite distinct, sharing very similar morphology particularlyfor body colour (thorax partially yellowish or yellow brown)(Kazmi & Hayat, 1998). Copidosoma noyesi was described byKazmi & Hayat (1998) from India. As one of the examinedspecies, C . sp2 near noyesi was collected in North China(Shanxi), further collections and characterizations are neededto understand the composition of this species group in theChinese fauna.

albipes group. This group was highly supported as sistergroup to varicorne group in the Bayesian analysis (Fig. 2).Morphologically, females of C. albipes are similar to those ofcoimbatorense for the shape of the sensorial part of clava atthe apex and the shallow sculpture on scutellum. Copidosomaalbipes was reported as a parasitoid of Anacampsis innocuella ,A. populella and Gelechia turpella (Lepidoptera: Gelechiidae)(Guerrieri & Noyes, 2005).

varicorne group. This group was identical in both analy-ses. It is split into two strongly supported lineages (PP = 1.00)in Bayesian analysis, with one represented by C . aretas ,C . fuscisquama and species near C . subalbicorne, and theother by C . varicorne and C . subalbicorne. In one lineage,C . aretas and C . fuscisquama were recovered as sister specieswith strong support (BS = 99, PP = 1.00). The morphologicalsimilarities between aretas and fuscisquama in antennal struc-ture, ovipositor, hypopygium of females and male genitaliaexplained their sister-species relationship in the trees. Further-more, both species are parasitoids of Tortricidae (Guerrieri &Noyes, 2005).

Our results corroborated the synonymy of Paralitomastixwith Copidosoma as suggested by different morphologicalrevisions of species (Kazmi & Hayat, 1998; Guerrieri & Noyes,2005). The genus Paralitomastix has been distinguished fromCopidosoma by the black and white flagellum and, to a lesserextent, by the elongated sculpture on scutellum (Noyes &Hayat, 1984; Kazmi & Hayat, 1998). Two species C . varicorneand C . subalbicorne, placed in previously Paralitomastix ,were recovered as sister with low posterior probability(PP = 0.77). Females of C . varicorne and C . subalbicorne canbe separated most easily on the colour of F5 (brown invaricorne, white in subalbicorne) (Guerrieri & Noyes, 2005).Biologically, C . varicorne and C . subalbicorne are reported asparasitoids of Gelechiidae.

thebe group. Our results suggest that this group could besister to the floridanum and exiguum species groups. Our

results extend the distribution of C. thebe to China; previouslyC . thebe has been reported only from European countries withunknown host (Guerrieri & Noyes, 2005). In North China(e.g. Beijing) and South China (e.g. Hainan) we have collectedsome species close to C. thebe that could well be placed inthis species group, suggesting that their exact distribution isnot fully known.

exiguum group. The exiguum group is sister to thefloridanum group in the Bayesian analysis (Fig. 2) and toC . lucidum in the MP analysis (Fig. 1). Kazmi & Hayat (1998)described Copidosoma exiguum from India as a parasitoid ofpod borer larva of Cassia tora . Many specimens collected inthe South of China (e.g. Yunnan) appeared morphologicallysimilar to exiguum and could be reasonably placed in thisgroup.

floridanum group. The position of the floridanum group wasrelatively consistent in both MP and Bayesian analyses. Itis split into strongly supported two lineages in the Bayesiantree (PP = 1.00), one including C. primulum , C. transversumand C . floridanum , and the other including C. truncatellum ,C . agrotis and species near C . agrotis . Species in this groupare morphologically very similar and frequently misidenti-fied. Only recently has it been possible to reliably sepa-rate C . floridanum and truncatellun (Noyes, 1988). In thisgroup, our phylogenetic results indicated that C . floridanum ,C. primulum and C. transversum shared a common ancestor,whereas C . truncatellum and C . agrotis are closer to each otherthan to species of the other subclade. Some morphologicaland biological considerations support this clade: its speciesshare a similar antennal and thoracic sculpture. Slight butconsistent differences can be found in the forewing venationand male genitalia between species of the two subclades. InC . floridanum , C. transversum and C . primulum , the marginalvein of the forewing is as long as the stigmal vein, whereas inC. truncatellum and C. agrotis it is distinctly shorter (Guerrieri& Noyes, 2005; Zhang & Huang, 2007). The male genitalia ofthe four species are also almost identical with phallobase nar-rowing proximally, digiti narrow and slender, and parameresreduced (Guerrieri & Noyes, 2005; Zhang & Huang, 2007).Differences also occur on the aedeagus with that of floridanumand primulum simple, whereas in truncatellum and agrotis it isapically bilaterally concave and pointed, with two button-likestructures in the apical third in addition to the spermatic pores(Guerrieri & Noyes, 2005; Zhang & Huang, 2007). Species ofthis group with confirmed host associations are polyembryonicendoparasitoids of Noctuidae.

Host specificity

All species of Copidosoma with known biology are pri-mary egg-larval endoparasitoids of Lepidoptera (Guerrieri& Noyes, 2005), ovipositing into the eggs of their hostsand emerging from their last larval instar. Within the tribeCopidosomatini, Ageniaspis , Copidosomopsis and Copido-soma have been reported to be polyembryonic parasitoids



of Lepidoptera. Ageniaspis is reported to attack Yponomeu-tidae, Nepticulidae and Gracillariidae (Noyes & Hayat, 1984).Species of Copidosoma are reported as parasitoids of mothsin 13 families belonging to seven superfamilies (Guerrieri &Noyes, 2005).

Each of the species groups recognized in this paper appearsto be associated with a restricted number of lepidopteranfamilies, if not to a single one (Fig. 3). The cervius group – themost basal clade in our analysis – attacks mainly Geometridae.In Europe, C . serricorne is reared from Larentiinae andEnnominae (Geometridae); C . cervius and C . chalconotum aregenerally reared from Larentiinae only (Guerrieri & Noyes,2005). Species of nacoleiae group (= Copidosomopsis) aremainly endoparasitoids of Pyralidae and Tortricidae (Noyes& Hayat, 1984). Although species of the boucheanumgroup are associated with different lepidopteran families(see Fig. 3 and Appendix S1 for details), they attackmembers of Gelechioidea, with the exception of C. phaloniaerecorded from larvae of Tortricidae (Zhang & Huang, 2007).Within the varicorne group, C . aretas and C . fuscisquama(belonging to subclade 1) are parasitoids of Tortricidae,whereas C . varicorne and C . subalbicorne (belonging tosubclade 2) are parasitoids of Gelechiidae. The large cladeof the floridanum group seems to comprise parasitoids ofNoctuidae exclusively, but each species is associated witha single subfamily (see Appendix S1 for details). Thisprobably reflects host-searching behaviour because the eggsof Plusiinae are laid well above ground level, whereas thoseof cutworms and hepialids are deposited at or near ground level(Noyes, 1988).

Our phylogenetic analysis has resulted in the first hypothesisfor the evolutionary relationships within the genus Copido-soma . However, additional taxonomic sampling will be neededto better resolve the phylogeny of this polyembryonic para-sitoid group. Because several groups of related species gener-ally attack hosts within the same family, these might indicatea certain level of coevolution with their hosts or perhaps rapiddiversification on related hosts (Guerrieri & Noyes, 2005). Acomprehensive phylogeny of Copidosoma will be a startingpoint for the analysis of the co-evolution of the genus andtheir lepidopteran hosts.

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article under the DOI reference:10.1111/syen.12057

Table S1. Specimens used in molecular phylogeneticanalysis with sequences GenBank accession numbers.

Table S2. List of primer sequences.

Table S3. Summary of number of taxa and characters, andsubstitution model for each partition.

Appendix S1. List of host association of species analysedin this study.

Acknowledgements

Thanks to R. Zhang, G. Zheng, F. Yuan and other kind indi-viduals for helping collect specimens. We are grateful to Prof.John M. Heraty, Department of Entomology, University of Cal-ifornia, Riverside, USA, for offering helpful suggestions onphylogenetic analysis and discussion. We sincerely thank threeanonymous referees for their comments on the manuscript.This project was supported by the National Natural ScienceFoundation of China (NSFC grant No. 31071950 and No.31272350), by Chinese Academy of Sciences (KSCX2-YW-NF-02) and partially by Project of the Department of Scienceand Technology of China (2012FY111100).

References

Burks, R.A., Heraty, J.M., Gebiola, M. & Hansson, C. (2011)Combined molecular and morphological phylogeny of Eulophidae(Hymenoptera: Chalcidoidea), with focus on the subfamily Ente-doninae. Cladistics , 27, 1–25.

Campbell, B.C., Steffen-Campbell, J.D. & Werren, J.H. (1993) Phy-logeny of the Nasonia species complex (Hymenoptera: Pteromal-idae) inferred from an internal transcribed spacer (ITS2) and 28SrDNA sequences. Insect Molecular Biology , 2, 225–237.

Campbell, B., Heraty, J.M., Rasplus, J.-Y., Chan, K., Steffen-Campbell, J. & Babcock, C. (2000) Molecular systematics of theChalcidoidea using 28S-D2 rDNA. The Hymenoptera: Evolution,Biodiversity and Biological Control (ed. by A.D. Austin and M.Dowton), pp. 57–71. CSIRO Publishing, Melbourne.

Cruaud, A., Jabbour-Zahab, R., Genson, G. et al. (2010) Laying thefoundations for a new classification of Agaonidae (Hymenoptera:Chalcidoidea), a multilocus phylogenetic approach. Cladistics , 26,359–387.

Darriba, D., Taboada, G.L., Doallo, R. & Posada, D. (2012) jModelTest2: more models, new heuristics and parallel computing. NatureMethods , 9, 772.

Donnell, D.M., Corley, L.S., Chen, G. & Strand, M.R. (2004) Castedetermination in a polyembryonic wasp involves inheritance of germcells. Proceedings of the National Academy of Science of the UnitedStates of America , 101, 10095–10100.

Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994)DNA primers for amplification of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates. MolecularMarine Biology and Biotechnology , 3, 294–299.

Gauthier, N., LaSalle, J., Quicke, D.L.J. & Godfray, H.C.J. (2000)Phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with areclassification of Eulophinae and the recognition that Elasmidaeare derived eulophids. Systematic Entomology , 25, 521–539.

Gillespie, J., Munro, J., Heraty, J., Yoder, M., Owen, A. & Carmichael,A. (2005a) A secondary structural model of the 28S rRNAexpansion segments D2 and D3 for chalcidoid wasps (Hymenoptera:Chalcidoidea). Molecular Biology and Evolution , 22, 1593–1608.

Gillespie, J.J., Yoder, M.J. & Wharton, R.A. (2005b) Predictedsecondary structure for 28S and 18S rRNA from Ichneumonoidea(Insecta: Hymenoptera: Apocrita): impact on sequence alignmentand phylogeny estimation. Journal of Molecular Evolution , 61,114–137.

Giron, D., Dunn, D., Hardy, I.C.W. & Strand, M.R. (2004) Aggressionby polyembryonic wasp soldiers correlates with kinship but notresource competition. Nature, 430, 676–679.

Goloboff, P.A., Farris, J.S. & Nixon, K.C. (2008) TNT, a free programfor phylogenetic analysis. Cladistics , 24, 774–786.


10 F. Yu et al.

Grbic, M., Ode, P.J. & Strand, M.R. (1992) Sibling rivalry and broodsex ratios in polyembryonic wasps. Nature, 360, 254–256.

Guerrieri, E. (1995) Influence of temperature on developmentand longevity of the adults of Copidosoma koehleri Blanchard(Hymenoptera: Encyrtidae), parasitoid of Phthorimaea operculella(Zeller) (Lepidoptera: Gelechiidae). Bollettino del Laboratorio diEntomologia agraria Filippo Silvestri , 50, 261–270.

Guerrieri, E. & Noyes, J.S. (2005) Revision of the European speciesof Copidosoma Ratzeburg (Hymenoptera: Encyrtidae), parasitoidsof caterpillars (Lepidoptera). Systematic Entomology , 30, 97–174.

Hain, F.B. & Wallner, W.E. (1973) The life history, biology, andparasites of the pine candle moth, Exoteleia nepheos (Lepidoptera:Gelechiidae), on Scotch pine in Michigan. Canadian Entomologist ,105, 157–164.

Hall, T.A. (1999) BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT.Nucleic Acids Symposium Series , 41, 95–98.

Harvey, J.A., Corley, L.S. & Strand, M.R. (2000) Competition inducesadaptive shifts in caste ratios of a polyembryonic wasp. Nature, 406,183–186.

Hayat, M. (1986) Family Encyrtidae. Oriental Insects , 20, 67–137.Hebert, P.D.N., Cywinska, A., Ball, S.L. & DeWaard, J.R. (2003)

Biological identifications through DNA barcodes. Proceedings ofthe Royal Society B , 270, 313–321.

Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H. & Hallwachs,W. (2004) Ten species in one: DNA barcoding reveals crypticspecies in the neotropical skipper butterfly Astraptes fulgerator .Proceedings of the National Academy of Science of the United Statesof America , 101, 14 812–14 817.

Heraty, J.M., Hawks, D.L., Kostecki, J.S. & Carmichael, A.C. (2004)Phylogeny and behaviour of the Gollumiellinae, a new subfamilyof the ant-parasitic Eucharitidae (Hymenoptera: Chalcidoidea).Systematic Entomology , 29, 544–559.

Heraty, J.M., Burks, R.A., Cruaud, A. et al. (2013) A phylogeneticanalysis of the megadiverse Chalcidoidea (Hymenoptera). Cladis-tics , 29, 466–542.

Horne, P.A. (1990) The influence of introduced parasitoids of thepotato moth, Phthorimaea operculella (Lepidoptera: Gelechiidae)in Victoria, Australia. Bulletin of Entomological Research , 80,159–163.

Huelsenbeck, J.P. & Ronquist, F. (2001) MrBayes: Bayesian inferenceof phylogenetic trees. Bioinformatics , 17, 754–755.

Japoshvili, G., Hansen, L.O. & Guerrieri, E. (2013) The Norwegianspecies of Copidosoma (Hymenoptera, Chalcidoidea, Encyrtidae).Zootaxa , 3619, 145–153.

Kazmi, S.I. & Hayat, M. (1998) Revision of the Indian Copidosomatini(Hymenoptera: Chalcidoidea: Encyrtidae). Oriental Insects , 32,287–362.

Li, W.J., Zhou, Y.F. & Liu, H.C. (1996) Effect of releasing Litomastixsp. in wheat fields for controlling a cotton bollworm population.Chinese Journal of Biological Control , 12, 43.

Li, Y.W., Zhou, X., Feng, G., Hu, H.Y., Niu, L.M., Hebert, P. &Huang, D.W. (2010) COI and ITS2 sequences delimit species,reveal cryptic taxa and host specificity of fig-associated Sycophila(Hymenoptera, Eurytomidae). Molecular Ecology Resources , 10,31–40.

Munro, J.B., Heraty, J.M., Burks, R.A. et al. (2011) A molecularphylogeny of the Chalcidoidea (Hymenoptera). PLoS ONE , 6,e27023.

Noyes, J.S. (1982) Collecting and preserving chalcid wasps(Hymenoptera: Chalcidoidea). Journal of Natural History , 16,315–334.

Noyes, J.S. (1988) Copidosoma truncatellum (Dalman) and C.floridanum (Ashmead) (Hymenoptera, Encyrtidae), two fre-quently misidentified polyembryonic parasitoids of caterpillars(Lepidoptera). Systematic Entomology , 13, 197–204.

Noyes, J.S. (2012) Universal Chalcidoidea Database [WWW docu-ment]. URL http://www.nhm.ac.uk/chalcidoids [accessed on 12 June2012].

Noyes, J.S. & Hayat, M. (1984) A review of the genera of Indo-Pacific Encyrtidae (Hymenoptera: Chalcidoidea). Bulletin of theBritish Museum of Natural History (Entomology), 48, 131–395.

Owen, A.K., George, J., Pinto, J.D. & Heraty, J.M. (2007) A molecularphylogeny of the Trichogrammatidae (Hymenoptera: Chalcidoidea),with an evaluation of the utility of their male genitalia for higherlevel classification. Systematic Entomology , 32, 227–251.

Ratzeburg, J.T.C. (1844) Die Ichneumonen der Forstinsekten inentomologischer und forstlicher Beziehung , Vol. 1. Nicolai’scheBuchhandlung, Berlin.

Schmidt, S. & Polaszek, A. (2007) Encarsia or Encar-siella? – redefining generic limits based on morphologicaland molecular evidence (Hymenoptera, Aphelinidae). SystematicEntomology , 32, 81–94.

Swezey, O.H. (1931) Litomastix floridana (Ashm.), a recent immigrantin Hawaii. Proceedings of the Hawaiian Entomological Society , 7,369–370, 390, 419–421.

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4: Molec-ular Evolutionary Genetics Analysis (MEGA) software version 4.0.Molecular Biology Evolution , 24, 1596–1599.

Trjapitzin, V.A. (1971) Problems of morphological evolution andclassification of the family Encyrtidae (Hymenoptera: Chalcidoidea).Proceedings, XIII International Congress of Entomology, Moscow ,1, 310–311.

Trjapitzin, V.A. (1973) Classification of the parasitic Hymenoptera ofthe family Encyrtidae (Chalcidoidea). Part II. Subfamily EncyrtinaeWalker, 1837. Entomologicheskoe Obozrenie, 52, 416–429.

Trjapitzin, V.A. (1977) The characteristic features of the morphol-ogy of adult encyrtids (Hymenoptera, Chalcidoidea, Encyrtidae) andtheir systematic significance. Trudy Vsesoyuznogo Entomologich-eskogo Obshchestva , 58, 145–199.

Trjapitzin, V.A. (1989) Parasitic Hymenoptera of the Fam. Encyrtidaeof Palaearctics. Opredeliteli po Faune SSSR, Vol. 158, pp. 1–489.Zoologicheskim Institutom Akademii Nauk SSR, Leningrad.

Whiteside, E.F. (1980) Biological control of the potato tuber moth(Phthorimaea operculella) in South Africa by two introducedparasites (Copidosoma koehleri and Apanteles subandinus). Journalof the Entomological Society of Southern Africa , 43, 239–256.

Whiting, M., Carpenter, J.C., Wheeler, Q.D. & Wheeler, W.C. (1997)The Strepsiptera problem: phylogeny of the holometabolous insectorders inferred from 18S and 28S ribosomal DNA sequences andmorphology. Systematic Biology , 46, 1–68.

Yu, F., Zhang, Y.Z., Zhu, C.D. & Tu, L.H. (2010) A taxonomicstudy of Chinese species of Copidosomopsis Girault (Hymenoptera:Encyrtidae). Zootaxa , 2490, 55–62.

Zhang, Y.Z. & Huang, D.W. (2007) Study of the Chinese speciesof Copidosoma (Hymenoptera: Encyrtidae). Insect Systematic &Evolution , 38, 105–119.

Zhang, Y.Z., Yu, F. & Zhu, C.D. (2008) A preliminary phylogeneticstudy of Copidosoma spp. (Hymenoptera: Encyrtidae) associatedwith Noctuidae (Lepidoptera) based on 28S rDNA D2 sequence.Acta Entomologica Sinica , 51, 992–996.

Zolnerowich, G. (1995) Systematics of the Copidosomatini: polyembry-onic parasites (Hymenoptera: Encyrtidae). PhD Thesis, Texas A&MUniversity, College Station, Texas.

Accepted 20 November 2013


Download - 2014 Preliminary phylogeny of the genus Copidosoma (Hymenoptera, Encyrtidae), polyembryonic parasitoids of Lepidoptera

Top Related