Systematic Entomology (2014), DOI: 10.1111/syen.12057
Preliminary phylogeny of the genus Copidosoma (Hymenoptera, Encyrtidae), polyembryonic parasitoids of Lepidoptera F A N G Y U 1,2 , F U - Q I A N G C H E N 1 , S H E N - H O R N Y E N 3 , L I - H O N G T U 2 , C H A O - D O N G Z H U 1 , E M I L I O G U E R R I E R I 4,5 and Y A N - Z H O U Z H ANG1 1
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, School of Life Sciences, Capital Normal University, Beijing, China, 3 Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, 4 Istituto per la Protezione delle Piante, CNR, Portici (Napoli), Italy and 5 Department of Life Sciences, The Natural History Museum, London, U.K. 2
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
Correspondence: Yan-Zhou Zhang, Institute of Zoology, Chinese Academy 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 delle Ricerche, Sez. Portici, Via Universita’ 133, 80055 Portici (NA), Italy. E-mail:
[email protected] 2014 The Royal Entomological Society
The genus Copidosoma was erected by Ratzeburg (1844) for the species Copidosoma boucheanum (Hymenoptera: Chalcidoidea: Encyrtidae). It is a diverse and cosmopolitan group currently with about 190 described species, developing as primary parasitoids of lepidopteran caterpillars (Noyes, 2012). Several species of Copidosoma play a key role in the control of 1
2 F. Yu et al. agricultural and forestry pests (Hain & Wallner, 1973; Guerrieri & Noyes, 2005). For example, Copidosoma floridanum (Ashmead) was introduced into Hawaii in 1898 for the control of Chrysodeixis chalcites (Esper) (Noctuidae) (Swezey, 1931); Copidosoma koehleri Blanchard has been widely used for the control of the potato tuber moth Phthorimaea poerculella (Zeller) (Gelechiidae) (e.g. Whiteside, 1980; Horne, 1990; Guerrieri, 1995). Copidosoma primulum (Mercet) (misidentified as Copidosoma heliothis Liao) has been released in China against Helicoverpa armigera (H¨ubner) (Noctuidae) in wheat fields (Li et al., 1996). Apart from potential biocontrol agents, species of Copidosoma are of great biological interest because of polyembryony (Grbi´c et al., 1992; Harvey et al., 2000). These polyembryonic parasitoids have evolved a caste system in which embryos from the same egg develop into either reproductive wasps or soldier caste larvae (Donnell et al., 2004; Giron et al., 2004). Despite obligate polyembryony found in several animal groups, little is known about its origin and evolution in parasitoid wasps. Although not all host associations of Copidosoma are reliable or even known, a total of 13 families distributed among seven superfamilies of the Lepidoptera are reported so far (Guerrieri & Noyes, 2005). Nonetheless, some species of Copidosoma appear to exhibit quite strict host specificity. 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 synonymy with Copidosomopsis Girault remains debatable. Besides the different number of funicular segments (five in Copidosomopsis, six in Copidosoma), Kazmi & Hayat (1998) suggested that females of the two genera also could be separated by the shape of hypopygium. Guerrieri & Noyes (2005) noted that this difference did not apply for at least two European species of Copidosoma but retained Copidosomopsis as valid genus on the basis of the elongated and pear-shaped propodeal spiracle found in some New World species. In his Ph.D. thesis, Zolnerowich (1995) proposed the synonymy of Raffaellia Girault and Apsilophrys De Santis with Copidosoma, but these two cannot be considered until formal publication. Several regional revisions of Copidosoma were finished over the last 20 years (Zolnerowich, 1995, North American species; Kazmi & Hayat, 1998, Indian species; Guerrieri & Noyes, 2005, European species; Zhang & Huang, 2007, Chinese species). Nevertheless, there is need of comprehensive phylogenetic analysis of this genus: so far, only one phylogenetic study of the tribe Copidosomatini has been conducted based on morphological characters (Zolnerowich, 1995) and another on four Copidosoma species associated with Noctuidae was carried out using 28S rDNA (Zhang et al., 2008). In this work, in order to understand the evolutionary relationships of the species in the genus Copidosoma, we infer a molecular phylogeny using 18S rDNA (18S ), the 28S rDNA (28S ) and the mitochondrial cytochrome c oxidase I (COI ) gene. These three genes have been frequently used
for phylogenetic reconstruction ranging from the superfamily to 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 of this preliminary phylogeny is to provide a framework for a more comprehensive phylogenetic analysis of the species of Copidosoma. The pattern of host use and host specificity related to the Lepidoptera is also discussed. Materials and methods Specimen handling Specimens were obtained from laboratory rearing and field collection (sweeping, yellow pan or Malaise trapping) (Noyes, 1982). All samples were preserved in 95–100% ethanol and kept at −20◦ C until DNA extraction. Identification was performed by the author Z.Y.Z. at species/genus level with the aid of available keys and type material. The sequenced specimens were deposited as voucher specimens in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. On the basis of biology and currently accepted tribal classification of Encyrtidae (Trjapitzin, 1973), the genus Ageniaspis was chosen as outgroup. A total of 58 taxa, including 56 ingroup taxa and two outgroup taxa (Ageniaspis citricola and A. sp.), were used for phylogenetic analysis. Details of the sequenced specimens and voucher information are listed in Table S1. DNA extraction, PCR and sequencing Parasitoids were removed from 95–100% ethanol and dried in open Eppendorf tubes prior to extraction. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s protocols. Primer sequences for PCR amplification of 18S , 28S and COI are listed in Table S2. The 28S sequences (D2 expansion segment) were generated using the primer pairs D2-3549 and D2-4068 (Campbell et al., 1993), or D2-3566 (Gillespie et al., 2005a) and D2-4057 (Heraty et al., 2004). Partial 18S sequences were amplified using the primer combinations 18S_H17-35F and 18S_H17-35R (Heraty et al., 2004), or Sai and Sbi (Whiting et al., 1997). The PCR program for both ribosomal DNA genes was as follows: 3 min at 94◦ C; 30 cycles of 45 s at 94◦ C, 45 s at 56◦ C, 1 min at 72◦ C; followed by a final extension at 72◦ C for 10 min. The COI gene fragment was amplified using the universal DNA barcoding primers LCO1490 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 approximately 500-bp internal sequence. The PCR cycle program for COI followed Hebert et al. (2003). Polymerase chain reactions (PCR) were carried out in 50-µL reaction volumes using TaKaRa ExTaq Polymerase kits (TaKaRa, Dalian, China). Final volumes contained 5 µL
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
Phylogeny of Copidosoma ten × Buffer, 25 mm MgCl2 , 2.5 mm dNTP mixture, 10 pmol of each primer, 1 U of ExTaq and 5 µL genomic DNA. All PCRs were performed on an Eppendorf Mastercycler gradient (Hamburg, Germany). Each PCR product was electrophoresed through 1% agarose gel and sequencing was performed directly from positive products on both directions using BigDye v3.1 on an ABI 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). All sequences have been deposited in GenBank (see Table S1 for accession numbers). Sequence alignment and phylogenetic analysis All nucleotide sequences were verified as Encyrtidae using BLAST searches of NCBI (http://blast.ncbi. nlm.nih.gov/Blast.cgi). Sequences of COI gene were aligned using 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. The ribosomal DNA sequences were aligned manually using secondary structure models following Gillespie et al. (2005a,b). Phylogenetic trees were reconstructed using the combined dataset of 28S , 18S and COI . The parsimony analysis of the combined dataset was conducted in TNT v1.1 (Goloboff et al., 2008) under New Technology Search. Gaps were coded as missing. Equally weighted heuristic searches were performed employing 1000 random addition sequence replicates with default sectorial, ratchet, drift and tree-fusing parameters. Nodal supports were evaluated with 1000 standard bootstrap replicates. For Bayesian analysis, the dataset included five partitions: 28S , 18S , COI first codon positon, COI second codon position, COI third codon position. The best-fitting model of nucleotide substitution was selected for each partition using jModelTest v2.1.3 (Darriba et al., 2012) based on the corrected Akaike information criterion (AICc). Bayesian analyses were conducted 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 every 100 generations and using four chains and default priors. Convergence between the two runs was assessed using the average standard deviation of split frequencies (below 0.01). The two runs were combined after the removal of the first 1 000 000 generations from each run as burn-in.
Results Alignments The final alignment of the combined dataset, including 28S sequences of Copidosoma floridanum (AY599319) and C . truncatellum (AY599320) from GenBank, was 2158 bp in length with 864 parsimony-informative characters. The numbers of taxa and characters of each gene (total and parsimony informative), and the best-fitting model of each partition are summarized in Table S3.
3
Phylogenetic analysis Parsimony analysis of the combined data resulted in a well-resolved phylogeny with several strongly supported clades. The parsimony analysis yielded two most parsimonious trees with a tree length of 4707 steps (Fig. 1). Bayesian analysis of the combined dataset resulted in a wellresolved and strongly supported phylogenetic tree, regardless 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). In both Bayesian and MP analysis, Copidosoma was not monophyletic, with the examined species of Copidosomopsis monophyletic and nested within Copidosoma. Although the relationships between some deeper nodes were problematic and poorly supported, the following nine clades (Figs 1, 2) (= groups) are recognized: Clade I: C. cervius, C . chalconotum, C . serricorne and C . sp. near notatum. This clade was strongly supported as the sister group to Copidosomopsis + the remaining Copidosoma (Figs 1, 2). Clade II: all sampled species of the genus Copidosomopsis, Co. nacoleiae, Co. meridionalis, and Co. sp. The sister group relationship between Copidosomopsis + the remaining Copidosoma had high support. Clade III: C . boucheanum, C . terebrator, C . peticus, C . phaloniae, C . ancharus, C . tibiale, C . sosares and species near C . peticus. The similar internal relationships were retrieved in both analyses (Figs 1, 2). Clade IV: C . noyesi and two species near C. noyesi . Despite sister group to the Clade III and Clades V–IX in the MP analysis (Fig. 1), this group was unresolved in the Bayesian analysis (Fig. 2). Clade V: C. albipes, C. coimbatorense and C . sp. near coimbatorense. This clade is strongly supported as sister group to Clade VI in the Bayesian analysis (Fig. 2). Clade VI: C . varicorne, C . subalbicorne (both species formerly included in the genus Paralitomastix ), C . fuscisquama, C . aretas and five species near C . subalbicorne. This clade was sister group to Clades VII–IX in the MP analysis (Fig. 1), but sister to Clade V in the Bayesian analysis (Fig. 2). 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. Clade VIII: C . exiguum, C . sp1 near exiguum and C . sp2 near exiguum. This clade was supported as sister group to Clade IX in the Bayesian analysis (Fig. 2), but sister to C . lucidum in the MP analysis. Clade IX: C . floridanum, C . primulum, C . transversum, C . truncatellum, C . agrotis and three species near C . agrotis. This group is the most apical clade in MP analysis, but unsupported. In both analyses, Clade I was the sister group to Copidosomopsis and the remaining Copidosoma, with strong support
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
4 F. Yu et al.
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.
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
Phylogeny of Copidosoma
5
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. 2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
6 F. Yu et al. (BS = 100, PP = 1.00). Copidosomopsis (Clade II) was monophyletic and strongly supported (BS = 92, PP = 1.00) as sister group to the remaining Copidosoma. The topology of the remaining clades was inconsistent, depending on analytical methods. In MP analysis, the relationship between clades was resolved but unsupported. In the Bayesian result Clade III was strongly supported as sister to the remaining clades in the genus (PP = 0.99), whereas Clade IV was sister group to Clade III and Clades V–IX in the MP tree. Clade IV was unresolved because of collapse in the Bayesian analysis, and Clade V was sister group to Clade VI with strong support (PP = 0.95). A sister relationship between Clade VII and remaining clades was consistent in both trees, but with moderate support only in the Bayesian analysis (PP = 0.80). Clade IX was sister group to Clade VIII in the Bayesian analysis (PP = 0.76), but sister to Clade VIII + C. lucidum in the MP result (Fig. 1). Additionally, the position of C . lucidum was unstable. On the one hand, C . lucidum was found sister to Clade VIII + Clade IX with poor support (PP = 0.61) in the Bayesian analysis, but on the other hand, C . lucidum appeared sister to Clade VIII without support in MP analysis. Discussion Phylogenetic relationships, taxonomic inferences and host ranges Our phylogenetic analysis confirmed the synonymy of Litomastix and Paralitomastix with Copidosoma as suggested in the previous revisions based on morphological characters (Kazmi & Hayat, 1998; Guerrieri & Noyes, 2005). Copidosomopsis was nested with strong support within Copidosoma in both MP and Bayesian analyses. Given some considerations on this controversial genus (Guerrieri & Noyes, 2005), we propose Copidosomopsis syn.n. as a new synonymy of Copidosoma on the basis of our reconstructed phylogenetic relationships. We further propose the recognition of the following speciesgroups: cervius group (Clade I), nacoleiae group (Clade II), boucheanum group (Clade III), noyesi group, (Clade IV), albipes group (Clade V), varicorne group (Clade VI), thebe group (Clade VII), exiguum group (Clade VIII) and floridanum group (Clade IX). cervius group. The cervius group (Clade I) was recovered as the most basal clade of Copidosoma in both analyses (Figs 1, 2). Morphologically, this group has a number of distinctive features, including forewing venation with postmarginal vein as long as stigmal vein, long antennal segments with clava transversally truncated at the apex, similar thoracic sculpture consisting of small rounded cells that are moderately deep on the mesoscutum and very superficial on scutellum, and male genitalia with long parameres and digiti (Guerrieri & Noyes, 2005). Trjapitzin (1971, 1977) considered an elongate postmarginal vein as plesiomorphic in Encyrtidae; as the only species group showing this feature, the cervius group 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 group of C. chalconotum + C. serricorne (BS = 99, PP = 1.00). The grouping of C . cervius, C. chalconotum + C. serricorne was first proposed based on morphology by Guerrieri & Noyes (2005). Females of C . cervius can be readily separated from the other two species by the shorter length of the funicular segments. Similarly, males of C . cervius can be separated by the relatively longer digiti and the gently curved inner margins of the parameres (sinuous in serricorne and chalconotum) (Guerrieri & Noyes, 2005). The sister species C . serricorne and C . chalconotum can be separated from each other by only slight but consistent differences in the shape of the apex of the ovipositor sheaths (gonostyli) and in the relative length of the clava (Guerrieri & Noyes, 2005). nacoleiae group. The examined species of Copidosomopsis were monophyletic and nested within Copidosoma in both analyses, with robust support values. Copidosomopsis is very 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 & Hayat (1998) separated Copidosomopsis from Copidosoma on the basis of the shape of hypopygium in females and male genitalia with sclerotized digiti without denticles and parameres reduced or absent. Subsequently, Guerrieri & Noyes (2005) noted that some of these characters were shared by European species of Copidosoma; in fact, they found that Copidosomopsis could be kept separated from Copidosoma only for New World species, showing a distinctive propodeal spiracle elongate and pear-shaped. A molecular characterization of these species would be of great help in assessing their correct taxonomic position because our analysis and biological features strongly support a formal synonymy of Copidosomopsis with Copidosoma. C. nacoleiae, C. plethorica and C. tanytmema, are indeed polyembryonic endoparasitoids of Lepidoptera, mainly Pyralidae and Tortricidae (Noyes & Hayat, 1984; Yu et al., 2010). boucheanum group. The boucheanum group was recovered as sister group to Clades IV–IX (see Fig. 2) with strong support (PP = 0.99) in the Bayesian analysis. Overall, the males of this group share similar genitalia with phallobase narrowing towards its base and parameres no longer than digiti, aedeagus long and slender, bilaterally concave and pointed at the apex. Biologically, species of this group were associated with different lepidopteran families; including Gelechiidae, Depressariidae, Coleophoridae, Blastodacnidae and Tortricidae (see Fig. 3 and Appendix S1). This group divided into two subclades in both analyses (BS = 100, PP = 1.00), including C. boucheanum, C. terebrator, C . phaloniae, C. peticus and species near C. peticus in one and C. ancharus, C. tibiale and C. sosares in the other. In the first subclade, two species, C . ancharus and C . tibiale were recovered as sister species with strong support (BS = 100, PP = 1.00). The morphological characters
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
Phylogeny of Copidosoma
7
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 share a similar antennal structure and a very superficial sculpture on scutellum, and can be separated on the relative length of F1, gonostyli and exerted part of the ovipositor (Guerrieri and Noyes, 2005). In both analyses, the two species were found close to C . sosares (BS = 82, PP = 1.00).
In the second subclade, the species relationships were relatively stable in both MP and Bayesian analyses. Copidosoma phaloniae was close to C . peticus and species near C . peticus. C. boucheanum was sister to C. terebrator, with strong support (PP = 0.99) in Bayesian analysis, confirming a morphological closeness (Guerrieri & Noyes, 2005) in
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
8 F. Yu et al. general habitus, thoracic sculpture and strongly exerted ovipositor. On the basis of morphological characters, we believe that the recently described species C. longicaudata Japoshvili & Guerrieri (Japoshvili et al., 2013) could reasonably fall within this subgroup. noyesi group. In the Bayesian analysis, the noyesi group was unresolved. However, the sister relationship between this group and remaining ones was recovered in the MP analysis, although this was not well supported (Fig. 1). Females of this group are quite distinct, sharing very similar morphology particularly for body colour (thorax partially yellowish or yellow brown) (Kazmi & Hayat, 1998). Copidosoma noyesi was described by Kazmi & Hayat (1998) from India. As one of the examined species, C . sp2 near noyesi was collected in North China (Shanxi), further collections and characterizations are needed to understand the composition of this species group in the Chinese fauna. albipes group. This group was highly supported as sister group to varicorne group in the Bayesian analysis (Fig. 2). Morphologically, females of C. albipes are similar to those of coimbatorense for the shape of the sensorial part of clava at the apex and the shallow sculpture on scutellum. Copidosoma albipes 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 analyses. 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 the other by C . varicorne and C . subalbicorne. In one lineage, C . aretas and C . fuscisquama were recovered as sister species with strong support (BS = 99, PP = 1.00). The morphological similarities between aretas and fuscisquama in antennal structure, ovipositor, hypopygium of females and male genitalia explained their sister-species relationship in the trees. Furthermore, both species are parasitoids of Tortricidae (Guerrieri & Noyes, 2005). Our results corroborated the synonymy of Paralitomastix with Copidosoma as suggested by different morphological revisions of species (Kazmi & Hayat, 1998; Guerrieri & Noyes, 2005). The genus Paralitomastix has been distinguished from Copidosoma by the black and white flagellum and, to a lesser extent, by the elongated sculpture on scutellum (Noyes & Hayat, 1984; Kazmi & Hayat, 1998). Two species C . varicorne and C . subalbicorne, placed in previously Paralitomastix , were recovered as sister with low posterior probability (PP = 0.77). Females of C . varicorne and C . subalbicorne can be separated most easily on the colour of F5 (brown in varicorne, white in subalbicorne) (Guerrieri & Noyes, 2005). Biologically, C . varicorne and C . subalbicorne are reported as parasitoids of Gelechiidae. thebe group. Our results suggest that this group could be sister to the floridanum and exiguum species groups. Our
results extend the distribution of C. thebe to China; previously C . thebe has been reported only from European countries with unknown host (Guerrieri & Noyes, 2005). In North China (e.g. Beijing) and South China (e.g. Hainan) we have collected some species close to C. thebe that could well be placed in this species group, suggesting that their exact distribution is not fully known. exiguum group. The exiguum group is sister to the floridanum group in the Bayesian analysis (Fig. 2) and to C . lucidum in the MP analysis (Fig. 1). Kazmi & Hayat (1998) described Copidosoma exiguum from India as a parasitoid of pod borer larva of Cassia tora. Many specimens collected in the South of China (e.g. Yunnan) appeared morphologically similar to exiguum and could be reasonably placed in this group. floridanum group. The position of the floridanum group was relatively consistent in both MP and Bayesian analyses. It is split into strongly supported two lineages in the Bayesian tree (PP = 1.00), one including C. primulum, C. transversum and C . floridanum, and the other including C. truncatellum, C . agrotis and species near C . agrotis. Species in this group are morphologically very similar and frequently misidentified. Only recently has it been possible to reliably separate C . floridanum and truncatellun (Noyes, 1988). In this group, 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 other than to species of the other subclade. Some morphological and biological considerations support this clade: its species share a similar antennal and thoracic sculpture. Slight but consistent differences can be found in the forewing venation and male genitalia between species of the two subclades. In C . floridanum, C. transversum and C . primulum, the marginal vein of the forewing is as long as the stigmal vein, whereas in C. truncatellum and C. agrotis it is distinctly shorter (Guerrieri & Noyes, 2005; Zhang & Huang, 2007). The male genitalia of the four species are also almost identical with phallobase narrowing proximally, digiti narrow and slender, and parameres reduced (Guerrieri & Noyes, 2005; Zhang & Huang, 2007). Differences also occur on the aedeagus with that of floridanum and primulum simple, whereas in truncatellum and agrotis it is apically bilaterally concave and pointed, with two button-like structures in the apical third in addition to the spermatic pores (Guerrieri & Noyes, 2005; Zhang & Huang, 2007). Species of this group with confirmed host associations are polyembryonic endoparasitoids of Noctuidae. Host specificity All species of Copidosoma with known biology are primary egg-larval endoparasitoids of Lepidoptera (Guerrieri & Noyes, 2005), ovipositing into the eggs of their hosts and emerging from their last larval instar. Within the tribe Copidosomatini, Ageniaspis, Copidosomopsis and Copidosoma have been reported to be polyembryonic parasitoids
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
Phylogeny of Copidosoma of Lepidoptera. Ageniaspis is reported to attack Yponomeutidae, Nepticulidae and Gracillariidae (Noyes & Hayat, 1984). Species of Copidosoma are reported as parasitoids of moths in 13 families belonging to seven superfamilies (Guerrieri & Noyes, 2005). Each of the species groups recognized in this paper appears to be associated with a restricted number of lepidopteran families, if not to a single one (Fig. 3). The cervius group – the most basal clade in our analysis – attacks mainly Geometridae. In Europe, C . serricorne is reared from Larentiinae and Ennominae (Geometridae); C . cervius and C . chalconotum are generally reared from Larentiinae only (Guerrieri & Noyes, 2005). Species of nacoleiae group (= Copidosomopsis) are mainly endoparasitoids of Pyralidae and Tortricidae (Noyes & Hayat, 1984). Although species of the boucheanum group are associated with different lepidopteran families (see Fig. 3 and Appendix S1 for details), they attack members of Gelechioidea, with the exception of C. phaloniae recorded 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 to subclade 2) are parasitoids of Gelechiidae. The large clade of the floridanum group seems to comprise parasitoids of Noctuidae exclusively, but each species is associated with a single subfamily (see Appendix S1 for details). This probably reflects host-searching behaviour because the eggs of Plusiinae are laid well above ground level, whereas those of cutworms and hepialids are deposited at or near ground level (Noyes, 1988). Our phylogenetic analysis has resulted in the first hypothesis for the evolutionary relationships within the genus Copidosoma. However, additional taxonomic sampling will be needed to better resolve the phylogeny of this polyembryonic parasitoid group. Because several groups of related species generally attack hosts within the same family, these might indicate a certain level of coevolution with their hosts or perhaps rapid diversification on related hosts (Guerrieri & Noyes, 2005). A comprehensive phylogeny of Copidosoma will be a starting point for the analysis of the co-evolution of the genus and their lepidopteran hosts.
Supporting Information Additional Supporting Information may be found in the online version of this article under the DOI reference: 10.1111/syen.12057
Table S1. Specimens used in molecular phylogenetic analysis with sequences GenBank accession numbers. Table S2. List of primer sequences. Table S3. Summary of number of taxa and characters, and substitution model for each partition. Appendix S1. List of host association of species analysed in this study.
9
Acknowledgements Thanks to R. Zhang, G. Zheng, F. Yuan and other kind individuals for helping collect specimens. We are grateful to Prof. John M. Heraty, Department of Entomology, University of California, Riverside, USA, for offering helpful suggestions on phylogenetic analysis and discussion. We sincerely thank three anonymous referees for their comments on the manuscript. This project was supported by the National Natural Science Foundation of China (NSFC grant No. 31071950 and No. 31272350), by Chinese Academy of Sciences (KSCX2-YWNF-02) and partially by Project of the Department of Science and 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 Entedoninae. Cladistics, 27, 1–25. Campbell, B.C., Steffen-Campbell, J.D. & Werren, J.H. (1993) Phylogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences. Insect Molecular Biology, 2, 225–237. Campbell, B., Heraty, J.M., Rasplus, J.-Y., Chan, K., SteffenCampbell, J. & Babcock, C. (2000) Molecular systematics of the Chalcidoidea 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 the foundations 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) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9, 772. Donnell, D.M., Corley, L.S., Chen, G. & Strand, M.R. (2004) Caste determination in a polyembryonic wasp involves inheritance of germ cells. Proceedings of the National Academy of Science of the United States of America, 101, 10095–10100. Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine 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 a reclassification of Eulophinae and the recognition that Elasmidae are 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 rRNA expansion 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) Predicted secondary structure for 28S and 18S rRNA from Ichneumonoidea (Insecta: Hymenoptera: Apocrita): impact on sequence alignment and phylogeny estimation. Journal of Molecular Evolution, 61, 114–137. Giron, D., Dunn, D., Hardy, I.C.W. & Strand, M.R. (2004) Aggression by polyembryonic wasp soldiers correlates with kinship but not resource competition. Nature, 430, 676–679. Goloboff, P.A., Farris, J.S. & Nixon, K.C. (2008) TNT, a free program for phylogenetic analysis. Cladistics, 24, 774–786.
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
10
F. Yu et al.
Grbi´c, M., Ode, P.J. & Strand, M.R. (1992) Sibling rivalry and brood sex ratios in polyembryonic wasps. Nature, 360, 254–256. Guerrieri, E. (1995) Influence of temperature on development and longevity of the adults of Copidosoma koehleri Blanchard (Hymenoptera: Encyrtidae), parasitoid of Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae). Bollettino del Laboratorio di Entomologia agraria Filippo Silvestri , 50, 261–270. Guerrieri, E. & Noyes, J.S. (2005) Revision of the European species of Copidosoma Ratzeburg (Hymenoptera: Encyrtidae), parasitoids of caterpillars (Lepidoptera). Systematic Entomology, 30, 97–174. Hain, F.B. & Wallner, W.E. (1973) The life history, biology, and parasites 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 sequence alignment 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 induces adaptive 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 of the 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 cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Science of the United States of 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 subfamily of the ant-parasitic Eucharitidae (Hymenoptera: Chalcidoidea). Systematic Entomology, 29, 544–559. Heraty, J.M., Burks, R.A., Cruaud, A. et al. (2013) A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera). Cladistics, 29, 466–542. Horne, P.A. (1990) The influence of introduced parasitoids of the potato moth, Phthorimaea operculella (Lepidoptera: Gelechiidae) in Victoria, Australia. Bulletin of Entomological Research, 80, 159–163. Huelsenbeck, J.P. & Ronquist, F. (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755. Japoshvili, G., Hansen, L.O. & Guerrieri, E. (2013) The Norwegian species 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 Litomastix sp. 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 molecular phylogeny 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 frequently misidentified polyembryonic parasitoids of caterpillars (Lepidoptera). Systematic Entomology, 13, 197–204. Noyes, J.S. (2012) Universal Chalcidoidea Database [WWW document]. URL http://www.nhm.ac.uk/chalcidoids [accessed on 12 June 2012]. Noyes, J.S. & Hayat, M. (1984) A review of the genera of IndoPacific Encyrtidae (Hymenoptera: Chalcidoidea). Bulletin of the British Museum of Natural History (Entomology), 48, 131–395. Owen, A.K., George, J., Pinto, J.D. & Heraty, J.M. (2007) A molecular phylogeny of the Trichogrammatidae (Hymenoptera: Chalcidoidea), with an evaluation of the utility of their male genitalia for higher level classification. Systematic Entomology, 32, 227–251. Ratzeburg, J.T.C. (1844) Die Ichneumonen der Forstinsekten in entomologischer und forstlicher Beziehung, Vol. 1. Nicolai’sche Buchhandlung, Berlin. Schmidt, S. & Polaszek, A. (2007) Encarsia or Encarsiella? – redefining generic limits based on morphological and molecular evidence (Hymenoptera, Aphelinidae). Systematic Entomology, 32, 81–94. Swezey, O.H. (1931) Litomastix floridana (Ashm.), a recent immigrant in Hawaii. Proceedings of the Hawaiian Entomological Society, 7, 369–370, 390, 419–421. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology Evolution, 24, 1596–1599. Trjapitzin, V.A. (1971) Problems of morphological evolution and classification 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 of the family Encyrtidae (Chalcidoidea). Part II. Subfamily Encyrtinae Walker, 1837. Entomologicheskoe Obozrenie, 52, 416–429. Trjapitzin, V.A. (1977) The characteristic features of the morphology of adult encyrtids (Hymenoptera, Chalcidoidea, Encyrtidae) and their systematic significance. Trudy Vsesoyuznogo Entomologicheskogo Obshchestva, 58, 145–199. Trjapitzin, V.A. (1989) Parasitic Hymenoptera of the Fam. Encyrtidae of 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 introduced parasites (Copidosoma koehleri and Apanteles subandinus). Journal of 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 insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Systematic Biology, 46, 1–68. Yu, F., Zhang, Y.Z., Zhu, C.D. & Tu, L.H. (2010) A taxonomic study of Chinese species of Copidosomopsis Girault (Hymenoptera: Encyrtidae). Zootaxa, 2490, 55–62. Zhang, Y.Z. & Huang, D.W. (2007) Study of the Chinese species of Copidosoma (Hymenoptera: Encyrtidae). Insect Systematic & Evolution, 38, 105–119. Zhang, Y.Z., Yu, F. & Zhu, C.D. (2008) A preliminary phylogenetic study of Copidosoma spp. (Hymenoptera: Encyrtidae) associated with Noctuidae (Lepidoptera) based on 28S rDNA D2 sequence. Acta Entomologica Sinica, 51, 992–996. Zolnerowich, G. (1995) Systematics of the Copidosomatini: polyembryonic parasites (Hymenoptera: Encyrtidae). PhD Thesis, Texas A&M University, College Station, Texas. Accepted 20 November 2013
2014 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12057
