Preliminary Phylogeny of the Genus Copidosoma (Hymenoptera

Systematic Entomology (2014), 39, 325–334 DOI: 10.1111/syen.12057

Preliminary phylogeny of the genus Copidosoma (Hymenoptera, Encyrtidae), polyembryonic parasitoids of Lepidoptera FANG YU1,2, FU-QIANG CHEN1, SHEN-HORN YEN3, LI-HONG TU2, CHAO-DONG ZHU1, EMILIO GUERRIERI4,5 and YAN-ZHOU ZHANG1

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-Sen University, Kaohsiung, Taiwan, 4Istituto per la Protezione delle Piante, CNR, Portici (Napoli), Italy and 5Department of Life Sciences, The Natural History Museum, London, U.K.

Abstract. The genus Copidosoma (Hymenoptera: Chalcidoidea: Encyrtidae) is a diverse group of polyembryonic parasitoids of Lepidoptera, including species that have the potential to control agricultural and forestry pests. Moreover, some species of Copidosoma display polyembryony. Despite their economic and scientific importance, little is known about the phylogeny of Copidosoma and its relations to other groups of Encyrtidae. Here we infer the phylogenetic relationships of this genus from nucleotide sequences of two nuclear (18S and 28S ) and one mitochondrial (COI ) genes. Forty-four species of Copidosoma and three species of Copidosomopsis plus two outgroup species are included in Maximum Parsimony and Bayesian analyses. Copidosomopsis syn. n. is proposed as a junior synonym of Copidosoma based on phylogenetic analysis results. Each of nine identical clades, resulting from both analyses, is proposed as informal species group: cervius group (cervius, chalconotum and serricorne), recovered as the basal lineage of Copidosoma; nacoleiae group (nacoleaie, meridionalis and an undescribed species, formerly belonging to the genus Copidosomopsis); boucheanum group (boucheanum, terebrator, peticus, phaloniae, ancharus, tibiale and sosares); noyesi group (noyesi and probably undescribed related species); albipes group (albipes and coimbatorense); varicorne group (including varicorne and subalbicorne in one subclade, and aretas and fuscisquama in the other); 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 genus and host specificity of recognized groups are discussed. The current work offers a foundation for a comprehensive phylogeny of Copidosoma and the possibility to reconstruct cophylogeny between Copidosoma and their lepidopteran hosts.

Introduction

The genus Copidosoma was erected by Ratzeburg (1844) for Correspondence: Yan-Zhou Zhang, Institute of Zoology, Chinese the species Copidosoma boucheanum (Hymenoptera: Chalci- Academy of Sciences, 1 Beichen West Road, Chaoyang District, doidea: Encyrtidae). It is a diverse and cosmopolitan group 100101, Beijing, China. E-mail: [email protected]; Emilio Guerrieri, currently with about 190 described species, developing as Istituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Sez. Portici, Via Universita’ 133, 80055 Portici (NA), Italy. primary parasitoids of lepidopteran caterpillars (Noyes, 2012). E-mail: [email protected] Several species of Copidosoma play a key role in the control of

© 2014 The Royal Entomological Society 325 326 F. Yu et al. agricultural and forestry pests (Hain & Wallner, 1973; Guerrieri for phylogenetic reconstruction ranging from the superfamily & Noyes, 2005). For example, Copidosoma floridanum (Ash- to the generic level in Chalcidoidea (Campbell et al., 2000; mead) was introduced into Hawaii in 1898 for the control of Gauthier et al., 2000; Heraty et al., 2004; Owen et al., 2007; Chrysodeixis chalcites (Esper) (Noctuidae) (Swezey, 1931); Schmidt & Polaszek, 2007; Cruaud et al., 2010; Burks et al., Copidosoma koehleri Blanchard has been widely used for 2011; Munro et al., 2011; Heraty et al., 2013). The aim of the control of the potato tuber moth Phthorimaea poerculella this preliminary phylogeny is to provide a framework for (Zeller) (Gelechiidae) (e.g. Whiteside, 1980; Horne, 1990; a more comprehensive phylogenetic analysis of the species Guerrieri, 1995). Copidosoma primulum (Mercet) (misidenti- of Copidosoma. The pattern of host use and host specificity fied as Copidosoma heliothis Liao) has been released in China related to the Lepidoptera is also discussed. against Helicoverpa armigera (Hubner)¨ (Noctuidae) in wheat fields (Li et al., 1996). Apart from potential biocontrol agents, species of Copidosoma are of great biological interest because Materials and methods of polyembryony (Grbic´ et al., 1992; Harvey et al., 2000). Specimen handling These polyembryonic parasitoids have evolved a caste system in which embryos from the same egg develop into either repro- Specimens were obtained from laboratory rearing and field ductive wasps or soldier caste larvae (Donnell et al., 2004; collection (sweeping, yellow pan or Malaise trapping) (Noyes, Giron et al., 2004). Despite obligate polyembryony found in 1982). All samples were preserved in 95–100% ethanol several animal groups, little is known about its origin and evo- ◦ and kept at −20 C until DNA extraction. Identification was lution in parasitoid wasps. Although not all host associations performed by the author Z.Y.Z. at species/genus level with of Copidosoma are reliable or even known, a total of 13 fam- the aid of available keys and type material. The sequenced ilies distributed among seven superfamilies of the Lepidoptera specimens were deposited as voucher specimens in the Institute are reported so far (Guerrieri & Noyes, 2005). Nonetheless, of Zoology, Chinese Academy of Sciences, Beijing, China. On some species of Copidosoma appear to exhibit quite strict host the basis of biology and currently accepted tribal classification specificity. of Encyrtidae (Trjapitzin, 1973), the genus Ageniaspis was In the most recent revision of Copidosoma (Guerrieri & chosen as outgroup. A total of 58 taxa, including 56 ingroup Noyes, 2005), a detailed history of synonymies is reported. taxa and two outgroup taxa (Ageniaspis citricola and A. sp.), The most significant ones include Litomastix (Noyes & Hayat, were used for phylogenetic analysis. Details of the sequenced 1984; Hayat, 1986; Trjapitzin, 1989; Guerrieri & Noyes, specimens and voucher information are listed in Table S1. 2005; Zhang & Huang, 2007) and Paralitomastix (Kazmi & Hayat, 1998; Guerrieri & Noyes, 2005). A potential synonymy with Copidosomopsis Girault remains debatable. Besides the DNA extraction, PCR and sequencing different number of funicular segments (five in Copidosomop- sis, six in Copidosoma), Kazmi & Hayat (1998) suggested Parasitoids were removed from 95–100% ethanol and dried that females of the two genera also could be separated by in open Eppendorf tubes prior to extraction. Genomic DNA the shape of hypopygium. Guerrieri & Noyes (2005) noted was extracted using the DNeasy Blood & Tissue Kit (Qia- that this difference did not apply for at least two European gen GmbH, Hilden, Germany) following the manufacturer’s species of Copidosoma but retained Copidosomopsis as valid protocols. genus on the basis of the elongated and pear-shaped propodeal Primer sequences for PCR amplification of 18S , 28S and spiracle found in some New World species. In his Ph.D. thesis, COI are listed in Table S2. The 28S sequences (D2 expansion Zolnerowich (1995) proposed the synonymy of Raffaellia segment) were generated using the primer pairs D2-3549 Girault and Apsilophrys De Santis with Copidosoma, but and D2-4068 (Campbell et al., 1993), or D2-3566 (Gillespie these two cannot be considered until formal publication. et al., 2005a) and D2-4057 (Heraty et al., 2004). Partial Several regional revisions of Copidosoma were finished 18S sequences were amplified using the primer combinations over the last 20 years (Zolnerowich, 1995, North American 18S_H17-35F and 18S_H17-35R (Heraty et al., 2004), or Sai species; Kazmi & Hayat, 1998, Indian species; Guerrieri & and Sbi (Whiting et al., 1997). The PCR program for both ◦ Noyes, 2005, European species; Zhang & Huang, 2007, Chi- ribosomal DNA genes was as follows: 3 min at 94 C; 30 cycles ◦ ◦ ◦ nese species). Nevertheless, there is need of comprehensive of 45 s at 94 C, 45 s at 56 C, 1 min at 72 C;followedbya ◦ phylogenetic analysis of this genus: so far, only one phylo- final extension at 72 C for 10 min. The COI gene fragment genetic study of the tribe Copidosomatini has been conducted was amplified using the universal DNA barcoding primers based on morphological characters (Zolnerowich, 1995) and LCO1490 and HCO2198 (Folmer et al., 1994). In some taxa, another on four Copidosoma species associated with Noctuidae the primer FWPTF1 (Li et al., 2010) paired with Lep-R1 was carried out using 28S rDNA (Zhang et al., 2008). (Hebert et al., 2004) was used to generate an approximately In this work, in order to understand the evolutionary 500-bp internal sequence. The PCR cycle program for COI relationships of the species in the genus Copidosoma,we followed Hebert et al. (2003). infer a molecular phylogeny using 18S rDNA (18S ), the 28S Polymerase chain reactions (PCR) were carried out in rDNA (28S ) and the mitochondrial cytochrome c oxidase I 50-μL reaction volumes using TaKaRa ExTaq Polymerase (COI ) gene. These three genes have been frequently used kits (TaKaRa, Dalian, China). Final volumes contained 5 μL

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 325–334 Phylogeny of Copidosoma 327 ten × Buffer, 25 mm MgCl2, 2.5 mm dNTP mixture, 10 pmol Phylogenetic analysis of each primer, 1 U of ExTaq and 5 μL genomic DNA. All PCRs were performed on an Eppendorf Mastercycler gradient Parsimony analysis of the combined data resulted in (Hamburg, Germany). Each PCR product was electrophoresed a well-resolved phylogeny with several strongly supported through 1% agarose gel and sequencing was performed clades. The parsimony analysis yielded two most parsimonious directly from positive products on both directions using trees with a tree length of 4707 steps (Fig. 1). BigDye v3.1 on an ABI 3730xl DNA Analyzer (Applied Bayesian analysis of the combined dataset resulted in a well- Biosystems, Carlsbad, CA, USA). All sequences have been resolved and strongly supported phylogenetic tree, regardless deposited in GenBank (see Table S1 for accession numbers). of a few weak posterior probability (< 0.70) nodes and some collapse in the more apical groups Clade IV, Clade V and Clade VI (Fig. 2). Sequence alignment and phylogenetic analysis In both Bayesian and MP analysis, Copidosoma was not monophyletic, with the examined species of Copidosomopsis All nucleotide sequences were verified as Encyr- monophyletic and nested within Copidosoma. Although the tidae using BLAST searches of NCBI (http://blast.ncbi. relationships between some deeper nodes were problematic nlm.nih.gov/Blast.cgi). Sequences of COI gene were aligned and poorly supported, the following nine clades (Figs 1, 2) using ClustalW implemented in Bioedit v7.1.3.0 (Hall, 1999) (= groups) are recognized: and translated into amino acid sequences using MEGA v4.0 (Tamura et al., 2007) to test the presence of stop codons. The Clade I: C. cervius, C . chalconotum, C . serricorne and ribosomal DNA sequences were aligned manually using sec- C . sp. near notatum. This clade was strongly supported ondary structure models following Gillespie et al. (2005a,b). as the sister group to Copidosomopsis + the remaining Phylogenetic trees were reconstructed using the combined Copidosoma (Figs 1, 2). dataset of 28S , 18S and COI . Clade II: all sampled species of the genus Copidosomop- The parsimony analysis of the combined dataset was sis, Co. nacoleiae, Co. meridionalis,andCo. sp. The sister conducted in TNT v1.1 (Goloboff et al., 2008) under New group relationship between Copidosomopsis + the remain- Technology Search. Gaps were coded as missing. Equally ing Copidosoma had high support. weighted heuristic searches were performed employing 1000 Clade III: C . boucheanum, C . terebrator, C . peticus, random addition sequence replicates with default sectorial, C . phaloniae, C . ancharus, C . tibiale, C . sosares and ratchet, drift and tree-fusing parameters. Nodal supports were species near C . peticus. The similar internal relationships evaluated with 1000 standard bootstrap replicates. were retrieved in both analyses (Figs 1, 2). For Bayesian analysis, the dataset included five partitions: Clade IV: C . noyesi and two species near C. noyesi. Despite 28S , 18S , COI first codon positon, COI second codon sister group to the Clade III and Clades V–IX in the MP position, COI third codon position. The best-fitting model of analysis (Fig. 1), this group was unresolved in the Bayesian nucleotide substitution was selected for each partition using analysis (Fig. 2). jModelTest v2.1.3 (Darriba et al., 2012) based on the corrected Clade V: C. albipes, C. coimbatorense and C . sp. near Akaike information criterion (AICc). Bayesian analyses were coimbatorense. This clade is strongly supported as sister conducted using MrBayes v3.2.1 (Huelsenbeck & Ronquist, group to Clade VI in the Bayesian analysis (Fig. 2). 2001) consisting of two Markov chain Monte Carlo (MCMC) Clade VI: C . varicorne, C . subalbicorne (both species analyses run for 4 000 000 generations sampling trees every formerly included in the genus Paralitomastix), 100 generations and using four chains and default priors. C . fuscisquama, C . aretas and five species near Convergence between the two runs was assessed using the C . subalbicorne. This clade was sister group to Clades average standard deviation of split frequencies (below 0.01). VII–IX in the MP analysis (Fig. 1), but sister to Clade V The two runs were combined after the removal of the first in the Bayesian analysis (Fig. 2). 1 000 000 generations from each run as burn-in. Clade VII: C . thebe, C . sp1 near thebe and C . sp2 near thebe. This group was sister to the remaining clades and C . lucidum in both analyses. Results Clade VIII: C . exiguum, C . sp1 near exiguum and C .sp2 near exiguum. This clade was supported as sister group to Alignments Clade IX in the Bayesian analysis (Fig. 2), but sister to C . lucidum in the MP analysis. The final alignment of the combined dataset, including Clade IX: C . floridanum, C . primulum, C . transversum, 28S sequences of Copidosoma floridanum (AY599319) and C . truncatellum, C . agrotis and three species near C . truncatellum (AY599320) from GenBank, was 2158 bp C . agrotis. This group is the most apical clade in MP in length with 864 parsimony-informative characters. The analysis, but unsupported. numbers of taxa and characters of each gene (total and parsimony informative), and the best-fitting model of each In both analyses, Clade I was the sister group to Copido- partition are summarized in Table S3. somopsis and the remaining Copidosoma, with strong support

Fig. 1. Strict consensus of two most-parsimonious trees from the combined molecular dataset with equally weighted characters. Values above the 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 the branches indicate posterior probabilities (≥ 0.50). Grey bars indicate the monophyletic clades from I to IX.

(BS = 100, PP = 1.00). Copidosomopsis (Clade II) was biology of the species of this group also appears homogeneous, monophyletic and strongly supported (BS = 92, PP = 1.00) as with species associated with Geometridae. sister group to the remaining Copidosoma. The topology of Within the cervius group, C . cervius was the sister group the remaining clades was inconsistent, depending on analytical of C. chalconotum + C. serricorne (BS = 99, PP = 1.00). The methods. In MP analysis, the relationship between clades was grouping of C . cervius, C. chalconotum + C. serricorne was resolved but unsupported. In the Bayesian result Clade III first proposed based on morphology by Guerrieri & Noyes was strongly supported as sister to the remaining clades in the (2005). Females of C . cervius can be readily separated from genus (PP = 0.99), whereas Clade IV was sister group to Clade the other two species by the shorter length of the funicular III and Clades V–IX in the MP tree. Clade IV was unresolved segments. Similarly, males of C . cervius can be separated by because of collapse in the Bayesian analysis, and Clade V the relatively longer digiti and the gently curved inner margins was sister group to Clade VI with strong support (PP = 0.95). of the parameres (sinuous in serricorne and chalconotum) A sister relationship between Clade VII and remaining clades (Guerrieri & Noyes, 2005). The sister species C . serricorne was consistent in both trees, but with moderate support only and C . chalconotum can be separated from each other by only in the Bayesian analysis (PP = 0.80). Clade IX was sister slight but consistent differences in the shape of the apex of the group to Clade VIII in the Bayesian analysis (PP = 0.76), but ovipositor sheaths (gonostyli) and in the relative length of the sister to Clade VIII + C. lucidum in the MP result (Fig. 1). clava (Guerrieri & Noyes, 2005). Additionally, the position of C . lucidum was unstable. On the one hand, C . lucidum was found sister to Clade VIII + Clade nacoleiae group. The examined species of Copidosomop- IX with poor support (PP = 0.61) in the Bayesian analysis, sis were monophyletic and nested within Copidosoma in both but on the other hand, C . lucidum appeared sister to Clade analyses, with robust support values. Copidosomopsis is very VIII without support in MP analysis. similar to Copidosoma (Noyes & Hayat, 1984; Kazmi & Hayat, 1998; Guerrieri & Noyes, 2005) and here we propose this genus as a junior synonym of Copidosoma.Kazmi& Discussion Hayat (1998) separated Copidosomopsis from Copidosoma on the basis of the shape of hypopygium in females and male gen- Phylogenetic relationships, taxonomic inferences and host italia with sclerotized digiti without denticles and parameres ranges reduced or absent. Subsequently, Guerrieri & Noyes (2005) noted that some of these characters were shared by European Our phylogenetic analysis confirmed the synonymy of Lit- species of Copidosoma; in fact, they found that Copidoso- omastix and Paralitomastix with Copidosoma as suggested mopsis could be kept separated from Copidosoma only for in the previous revisions based on morphological characters New World species, showing a distinctive propodeal spira- (Kazmi & Hayat, 1998; Guerrieri & Noyes, 2005). Copidoso- cle elongate and pear-shaped. A molecular characterization mopsis was nested with strong support within Copidosoma in of these species would be of great help in assessing their both MP and Bayesian analyses. Given some considerations on correct taxonomic position because our analysis and biolog- this controversial genus (Guerrieri & Noyes, 2005), we propose ical features strongly support a formal synonymy of Copido- Copidosomopsis syn.n. as a new synonymy of Copidosoma somopsis with Copidosoma. C. nacoleiae, C. plethorica and on the basis of our reconstructed phylogenetic relationships. C. tanytmema, are indeed polyembryonic endoparasitoids of We further propose the recognition of the following species- Lepidoptera, mainly Pyralidae and Tortricidae (Noyes & Hayat, groups: cervius group (Clade I), nacoleiae group (Clade II), 1984; Yu et al., 2010). boucheanum group (Clade III), noyesi group, (Clade IV), albipes group (Clade V), varicorne group (Clade VI), thebe boucheanum group. The boucheanum group was recovered group (Clade VII), exiguum group (Clade VIII) and floridanum as sister group to Clades IV–IX (see Fig. 2) with strong group (Clade IX). support (PP = 0.99) in the Bayesian analysis. Overall, the males of this group share similar genitalia with phallobase cervius group. The cervius group (Clade I) was recovered narrowing towards its base and parameres no longer than digiti, as the most basal clade of Copidosoma in both analyses (Figs aedeagus long and slender, bilaterally concave and pointed at 1, 2). Morphologically, this group has a number of distinctive the apex. Biologically, species of this group were associated features, including forewing venation with postmarginal vein with different lepidopteran families; including Gelechiidae, as long as stigmal vein, long antennal segments with clava Depressariidae, Coleophoridae, Blastodacnidae and Tortricidae transversally truncated at the apex, similar thoracic sculpture (see Fig. 3 and Appendix S1). consisting of small rounded cells that are moderately deep This group divided into two subclades in both anal- on the mesoscutum and very superficial on scutellum, and yses (BS = 100, PP = 1.00), including C. boucheanum, male genitalia with long parameres and digiti (Guerrieri C. terebrator, C . phaloniae, C. peticus and species near & Noyes, 2005). Trjapitzin (1971, 1977) considered an C. peticus in one and C. ancharus, C. tibiale and C. sosares elongate postmarginal vein as plesiomorphic in Encyrtidae; in the other. In the first subclade, two species, C . ancharus as the only species group showing this feature, the cervius and C . tibiale were recovered as sister species with strong group confirmed this character assumption. Where known, the support (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 tibiale In the second subclade, the species relationships were rel- share a similar antennal structure and a very superﬁcial atively stable in both MP and Bayesian analyses. Copido- sculpture on scutellum, and can be separated on the relative soma phaloniae was close to C . peticus and species near length of F1, gonostyli and exerted part of the ovipositor C . peticus. C. boucheanum was sister to C. terebrator, with (Guerrieri and Noyes, 2005). In both analyses, the two species strong support (PP = 0.99) in Bayesian analysis, conﬁrming were found close to C . sosares (BS = 82, PP = 1.00). a morphological closeness (Guerrieri & Noyes, 2005) in

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 325–334 332 F. Yu et al. general habitus, thoracic sculpture and strongly exerted ovipos- results extend the distribution of C. thebe to China; previously itor. On the basis of morphological characters, we believe that C . thebe has been reported only from European countries with the recently described species C. longicaudata Japoshvili & unknown host (Guerrieri & Noyes, 2005). In North China Guerrieri (Japoshvili et al., 2013) could reasonably fall within (e.g. Beijing) and South China (e.g. Hainan) we have collected this subgroup. some species close to C. thebe that could well be placed in this species group, suggesting that their exact distribution is noyesi group. In the Bayesian analysis, the noyesi group was not fully known. unresolved. However, the sister relationship between this group and remaining ones was recovered in the MP analysis, although exiguum group. The exiguum group is sister to the this was not well supported (Fig. 1). Females of this group floridanum group in the Bayesian analysis (Fig. 2) and to are quite distinct, sharing very similar morphology particularly C . lucidum in the MP analysis (Fig. 1). Kazmi & Hayat (1998) for body colour (thorax partially yellowish or yellow brown) described Copidosoma exiguum from India as a parasitoid of (Kazmi & Hayat, 1998). Copidosoma noyesi was described by pod borer larva of Cassia tora. Many specimens collected in Kazmi & Hayat (1998) from India. As one of the examined the South of China (e.g. Yunnan) appeared morphologically species, C . sp2 near noyesi was collected in North China similar to exiguum and could be reasonably placed in this (Shanxi), further collections and characterizations are needed group. to understand the composition of this species group in the Chinese fauna. floridanum group. The position of the floridanum group was relatively consistent in both MP and Bayesian analyses. It albipes group. This group was highly supported as sister is split into strongly supported two lineages in the Bayesian = group to varicorne group in the Bayesian analysis (Fig. 2). tree (PP 1.00), one including C. primulum, C. transversum Morphologically, females of C. albipes are similar to those of and C . floridanum, and the other including C. truncatellum, coimbatorense for the shape of the sensorial part of clava at C . agrotis and species near C . agrotis. Species in this group the apex and the shallow sculpture on scutellum. Copidosoma are morphologically very similar and frequently misidenti- albipes was reported as a parasitoid of Anacampsis innocuella, fied. Only recently has it been possible to reliably sepa- A. populella and Gelechia turpella (Lepidoptera: Gelechiidae) rate C . floridanum and truncatellun (Noyes, 1988). In this (Guerrieri & Noyes, 2005). group, our phylogenetic results indicated that C . floridanum, C. primulum and C. transversum shared a common ancestor, varicorne group. This group was identical in both analy- whereas C . truncatellum and C . agrotis are closer to each other ses. It is split into two strongly supported lineages (PP = 1.00) than to species of the other subclade. Some morphological in Bayesian analysis, with one represented by C . aretas, and biological considerations support this clade: its species C . fuscisquama and species near C . subalbicorne, and the share a similar antennal and thoracic sculpture. Slight but other by C . varicorne and C . subalbicorne. In one lineage, consistent differences can be found in the forewing venation C . aretas and C . fuscisquama were recovered as sister species and male genitalia between species of the two subclades. In with strong support (BS = 99, PP = 1.00). The morphological C . floridanum, C. transversum and C . primulum, the marginal similarities between aretas and fuscisquama in antennal struc- vein of the forewing is as long as the stigmal vein, whereas in ture, ovipositor, hypopygium of females and male genitalia C. truncatellum and C. agrotis it is distinctly shorter (Guerrieri explained their sister-species relationship in the trees. Further- & Noyes, 2005; Zhang & Huang, 2007). The male genitalia of more, both species are parasitoids of Tortricidae (Guerrieri & the four species are also almost identical with phallobase nar- Noyes, 2005). rowing proximally, digiti narrow and slender, and parameres Our results corroborated the synonymy of Paralitomastix reduced (Guerrieri & Noyes, 2005; Zhang & Huang, 2007). with Copidosoma as suggested by different morphological Differences also occur on the aedeagus with that of floridanum revisions of species (Kazmi & Hayat, 1998; Guerrieri & Noyes, and primulum simple, whereas in truncatellum and agrotis it is 2005). The genus Paralitomastix has been distinguished from apically bilaterally concave and pointed, with two button-like Copidosoma by the black and white flagellum and, to a lesser structures in the apical third in addition to the spermatic pores extent, by the elongated sculpture on scutellum (Noyes & (Guerrieri & Noyes, 2005; Zhang & Huang, 2007). Species of Hayat, 1984; Kazmi & Hayat, 1998). Two species C . varicorne this group with confirmed host associations are polyembryonic and C . subalbicorne, placed in previously Paralitomastix, endoparasitoids of Noctuidae. were recovered as sister with low posterior probability (PP = 0.77). Females of C . varicorne and C . subalbicorne can Host specificity be separated most easily on the colour of F5 (brown in varicorne, white in subalbicorne) (Guerrieri & Noyes, 2005). All species of Copidosoma with known biology are pri- Biologically, C . varicorne and C . subalbicorne are reported as mary egg-larval endoparasitoids of Lepidoptera (Guerrieri parasitoids of Gelechiidae. & Noyes, 2005), ovipositing into the eggs of their hosts and emerging from their last larval instar. Within the tribe thebe group. Our results suggest that this group could be Copidosomatini, Ageniaspis, Copidosomopsis and Copido- sister to the floridanum and exiguum species groups. Our soma have been reported to be polyembryonic parasitoids

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 325–334 Phylogeny of Copidosoma 333 of Lepidoptera. Ageniaspis is reported to attack Yponomeu- Acknowledgements tidae, Nepticulidae and Gracillariidae (Noyes & Hayat, 1984). Species of Copidosoma are reported as parasitoids of moths Thanks to R. Zhang, G. Zheng, F. Yuan and other kind indi- in 13 families belonging to seven superfamilies (Guerrieri & viduals for helping collect specimens. We are grateful to Prof. Noyes, 2005). John M. Heraty, Department of Entomology, University of Cal- Each of the species groups recognized in this paper appears ifornia, Riverside, USA, for offering helpful suggestions on to be associated with a restricted number of lepidopteran phylogenetic analysis and discussion. We sincerely thank three families, if not to a single one (Fig. 3). The cervius group – the anonymous referees for their comments on the manuscript. most basal clade in our analysis – attacks mainly Geometridae. This project was supported by the National Natural Science In Europe, C . serricorne is reared from Larentiinae and Foundation of China (NSFC grant No. 31071950 and No. Ennominae (Geometridae); C . cervius and C . chalconotum are 31272350), by Chinese Academy of Sciences (KSCX2-YW- generally reared from Larentiinae only (Guerrieri & Noyes, NF-02) and partially by Project of the Department of Science 2005). Species of nacoleiae group (= Copidosomopsis)are and Technology of China (2012FY111100). mainly endoparasitoids of Pyralidae and Tortricidae (Noyes & Hayat, 1984). Although species of the boucheanum References group are associated with different lepidopteran families (see Fig. 3 and Appendix S1 for details), they attack Burks, R.A., Heraty, J.M., Gebiola, M. & Hansson, C. (2011) members of Gelechioidea, with the exception of C. phaloniae Combined molecular and morphological phylogeny of Eulophidae recorded from larvae of Tortricidae (Zhang & Huang, 2007). 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