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A pilot study on the molecular phylogeny of Drepanoidea (Insecta: Lepidoptera) inferred from the nuclear gene EF-1α and the mitochondrial gene COI

Published online by Cambridge University Press:  07 July 2009

C.G. Wu ,
H.X. Han  and
D.Y. Xue
C.G. Wu
Affiliation:
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing100101, China Graduate University of the Chinese Academy of Sciences, Beijing100049, China
H.X. Han
Affiliation:
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing100101, China
D.Y. Xue*
Affiliation:
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing100101, China
*
*Author for correspondence Fax: +86-10-64807099 E-mail: xuedy@ioz.ac.cn
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Abstract

A molecular phylogenetic study of the Drepanoidea based on the EF-1α sequences and combined EF-1α and COI sequences was carried out in order to infer higher classification at and above the subfamily level. The sample contained 14 taxa representing 13 genera recognized in the Drepanoidea. The results revealed that the Drepaninae, Thyatirinae and Cyclidiinae respectively form monophyletic groups. The sister relationship between the Drepaninae and the Thyatirinae was validated. The monophyly of the Cyclidiinae with the Drepaninae+Thyatirinae was supported robustly. Hypsomadius insignis and Oreta vatama within the traditional definition of the Drepaninae formed an individual clade with robust support (100%) and constitutes a sister relationship to a clade containing the rest of the Drepaninae in all the topologies, which means that the subfamily Oretinae of the Drepanidae should be restored. The family Drepanidae is divided into four subfamilies: Drepaninae, Oretinae, Thyatirinae and Cyclidiinae in this work. The family Epicopeiidae formed a monophyly with high bootstrap values. The result of combined analysis of EF-1α and COI showed that the Epicopeiidae have a closer phylogenetic relationship with the Geometridae than with the Drepanidae and belong to neither the Drepanoidea nor the Geometroidea.

Keywords

Drepanoideamolecular phylogenysubfamily
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 100 , Issue 2 , April 2010 , pp. 207 - 216
Copyright
Copyright © Cambridge University Press 2009

Introduction

The superfamily Drepanoidea is currently composed of two families, the Epicopeiidae and the Drepanidae (Minet, Reference Minet1991; Minet & Scoble, Reference Minet, Scoble and Kristensen1999). The family Drepanidae is divided into three subfamilies: the Drepaninae, Thyatirinae and Cyclidiinae. The family Epicopeiidae is not divided into subfamilies owing to the relative homogeneity of the limited number of genera. The members of the Drepanoidea were once placed in the Geometroidea in the early years because most of them have abdominal tympanal organs (Imms, Reference Imms1934). Since that time, different authors have had different viewpoints about the content of the drepanoids. Inoue (Reference Inoue1954) recorded them as including the Drepanidae, Thyatiridae and Callidulidae, mainly following McDunnough (Reference McDunnough1938). Nakamura (Reference Nakamura1981) considered that the superfamily Drepanoidea consists of the Drepanidae, Thyatiridae, Cyclidiidae and Epicopeiidae. Minet (Reference Minet1983) considered the superfamily as only including the Drepanidae, based on the study of the tympanal organs, and regarded the Thyatiridae and Cyclidiidae as two subfamilies of the Drepanidae. The same author (Minet, Reference Minet1991) assigned the Epicopeiidae, unplaced by him in 1983, to the Drepanoidea, mainly based on the Epicopeiidae and Drepanidae sharing four apomorphies: setae of the larval mandible inserted on a large, flat, lateral area delimited ventrally by a projecting line; at least one secondary seta associated with L3 on segments A1-8 of the larva; the femur of the pupal foreleg concealed or very slightly exposed; and adult abdomen with prespiracular sclerite laterally, connecting the first tergite with the first sternite, and modified into tympanal organs in the Drepanidae. Scoble (Reference Scoble1992) still doubted the taxonomic position of the Epicopeiidae and tentatively placed them in the Uranioidea. However, he added that there was no similarity with other uraniid groups because the Epicopeiidae lacked tympanal organs; and he concluded that the treatment of Minet (Reference Minet1991), placing the Epicopeiidae in the Drepanoidea, might be more appropriate. Subsequently, the definition of Drepanoidea in Minet (Reference Minet1991) has been widely accepted and used (Holloway, Reference Holloway1998; Minet & Scoble, Reference Minet, Scoble and Kristensen1999; Holloway et al., Reference Holloway, Kirby and Peggie2001; Kristensen et al., Reference Kristensen, Scoble and Karsholt2007). But Minet & Scoble (Reference Minet, Scoble and Kristensen1999) also included some epicopeiid characters which are different from the Drepanidae when they summarized the characteristics of the group, e.g. the abdomen lacking tympanal organs; the tongue being well developed; frenulum and subcostal retinaculum usually present in males, female frenulum absent or strongly reduced; M2 rarely arising nearer M3 than M1 in both forewing and hindwing; Sc+R1 close to Rs in the base of cell and far away beyond cell in hindwing.

Some other researchers did not support the viewpoint of placing the Epicopeiidae in the Drepanoidea. In Imms' family system, the family Epicopeiidae was always a part of the Uraniidae, although he added that, “The Asiatic genus Epicopeia has a vestigial frenulum and is often relegated to a separate family—the Epicopeiidae” (Imms, Reference Imms1934). Inoue (Reference Inoue1954) listed the Epicopeiidae under the superfamily Uranioidea. Zhu & Wang (Reference Zhu and Wang1991) performed a phylogenetic analysis at family level on the Geometroidea based on morphologic characters in the same year in which Minet defined the Drepanoidea. The following characters were used in their analysis: antenna filiform or bipectinate; apex of forewing falcate or not; forewing R5 or R4+5 connected or stalked with R2+3 or far apart; hindwing Sc+R1 close to Rs or far apart beyond cell; the base of Sc+R1 in hindwing forked or not; one or two A veins present in hindwing; M2 located in the middle of M1 and M3 or other ways in both wings; frenulum present or not; a pair of hair clusters in the second abdomen segment present or not; abdominal tympanal organs located on the dorsal or ventral side. The analysis results showed (fig. 1a) that the Epicopeiidae formed a sister group with the Epiplemidae+Uraniidae, and the relationship between the Epicopeiidae and the Geometridae was closer than that between the Epicopeiidae and the Thyatiridae+(Drepanidae+Cyclidiidae). Kuznetzov & Stekolnikov (Reference Kuznetzov and Stekolnikov2001) also performed an analysis based on morphological characters and obtained almost the same result (fig. 1b) as those of Zhu & Wang (Reference Zhu and Wang1991), namely that the Epicopeiidae and Uraniidae+Epiplemidae formed a sister group. The author considered this group as a part of the Uranioidea and, furthermore, that the Uranioidea and Geometroidea+Drepanoidea were a sister group. The result shows that the relationship between the Epicopeiidae and Drepanidae is not closer than that between the Geometridae and Drepanidae. Therefore, it is clear that the monophyly of the Drepanoidea needs to be confirmed and that the taxonomic status of the Epicopeiidae should be reconsidered and further validated.

Fig. 1. Phylogeny trees (a) Imms' Geometroidea (Zhu & Wang, Reference Zhu and Wang1991); (b) Uranioidea, Geometroidea and Drepanoidea (Kuznetzov & Stekolnikov, Reference Kuznetzov and Stekolnikov2001).

The phylogenetic analysis results of Zhu & Wang (Reference Zhu and Wang1991) (fig. 1a) showed that the Drepanidae and Cyclidiidae formed a sister group, and formed a monophyly together with the Thyatiridae. But Minet (Reference Minet2002) considered that the first dichotomy was likely to lie between the Cyclidiinae and the Thyatirinae+Drepaninae within the Drepanidae. His viewpoint is supported by the following three apomorphies: a male frenulum with clubbed apex (e.g. Scoble & Edwards, Reference Scoble and Edwards1988: fig. 17), a small tympanal chamber provided with a fairly broad dorsal sclerotized wall (e.g. Gohrbandt, Reference Gohrbandt1937: fig. 14), and a large tympanal chamber that is distinctly fused with sternum A2 mesad of the apodemal protrusion (e.g. Minet, Reference Minet1983: fig. 95). Unlike that of most Thyatirinae and Drepaninae, the small tympanal chamber of the Cyclidiinae has a dorsal sclerotized wall, which varies from extremely narrow to entirely absent.

Different authors divided the Drepaninae into different subgroups based on a set of characters: adult body colour, tongue and frenulum, forewing colour and shape (falcate or not), hind tibial spurs, larval secondary setae and supracoxal vesicle. Many taxonomists considered that the Drepanidae (present Drepaninae) should be divided into two subfamilies: the Drepaninae and Oretinae (Inoue, Reference Inoue1962; Nakajima, Reference Nakajima1970; Wilkinson, Reference Wilkinson1972; Zhu & Wang, Reference Zhu and Wang1991; Smetacek, Reference Smetacek2002). However, other authors considered that the Drepaninae should be divided into two subgroups rather than subfamilies (e.g. Watson, Reference Watson1965, Reference Watson1967; Minet, Reference Minet1985; Scoble, Reference Scoble1992). Holloway (Reference Holloway1998) considered that the subgroups of Drepaninae did not reach the subfamily level and placed them at tribal level: Oretini and Drepanini. Minet & Scoble (Reference Minet, Scoble and Kristensen1999) further divided the Drepaninae into three tribes: Nidarini, Oretini and Drepanini.

Therefore, it is necessary to further investigate the monophyly of each subfamily within the Drepanidae and the relationships between the different subfamilies, especially the taxonomic status of the Oreta group.

Currently, many genes are available for phylogenetic analysis. Because the cytochrome oxidase I (COI), 16S rRNA, 18S rRNA and elongation factor-1α (EF-1α) genes have been widely used and are informative across a broad range of divergences. Caterino et al. (Reference Caterino, Cho and Sperling2000) advocate their use as standards for insect molecular phylogenetics. Since the amino acid sequences of EF-1α are highly conserved, nonsynonymous changes are rare, especially in the Lepidoptera, which have lost all introns and only have a single copy of the gene. These properties render the gene a useful marker for resolving the phylogenetic relationships of the higher classification of insects (Friedlander et al., Reference Friedlander, Regier and Mitter1992, Reference Friedlander, Regier and Mitter1994; Brower & DeSalle, Reference Brower and DeSalle1994; Cho et al., Reference Cho, Mitchell, Regier, Mitter, Poole, Friedlander and Zhao1995; Belshaw & Quicke, Reference Belshaw and Quicke1997; Mitchell et al., Reference Mitchell, Cho, Regier, Mitter, Poole and Matthews1997; Danforth & Shuqing, Reference Danforth and Shuqing1998). In the Lepidoptera, the EF-1α gene should give phylogenetic information and has been proved useful in reconstructing phylogenies at subfamily or lower levels, such as generic and tribal levels (Cho et al., Reference Cho, Mitchell, Regier, Mitter, Poole, Friedlander and Zhao1995; Mitchell et al., Reference Mitchell, Cho, Regier, Mitter, Poole and Matthews1997; Friedlander et al., Reference Friedlander, Horst, Regier, Mitter, Peigler and Fang1998; Reed & Sperling, Reference Reed and Sperling1999; Mitchell et al., Reference Mitchell, Mitter and Regier2000; Caterino et al., Reference Caterino, Reed, Kuo and Sperling2001; Monteiro & Pierce, Reference Monteiro and Pierce2001; Morinaka et al., Reference Morinaka, Miyata and Tanaka2002; Wahlberg & Nylin, Reference Wahlberg and Nylin2003; Braby et al., Reference Braby, Vila and Pierce2006).

COI is a widely used mitochondrial protein-encoding gene. In molecular phylogenetic studies on the Lepidoptera, the gene has shown great utility for resolving the phylogenetic relationships within closely related groups (Caterino et al., Reference Caterino, Cho and Sperling2000; Sperling, Reference Sperling, Boggs, Watt and Ehrlich2003). Different authors have different ideas about whether the saturated third codon positions should be removed when using COI to perform phylogenetic analysis (Gleeson et al., Reference Gleeson, Rowell, Tait, Briscoe and Higgins1998; Söller et al., Reference Söller, Wohltmann, Witte and Blohm2001; Wares, Reference Wares2001; Ros & Breeuwer, Reference Ros and Breeuwer2007; Zhang et al., Reference Zhang, Cao, Zhang, Guo, Duan and Ma2007; Ketmaier et al., Reference Ketmaier, Joyce, Horton and Mariani2008).

Because the two genes evolve at different rates, combining both genes will probably increase the reliability of phylogenetic analysis results. Furthermore, it may provide consistent information on nodes (Caterino et al., Reference Caterino, Cho and Sperling2000). In the Lepidoptera, several recent studies have demonstrated improved resolution of nodal support at both higher and intermediate systematic categories of divergence in a combined analysis of nuclear and mitochondrial genes (Caterino et al., Reference Caterino, Reed, Kuo and Sperling2001; Monteiro & Pierce, Reference Monteiro and Pierce2001; Wahlberg & Nylin, Reference Wahlberg and Nylin2003; Kandul et al., Reference Kandul, Lukhtanov, Dantchenko, Coleman, Sekercioglu, Haig and Pierce2004; Zakharov et al., Reference Zakharov, Caterino and Sperling2004).

The purpose of this study is to reconstruct the phylogeny of the Drepanoidea based on the analysis of EF-1α sequences and combined EF-1α and COI sequences. It also investigates the taxonomic system of higher categories above the subfamily level and the phylogenetic relationships of different subfamilies. It proved that the Oretinae should be restored, that the sister relationship between the Thyatirinae and Drepaninae+Oretinae was well formed and that the Epicopeiidae did not belong to the Drepanoidea.

Materials and methods

Taxa examined

The collection localities of the material examined in this study and GenBank Accession numbers of all sequences are given in table 1. For the phylogenetic analysis of the Drepanoidea, the EF-1α and COI sequences of 14 taxa belonging to two families, three subfamilies and 13 genera were obtained and used as ingroups. Two sequences were obtained from GenBank based on the published work of Yamamoto & Sota (Reference Yamamoto and Sota2007) and Cho et al. (Reference Cho, Mitchell, Mitter, Regier, Matthews and Robertson2008) (table 1). Two representatives of the Geometridae, which is regarded as the sister group of the Drepanidae based on morphology (Minet, Reference Minet1983; Zhu & Wang, Reference Zhu and Wang1991; Minet & Scoble, Reference Minet, Scoble and Kristensen1999; Xue & Zhu, Reference Xue and Zhu1999; Young, Reference Young2006; Kristensen et al., Reference Kristensen, Scoble and Karsholt2007) and molecular phylogenetics (Abraham et al., Reference Abraham, Ryrholm, Wittzell, Holloway, Scoble and Löfstedt2001), and two representatives of the Noctuidae, were used as outgroups.

Table 1. Species information and GenBank accession numbers.

Molecular techniques

The following protocol was adopted to obtain DNA sequences EF-1α and COI.

Specimen preparation

Fresh adult specimens were collected by using light traps and killed in cyanide bottles. Wings were immediately excised and stored in paper envelopes as vouchers for identification, and the bodies or only three legs on the same side were preserved in 100% ethylalcohol. The specimens were stored at −20°C for laboratory use. A few of the specimens were collected and preserved as dried adults. All DNA samples and voucher specimens were deposited in the Zoological Museum, the Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from single moth thorax or legs by using QIAgen's DNEasy extraction kit according to the manufacturer's protocols and with some slight improvements. Abdomens and wings were conserved in microtubes and paper envelopes, respectively, as vouchers and for confirmation of specimen identification by dissection of the genitalia.

The nuclear gene EF-1α and mitochondrial gene COI were amplified by polymerase chain reaction (PCR) using published primers. The primer sequences were as follows: EF1aF2 (sense) (5′-ACAAATGCGGTGGTATCGACAA-3′) and EF1aR (antisense) (5′-GATTTACCRGWACGACGRTC-3′) (see Yamamoto & Sato, Reference Yamamoto and Sota2007; Kawakita et al., Reference Kawakita, Takimura, Terachi, Sota and Kato2004) for EF-1α; LCO1490 (sense) (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (antisense) (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (see Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994) for COI. PCR reactions were performed in a 50 μl volume, containing 10 μl 5×PrimeSTAR™ buffer (5 mM of MgCl2); 4 μl dNTP mixture (each of 2.5 mM); 0.5 μl PrimeSTAR™ HS DNA polymerase (2.5 U μl−1), 2 μl of each primer (10 pM); 3 μl DNA template and 28.5 μl distilled water up to 50 μl. The reactions were done on a GeneAmp PCR System 9700 (Applied Biosystem, USA) with the following conditions for EF-1α: 95°C for 2 min, 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 1 min and a final extension period of 72°C for 10 min. The reaction cycle profile of COI PCR amplification was 95°C for 2 min, 30 cycles of 95°C for 30 s, 53°C for 30 s, 72°C for 1 min, and a final extension period of 72°C for 10 min. These two protocols and the two pairs of primers worked well for all examined species. Each PCR product was subsequently gel purified using the AxyPrep™ DNA Gel Extraction Kit (Axygen).

Sequencing reactions were performed with the corresponding amplifying primers from both directions and run with ABI 3730 automated sequencer (Applied Biosystem, USA).

Assembling and alignment of sequences

Chromatograms, including sense and antisense, were edited and assembled using DNASTAR 5.0 (DNASTAR, Madison, Wisconsin, USA, Inc.) to obtain single consensus sequences. The nucleotide sequences were translated into amino acid sequences to check for the presence of stop codons that might indicate that pseudogenes had been amplified (Sanders et al., Reference Sanders, Malhotra and Thorpe2006). Multiple alignments were done with Clustal version 1.81 (Thompson et al., Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997) with default parameter settings and verified by eye. For EF-1α, the consensus sequence of each sample was aligned against the published sequence for Bombyx mori (Kamie et al., Reference Kamie, Taira, Ooura, Kakuta, Matsumoto, Ejiri and Katsumata1993) and primer ends were removed, resulting in 960 bp (corresponding to positions 187–1146). For COI, the consensus sequence of each sample was aligned against the published sequence for Drosophila yakuba (Clary & Wolstenholme, Reference Clary and Wolstenholme1985) and/or other Lepidoptera sequences on GenBank; the final fragment was 617 bp (corresponding to positions 1556–2172 of Drosophila).

Aligned sequence data were imported into MEGA 3.1 (Kumar et al., Reference Kumar, Tamura and Nei2004) for analyses of nucleotide composition. Nucleotide saturation was analyzed by plotting the number of transitions and transversions on each codon position against the Tamura & Nei (Reference Tamura and Nei1993) (TN93) genetic distance using DAMBE (Xia & Xie, Reference Xia and Xie2001). Saturation was considered to have occurred if the scatter of points showed leveling off mutations as sequence divergence increased.

Phylogenetic analysis

Simultaneous analyses of Nuclear EF-1α gene and combined data of EF-1α and mitochondrial COI gene were attempted because phylogenetic resolution from an individual gene was obviously limited. In the search for optimal trees, maximum parsimony (MP), Bayesian and maximum likelihood (ML) analyses for each of the data sets were used. All phylogenetic analyses were performed with PAUP*4.0b10 (Swofford, Reference Swofford2003) and MrBayes 3.1.2 (Ronquist & Huelsenbeck, Reference Ronquist and Huelsenbeck2003).

A maximum parsimony analysis was carried out first, with all sites weighted equally, using 1000 random additions of sequences and tree bisection reconnection (TBR) branch swapping. The command of ‘contree’ was used to yield the strict consensus tree. To assess the support for branching events, non-parametric bootstrapping was performed with 1000 pseudo-replicates under the heuristic search strategy and 100 random addition sequences in each pseudo-replicate. A node was interpreted as strongly supported if the bootstrap percentage (BP) was ⩾70% (Hillis & Bull, Reference Hillis and Bull1993).

Bayesian analysis was conducted using MrBayes 3.1.2, based on the model selected by ModelTest 3.7 (Posada & Crandall, Reference Posada and Crandall1998). Model parameter values were treated as unknown variables with uniform prior probabilities and were estimated during the analysis. Four chains (three heated and one cold) were run, starting from a random tree and proceeding for 1,000,000 Markov chain Monte Carlo generations, sampling the chains every 100 generations. Two independent runs were conducted to verify the results. For all runs, 1000 trees were discarded as burn-in samples. Remaining trees were used to generate a majority-rule consensus tree, in which the percentage of trees recovering a clade portrayed the clade's posterior probability (PP) (Huelsenbeck et al., Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001) or the probability that the clade is correct in the given data and model parameters. The combined data set was treated as two partitions with different models accounted for their heterogeneity. The prior models of sequence evolution employed for both COI and EF-1α data sets were also determined using Modeltest 3.7 (Posada & Crandall, Reference Posada and Crandall1998) based on the likelihood ratio tests. The test indicated that GTR+I+G model was the most appropriate model for both of the two data sets. The ‘unlink’ command was utilized to unlink the following parameters: ‘unlink shape=(all) pinvar=(all) statefreq=(all) revmat=(all)’. Probabilities of 95% or more were considered to indicate significant support (Reeder, Reference Reeder2003; Zakharov et al., Reference Zakharov, Caterino and Sperling2004).

The best-fit nucleotide substitution model used in maximum likelihood analysis was selected by using ModelTest 3.7 based on the Akaike Information Criterion (AIC). Maximum likelihood analysis was performed in PAUP* with the selected optional model under the heuristic search strategy with 100 random additions of sequences and TBR branch swapping. Bootstrap analysis was performed under the same model, with 100 pseudo-replicates, ten random additions of sequences per replicate and TBR branch swapping.

Results

Sequences characteristics and saturation analysis

For all the taxa, including outgroups and download sequence directly from GenBank (accession no. AB265512), approximately 960 bp were sequenced for EF-1α. Because transitions and transversions in the nuclear EF-1α were accumulated linearly and showed no saturation patterns at any position (fig. 2), all nucleotide positions were employed in the subsequent analysis. Of a total of 960 characters, 702 sites were conserved, 258 variable and 203 parsimony-informative (717 sites were constant, 243 variable and 185 parsimony-informative for the ingroup only), and average base frequencies were well proportioned with 21.1% T, 28.2% C, 24.7% A and 26.0% G. Nucleotide frequencies average Ti/Tv ratio=1.9.

Fig. 2. Saturation plots of Nuclear gene EF-1α. The number of transitions and transversions of each pairwise comparison of taxa are plotted against the TN93 model; corrected distance and the broken lines show the mean value of transition and transversion, respectively (×, Ts; Δ, Tv).

As for COI, 14 ingroup taxa were sequenced, and one outgroup sequence of Helicoverpa armigera was downloaded directly from GenBank (accession no. EU768941). Because the third codon positions of mitochondrial gene COI exhibited a tendency towards saturation, individual analysis was not performed based on this gene. A sequence of 617 bp for phylogenetic analysis was acquired, with 412 sites conserved, 205 variable and 160 parsimony-informative (for ingroup taxa only, 424 sites conserved, 193 variable and 140 parsimony-informative). These sequences were heavily biased toward A and T nucleotides, as expected from previous studies (Simon et al., Reference Simon, Fraiti, Beckenbach, Crespi, Liu and Flook1994; Lunt et al., Reference Lunt, Zhang, Szymura and Hewitt1996). Base-composition averages were 38.6% T, 15.8% C, 31.4% A and 14.2% G. Nucleotide frequencies average Ti/Tv ratio=0.8.

For combined data of EF-1α and COI sequences, altogether 18 taxa were included. The combined data matrix comprises 1577 characters, and all nucleotide positions were employed in the subsequent analysis. Of the total characters, variable sites accounted for about 29.4%, nucleotide substitution mainly with transition, transition/transversion ratios=1.3. The average distance was 11.5% in all sequences. Within the ingroup, the minimum distance was 5.8% (between Hypsomadius insignis and Oreta vatama), and the maximum distance was 14.5% (between Cyclidia substigmaria and Callidrepana patrana).

Phylogenetic analysis

MP, ML and Bayesian analysis were performed on both the EF-1α gene data set and the combined data set of EF-1α and COI using PAUP* and Mrbayes. The MP, ML and Bayesian trees were obtained, but only the Bayesian trees are shown below. The selected optimal models for each data set and the corresponding parameters for ML and MP analysis are listed in table 2. Topologies of these trees are very similar (figs 3 and 4). In the defined outgroup taxa, only two taxa, Helicoverpa armigera and Catocala fraxini, in the Noctuidae are at the base of the trees, whereas another two defined outgroup taxa, Odontopera bilinearia coryphodes and Tanaorhinus viridiluteata, in the Geometridae cluster together with defined ingroup taxa and form a monophyly with robust support (97% support value in combined Bayesian trees, 89% in EF-1α Bayesian trees).

Table 2. Parameters for ML and MP analysis.

I, proportion of invariable sites; G, gamma distribution shape parameter; CI, consistency index; RI, retention index.

Fig. 3. Bayesian tree of Drepanoidea reconstructed from nuclear EF-1α sequences. Bootstrap percentages of consensus clades with Bayesian tree from maximum parsimony/maximum likelihood (50% and greater) are shown above the branches, and Bayesian posterior probabilities (50% and greater) are shown below the branches.

Fig. 4. Bayesian tree of Drepanoidea reconstructed from nuclear EF-1α and mitochondrial COI sequences. Bootstrap percentages of consensus clades with Bayesian tree from maximum parsimony/maximum likelihood (50% and greater) are shown above the branches, and Bayesian posterior probabilities (50% and greater) are shown below the branches.

Nuclear EF-1α gene analysis

MP, ML and Bayesian analysis were performed on the EF-1α gene data sets. Topologies of these trees (fig. 3; Bayesian tree, single tree of MP and ML not shown, only the bootstrap percentages of consensus clades shown with Bayesian tree) support the monophyletic groupings of the Drepaninae and Thyatirinae respectively with high bootstrap values (70%, 86% support value in MP tree, 86%, 95% support value in ML tree, both clades with 100% support value in Bayesian tree). Drepaninae and Thyatirinae are sister taxa with a high bootstrap value (98%) in the Bayesian tree. The Cyclidiinae also constitute a monophyly. The Epicopeiidae constitute a monophyly with high bootstrap values (73% support value in MP tree, 67% support value in ML tree, 100% support value in Bayesian tree). Hypsomadius insignis and Oreta vatama in the Drepaninae form a monophyly with robust support (100%) in three topologies. The relationship among Cyclidiinae, Epicopeiidae, Geometridae and Drepaninae+Thyatirinae is not certain in nuclear EF-1α gene analysis.

Combined analysis of EF-1α and COI

The partition-homogeneity test (Farris et al., Reference Farris, Källersjö, Kluge and Bult1995) revealed significant heterogeneity between EF-1α and COI (P=0.01). The combined data of EF-1α and COI was used to perform MP, ML and Bayesian analysis in the following analysis. In MP, ML and Bayesian trees resulting from combined gene data of EF-1α with COI, topologies of these trees (fig. 4; Bayesian tree, single tree of MP and ML not shown, with only bootstrap percentages of consensus clades shown with Bayesian tree) all support both the Drepaninae and Thyatirinae as a monophyletic group respectively with high bootstrap values (74%, 84% support value in MP tree, 80%, 95% support value in ML tree, both clades with 100% support value in Bayesian tree). The Drepaninae and Thyatirinae emerge as sister taxa with high bootstrap value (93%) in the Bayesian tree. The Cyclidiinae with Drepaninae+Thyatirinae form a monophyly with 59% bootstrap value in the Bayesian tree. Hypsomadius insignis and Oreta vatama in the Drepaninae form a monophyly with robust support (100%) in all the topologies. The Epicopeiidae also form a monophyly with high bootstrap values (84% support value in MP tree, 55% support value in ML tree, 99% support value in Bayesian tree). The relationship between the Epicopeiidae and Geometridae is closer than that between the Epicopeiidae and Cyclidiinae+(Drepaninae+Thyatirinae) from combined analysis of EF-1α and COI genes.

Discussion

Subfamilies of the Drepanidae and their relationship

The Drepanidae have been defined on the basis that the three currently recognized subfamilies (Drepaninae, Thyatirinae and Cyclidiinae) share a distinctive synapomorphy in the adult, namely the possession of abdominal tympanal organs associated with the tergosternal sclerites, which connect tergum 1 with sternum 2 (Minet, Reference Minet1991; Minet & Scoble, Reference Minet, Scoble and Kristensen1999). This study investigated the phylogenetic relationship among the subfamilies of the Drepanidae by using molecular data from EF-1α and COI. The results showed that the monophyly of the Drepaninae, Thyatirinae and Cyclidiinae respectively was well supported (figs 3 and 4), and the sister relationship between the Drepaninae and Thyatirinae of Minet (Reference Minet2002) was validated. It did not support the sister relationship between the Drepanidae (=Drepaninae) and Cyclidiidae (=Cyclidiinae) postulated by Zhu & Wang (Reference Zhu and Wang1991).

Hypsomadius insignis and Oreta vatama in the Oreta group in the traditional definition of the Drepaninae form an independent clade with robust support (100%) in all the topologies. Additional evidence from morphology are: both body and wings coloured brown; body stout; tongue undeveloped; labial palpus short, broad and with dense hair, only reaching the underside of face; frenulum undeveloped; hind tibia with only one pair of spurs which have the same length; uncus of male genitalia flat and broad, turtle-head like, socii absent; tergum of metathorax in larva extended and with spinose process; fourth segment of abdomen with one pair of processes. While in other traditional Drepaninae, both body and wings are white or yellow in colour; body is slender; tongue is well developed; labial palpus is slender, reaching the lower edge of the face, the third segment is visible; frenulum is well developed; hind tibia has two pairs of spurs; uncus of male genitalia is stick- or fork-like, socii are present; tergum of the metathorax and fourth segment of the abdomen in larva are without processes. Both morphological characters and the molecular phylogenetic evidence strongly support that the Oreta group should be separated from the original Drepaninae and constitutes a sister group with it, and that the Oretinae should be restored as a separate subfamily. We propose a revised higher classification of the Drepanidae. In this classification, the Drepanidae should be divided into four subfamilies (Drepaninae, Oretinae, Thyatirinae and Cyclidiinae). The result of restoring the Oreta group to Oretinae perhaps has limitation because the taxa selected were available from China. Therefore, the results in the present analysis should need further studies, which will cover more genera in the Drepanidae and use more molecular markers to test further the strength of support for Oretinae and Drepaninae.

The taxonomic status of the Epicopeiidae

The taxonomic position of the Epicopeiidae has long been disputed. Using the traditional morphology division, it was found to belong to the Geometroidea when Zhu & Wang (Reference Zhu and Wang1991) performed a phylogenetic analysis on the latter group. Minet (Reference Minet1983) ascribed it to the Uranioidea, but later (Minet, Reference Minet1991) attributed it to the Drepanoidea based on four apomorphies shared between the Epicopeiidae and Drepanidae. However, Kuznetzov & Stekolnikov (Reference Kuznetzov and Stekolnikov2001) still thought the Epicopeiidae belonged to the Uranioidea. In the present work, all available data analysis (figs 3 and 4) supports the monophyly of the Epicopeiidae, while evidence on the relationship between Epicopeiidae, Drepanidae and Geometridae, derived from the combined analysis between the EF-1α and COI genes, showed that the Epicopeiidae and Geometridae have a closer phylogenetic relationship than that between the Epicopeiidae and Drepanidae (fig. 4). The following additional evidence of these relationships derived from morphology are: the absence in the Epicopeiidae of tympanal organs, which are well developed in the Drepanidae and Geometridae; the fact that in the Epicopeiidae the hindwing vein M2 is closer to M1 than to M3, and in the Geometridae M2, if present, is never closer to M3 than to M1, whereas in the Drepanidae M2 is close to M3; the fact that in the Epicopeiidae hindwing Sc+R1 is close to Rs in the base of the cell and distant from it beyond the cell, as in the Geometridae, while in the Drepanidae hindwing Sc+R1 is close to Rs at or beyond the end of the cell (Zhu & Wang, Reference Zhu and Wang1991; Minet & Scoble, Reference Minet, Scoble and Kristensen1999). Putting together the evidence from the morphology and molecular analysis results in the present work, we think that the family Epicopeiidae could not belong within the Drepanoidea. This result differs from the provisional phylogenetic hypothesis based on morphological characters alone (Minet, Reference Minet1991; Minet & Scoble, Reference Minet, Scoble and Kristensen1999). The relationship between the Epicopeiidae and Geometridae is closer than that between the Epicopeiidae and Drepanidae and accords with the phylogenetic analysis on the Geometroidea performed by Zhu & Wang (Reference Zhu and Wang1991) based on morphological characters. However, our results, especially the combined analysis result from the EF-1α and COI genes, did not support the placing of the Epicopeiidae within the Geometroidea. There are perhaps three possible conclusions. Either the selected gene segments in this paper are insufficient to distinguish or reconstruct the relationship between the Epicopeiidae and other groups; or the two species of Geometridae in this study are an insufficiently representative outgroup and do not reflect the range of the Geometridae sufficiently well to express the relationship between the Geometridae and Epicopeiidae; or, finally, the family Epicopeiidae belongs to neither the Drepanoidea nor the Geometroidea and should be placed in another superfamily. It has been ascribed to the Uranioidea many times in the past, which might be a clue to a realistic phylogenetic relationship (Inoue, Reference Inoue1954; Kuznetzov & Stekolnikov, Reference Kuznetzov and Stekolnikov2001). However, Minet sunk the Uranioidea in 1991 and placed its members in either the Geometroidea or the Drepanoidea. The phylogenetic relationships of the Geometroidea were outside the scope of this paper. Further work needs to be done as Wahlberg & Wheat (Reference Wahlberg and Wheat2008) proposed, preferably using the other molecular markers and longer gene sequences, to reconstruct the phylogenetics among the Epicopeiidae, Geometridae and Uraniidae and to verify the results inferred in this paper from EF-1α and the combined EF-1α and COI analysis. On the basis of this, it might be possible to establish whether the Uranioidea should be resurrected to contain the Epicopeiidae and part or all of the Uraniidae or, possibly, whether a separate superfamily, the Epicopeiioidea, should be established.

Acknowledgements

We warmly thank Prof. Le Kang and the Evolutionary Ecology Group in the State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS), who provided extremely good molecular experimental conditions to obtain molecular data. We also thank Sir Antony Galsworthy, the Natural History Museum, London, and Prof. Xuexin Chen in Zhejiang University, China, for reading the manuscript and providing valuable comments and suggestions. Thanks are also due to our colleagues Xuejian Wang, Songyun Lang, Jing Li, Wenhui Song, Fuqiang Chen and Nan Jiang for help in collecting samples. We also express our hearty thanks to two referees for their valuable suggestions and comments in that they checked and corrected the manuscript very carefully and their work made our manuscript much better. This project was supported by the National Science Foundation of China (no. 30670238) and Chinese Academy of Sciences Innovation Program (no. KSCX3-IOZ-0810).

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Figure 0

Fig. 1. Phylogeny trees (a) Imms' Geometroidea (Zhu & Wang, 1991); (b) Uranioidea, Geometroidea and Drepanoidea (Kuznetzov & Stekolnikov, 2001).

Figure 1

Table 1. Species information and GenBank accession numbers.

Figure 2

Fig. 2. Saturation plots of Nuclear gene EF-1α. The number of transitions and transversions of each pairwise comparison of taxa are plotted against the TN93 model; corrected distance and the broken lines show the mean value of transition and transversion, respectively (×, Ts; Δ, Tv).

Figure 3

Table 2. Parameters for ML and MP analysis.

Figure 4

Fig. 3. Bayesian tree of Drepanoidea reconstructed from nuclear EF-1α sequences. Bootstrap percentages of consensus clades with Bayesian tree from maximum parsimony/maximum likelihood (50% and greater) are shown above the branches, and Bayesian posterior probabilities (50% and greater) are shown below the branches.

Figure 5

Fig. 4. Bayesian tree of Drepanoidea reconstructed from nuclear EF-1α and mitochondrial COI sequences. Bootstrap percentages of consensus clades with Bayesian tree from maximum parsimony/maximum likelihood (50% and greater) are shown above the branches, and Bayesian posterior probabilities (50% and greater) are shown below the branches.