a molecular phylogeny of the tribe aphidini (insecta: hemiptera: aphididae) based on the...

Systematic Entomology (2008), 33, 711–721

A molecular phylogeny of the tribe Aphidini (Insecta:Hemiptera: Aphididae) based on the mitochondrial tRNA/COII, 12S/16S and the nuclear EF1a genes

HYO JOONG K IM and S EUNGHWAN LEEProgram in Entomology, School of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea

Abstract. A phylogeny of the tribe Aphidini (Hemiptera: Aphididae) wasreconstructed from three gene fragments: two mitochondrial regions, partialtRNA-leucine þ cytochrome oxidase II (tRNA/COII), partial 12S rRNA þtRNA-valine þ 16S rRNA (12S/16S) and one nuclear gene, the elongationfactor-1 alpha (EF1a). Bayesian phylogenetic (BP) analyses were performed oneach individual dataset of tRNA/COII, 12S/16S and EF1a, and maximumparsimony (MP), Bremer support test, maximum likelihood (ML) and BP analysiswere performed on the combined dataset. After comparing our molecularphylogenetic results with the classic classification based on morphological andecological data, we analysed three main issues: the monophyletic relationshipsamong tribes and subtribes, the validities of the latest taxonomic positions ofgenera and species and the status of certain Aphis species groups. Our resultsindicate that 36 of the species analysed, with the exception of Cryptosiphumartemisiae, are clustered within the clade of Aphidini. Also, the 28 speciesrepresentative of the subtribe Aphidina were separated from the eight speciesrepresentative of Rhopalosiphina; each monophyletic subtribe was supported bysignificant P-values in the combined analysis. According to our results, Crypto-siphum should be moved to Macrosiphini because it is more closely related to thegenera Lipaphis and Brevicoryne. The genus Toxoptera was recovered as non-monophyletic. In Rhopalosiphina, three genera, Hyalopterus, Rhopalosiphum andSchizaphis, were relatively closer to each other than to the genusMelanaphis. In therelationships between species-groups among Aphis, most species were separatedinto two main lineages; the fabae group seemed to be more closely related to thespiraecola and craccivora group rather than to the gossypii group.

Introduction

More than 4700 valid species of aphids (Hemiptera: Aphi-didae) have been reported throughout the world, approxi-

mately 2660 of which are included in the subfamilyAphidinae. This subfamily comprises two tribes: Aphidini,with approximately 730 species and Macrosiphini, withapproximately 1930 species (Remaudiere & Remaudiere,

1997).

The tribe Aphidini includes some major agricultural crop

pests that result in considerable economic losses throughoutthe world (Blackman & Eastop, 2006). Aphids belonging toAphidini have host ranges varying from grasses to woody

plants; representative pestiferous species include the cottonaphid, Aphis gossypii Glover 1877 and the green citrusaphid, A. spiraecola Patch 1914. Aphids belonging to thesubtribe Rhopalosiphina live mainly on Prunus spp. as the

primary hosts, and migrate to Poaceae spp. as the secondaryhosts. Pestiferous examples include the mealy plum aphid,Hyalopterus pruni (Geoffroy, 1762) and the bird cherry-oat

aphid, Rhopalosiphum padi (Linnaeus, 1758).Of the�640 valid species of the subtribe Aphidina, almost

600 species are included in the largest genus, Aphis. Subtribe

Correspondence: Seunghwan Lee, Program in Entomology,

School of Agricultural Biotechnology, Seoul National University,

San 56-1, Sillim9-dong, Gwanak-gu, Seoul, 151-921, Republic of

Korea. E-mail: [email protected]

# 2008 The AuthorsJournal compilation # 2008 The Royal Entomological Society 711

SystematicEntemologySystematic

Entomology

Rhopalosiphina is smaller, with about 90 species (Remau-diere & Remaudiere, 1997). Since Borner (1952) defined the

subtribe Aphidina using European genera, Eastop (1979)and Heie (1986) adopted the sub-tribal concept in Aphidini.The subtribe Rhopalosiphina is morphologically very sim-

ilar to Aphidina, but has some different characteristics:marginal tubercle on abdominal segment VII placed abovethe level of stigmal porus, processus terminalis longer than4.0 � basal part of ultimate antennal segment (if not, no

marginal tubercle on abdominal segment VII or processusterminalis 1.5 � basal part of ultimate antennal segment),different shape of siphunculus – triangular and shorter than

cauda in Melanaphis van der Goot, swollen at 1/3 apex andwell-developed flange in Rhopalosiphum Koch, slender andrather long likeMacrosiphini in Schizaphis Borner (Stroyan,

1984; Heie, 1986). These characteristics have been utilizedbroadly in the present morphological taxonomy to discrim-inate two groups, Aphidina and Rhopalosiphina. Since Heie(1986) and Stroyan (1984) suggested the systematics of

Aphidini based on morphological characters, almost all aphidtaxonomists have adopted the systematic concept of Aphidini.Most taxonomists have accepted the mutually mono-

phyletic relationship between the Aphidini and the Macro-siphini, within the subfamily Aphidinae (Shaposhnikov,1964; Stroyan, 1984; Remaudiere & Remaudiere, 1997;

von Dohlen & Moran, 2000; Blackman & Eastop, 2006).However, phylogenetic relationships involving the subtribesAphidina and Rhopalosiphina have been controversial.

Contrary to the common view that the two subtribes arelocated within a monophyletic Aphidini (Boner, 1952;Shaposhnikov et al., 1998), on the basis of morphologicaland biological characters, Heie (1992, 1994, hypothesized

that Aphidini might be a non-monophyletic group, withRhopalosiphina forming a sister group to Macrosiphini.von Dohlen and Teulon’s study (2003) was the first trial

to reconstruct relationships within Aphidini based on DNAsequences, which included 12 New Zealand and Australianspecies indigenous to the southern hemisphere and nine

northern-hemisphere species among Aphidini: they recov-ered the two subtribes as monophyletic. Also, recent molec-ular data, using seven representative species of Aphidini

(von Dohlen et al., 2006), strongly supported the mono-phyletic relationships between Aphidina and Rhopalosiphina.However, these results did not fully explain the relationshipsat generic and species levels within Aphidini. Although

these previous results indicated that the two subtribes havea sister group relationship, more genera and species shouldbe sampled for a better resolved phylogeny of Aphidini.

Based upon the previously published information, weused two mitochondrial gene regions, the tRNA-leucine þcytochrome oxidase II (tRNA/COII) region and the partial

12S rRNA þ tRNA-valine þ 16S rRNA (12S/16S) region,and one nuclear gene, the elongation factor-1 alpha (EF1a).Mitochondrial DNA is often used in insect molecularphylogenetics because many useful primers and informative

regions have been investigated (Simon et al., 1994). Mito-chondrial COII has been used in many phylogenetic worksof aphid subfamilies: Hormaphidinae (Stern, 1994, 1998;

Stern et al., 1997; von Dohlen et al., 2002), Lachninae(Normark, 1999, 2000) and Aphidinae (von Dohlen et al.,

2006). Mitochondrial 16S rDNA has also been used instudying the phylogeny of the superfamily Aphidoidea atthe tribal level (von Dohlen & Moran, 2000), but its

usefulness has not been validated for lower-level taxa (e.g.subtribe, genus, and species). Phylogenetic reconstructionusing EF1a was useful for the higher-level studies of insectphylogenetics (Friedlander et al., 1992, 1994, 1997). Phylo-

genetic works using the EF1a combined with some mito-chondrial regions were performed in the subfamilyLachninae (Normark, 1999, 2000) and in the genus Uroleu-

con (Moran et al., 1999). Recently, von Dohlen & Teulon(2003) and von Dohlen et al. (2002, 2006) used the EF1a þtRNA/COII combination in phylogenetic studies of the

subfamily Aphididae.The aim of the present study was to reconstruct enhanced

relationships among major taxa (e.g. Aphis, Toxoptera,Rhopalosiphum and Schizaphis) within Aphidini and to

improve the previously-published higher classification basedon morphological characteristics (Borner, 1952; Eastop,1979; Stroyan, 1984; Heie, 1986, 1994). Further, we tested

whether Aphidina and Rhopalosiphina are mutually mono-phyletic within the tribe Aphidini.

Materials and methods

Taxon sampling

A total of 37 ingroup species and four outgroup specieswere selected for molecular analysis (Supporting Informa-

tion ST1). Within the tribe Aphidini, we selected 29 speciesin four genera of Aphidina and eight species in four generaof Rhopalosiphina. Although sampling of taxa was limited

geographically, we included many species that are Holarcticin distribution.In the subtribe Aphidina, four genera, Aleurosiphon,

Aphis, Cryptosiphum and Toxoptera found commonly inthe Palearctic, were selected. The genus Aleurosiphon (Al.smilacifoliae) is characterized by being covered with heavy

wax in life. The genus Aphis, which is the largest genus inAphidinae and containing 90% of the species of Aphidina(Remaudiere & Remaudiere, 1997), is represented by fourmajor cosmopolitan species (A. craccivora, A. fabae, A.

gossypii and A. spiraecola) and four other morphologicallydistinct species (A. crinosa,A. farinosa,A. horii andA. nerii).To measure the distance between two subgenera [Aphis (s.

str.) and Aphis (Bursaphis)], A. (Bursaphis) oenotherae of thesubgenus Bursaphis was included. Two genera, Cryptosiphumand Toxoptera, which are the next largest Aphidina genera

after Aphis, were included in this study. In the subtribeRhopalosiphina, four major genera, Hyalopterus, Melana-phis,Rhopalosiphum and Schizaphis, were selected. These fourgenera contribute 90% of the species of Rhopalosiphina

(Remaudiere & Remaudiere, 1997). Hyalopterus pruni wasselected from the genus Hyalopterus, which is Holarctic indistribution and pestiferous on Peach (Prunus persica). The

712 H. Kim and S. Lee

# 2008 The AuthorsJournal compilation # 2008 The Royal Entomological Society, Systematic Entomology, 33, 711–721

genus Melanaphis, occurring mainly in far eastern Asia, isrepresented by Melanaphis japonica, which mainly lives on

Miscanthus. Four cosmopolitan species (Rhopalosiphummaidis, R. nymphaeae, R. padi and R. rufiabdominale) livingon Prunus as primary host, were selected from the genus

Rhopalosiphum. In the genus Schizaphis, two subgenera,Schizaphis (S. graminum) and Paraschizaphis (S. scirpi),were included.As outgroup taxa, four representative species, which have

been used frequently in molecular phylogenies, were selectedfrom the tribe Macrosiphini, the sister group of Aphidini.The Macrosiphini was selected as the sister group of

Aphidini in view of historical taxonomy of the subfamilyAphidinae (Heie, 1986); the sequence distances betweenAphidini and Macrosiphini were expected to be appropriate

for comparing each taxon in this study. Four macrosiphinespecies occur world-wide, and among them, three specieswere classified into dactynotine (Ac. pisum) and myzine (M.persicae and B. brassicae) groups within Macrosiphini (von

Dohlen et al., 2006). Even although we should have includedthe genus Macrosiphum in order to confirm Heie’s hypoth-eses (1992, 1994), the genusAcyrthosiphon is available to test

the hypotheses because it is closely related to Macrosiphumin the dactynotine lineage (von Dohlen et al., 2006).To acquire DNA samples, all aphid samples for molecular

work were preserved in 95% or 99% ethanol; samples foridentification (macerated specimens) were preserved in 80%ethanol. All samples and specimens are deposited in the

Insect Museum of College of Agriculture and Life Sciences(CALS), Seoul National University (SNU).

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from single individualsusing DNAzol� (Molecular Research Centre, Inc., Cincin-nati, OH) according to the manufacturer’s protocol. The

primers used for PCR amplification are listed in SupportingInformation ST2. Primers 2993þ (Stern, 1994) and A3772(Normark, 1996) were used to amplify tRNA/COII. PrimersEF3 coupled with EF2 (Palumbi, 1996) and EF6 (von Dohlen

et al., 2002) were used to amplify EF1a. Primer 12Sai (Simonet al., 1994) and 1473 (von Dohlen & Moran, 2000) wereused to amplify 12S/16S. Three primers, 12Sfr [a reverse of

12Sfi (Simon et al., 1991)], 1470a and 1472 (von Dohlen &Moran, 2000) were used as internal primers for sequencing.Gene fragments were amplified using BD Advantage�

two PCR Taq polymerase (BD Biosciences, San Jose, CA)and the reactionmixture in 50 mL volumes containing 1 pmol/mL of each primer, 0.25 nM/mL of each dNTP, 2.5 mM

MgCl2 and 0.1 ng/mL of genomic DNA. PCR was per-formed using a PTC-100 thermo-cycler (MJ Research, Inc.,Waltham, MA) employing the following protocol: an initialdenaturation at 958C for 5 min, followed by 35 cycles of

958C for 1 min; annealing temperatures (42–558C depend-ing on the primer sets) for 1 min; extension at 728C for1 min; final extension at 728C for 5 min. The primer-specific

annealing temperatures of each primer set were used as

follows: 42–458C for tRNA/COII, 438C for 12S/16S and 52–558C for EF1a. PCR products were purified by Microcon

YM-100 (Millipore, Temecula, CA), and sequenced directlyusing the automated sequencer (ABI Prism 3730 XL DNAAnalyzer; ABI Prism, Foster City, CA) in the NICEM (the

National Instrumentation Center for Environment Man-agement, SNU). All sequences acquired in this study havebeen deposited in GenBank under the accession numbersgiven in Supporting Information ST3.

Alignment and characterization of gene fragments

For the alignments, reference sequences of three generegions (von Dohlen & Moran, 2000; von Dohlen &Teulon,

2003) used to compare with our sequences were retrievedfrom the GenBank. Raw sequences were examined andcorrected using SeqMan�II (version 5.01, 2001; DNA-star�). All DNA sequences of three gene fragments were

aligned using CLUSTAL X (version 1.83, 2003; Thompsonet al., 1997; used default setting). The intron splicingjunctions of nuclear EF1a sequences were identified and

compared using MEGA 3.1 (Kumar et al., 2004), and wedecided to include three intron regions in which some in-formative sites were found among taxa in this study. Some

ambiguous sites on 12S/16S and EF1a were removed usingGBLOCKS 0.91b (Castresana, 2002; used the default settingexcept for allowing the gap option by ‘with half’).

Phylogenetic analyses

Maximum parsimony (MP) analyses were performed withPAUP*4.0b10 (Swofford, 1998), using the heuristic searchprocedure, with 1000 random additions of sequences and

ten trees held at each pseudoreplicate, and the tree bisectionreconnection (TBR) branch swapping method. All charac-ters were treated as unordered and equally weighted for MP

analysis. Bootstrapping of the MP analysis was conductedunder the heuristic search procedure, with 1000 replicatesand a maxtree setting of 200 trees. Branch support was alsoassessed by Decay Index values (Bremer, 1988).

For maximum likelihood (ML) analysis, MODELTEST

Version 3.06 (Posada & Crandall, 1998) was used to selectthe significantly best-fitting nucleotide substitution model,

and then PAUP* settings were optimized from the data of theselected model before searching. ML analyses were alsoperformed with PAUP*, using the heuristic search procedure,

with 100 random additions of sequences and ten trees held ateach pseudoreplicate and the TBR branch swappingmethod. Bootstrapping of the ML analysis was also con-

ducted under the heuristic search procedure, with 100replicates and a maxtree setting of 200 trees. The parti-tion-homogeneity test (Farris et al., 1994), as implementedin PAUP*, was performed to test for significant phylogenetic

conflict between the three gene fragments.Bayesian phylogenetic (BP) analysis was performed using

MRBAYES (version 3.1.1; Ronquist &Huelsenbeck, 2003) for

the single and combined datasets. When the combined

Molecular phylogeny of the tribe Aphidini 713


analysis was performed, the data were partitioned intotRNA/COII, 12S/16S and EF1a. Four chains were run,

starting from a random tree, with the best-fit model ofmolecular evolution also estimated by the MODELTEST foreach analysis. The number of generations of Markov Chain

Monte Carlo and tree sampling frequency were 5 000 000generations and 100 generations, respectively. The chainappeared to reach the stationary phase by about the25 000th generation, thus the first 250 trees were ‘the burn

in’ of the chain and phylogenetic relationships were basedon the subsequent 99 502 trees. After the summarizing treecommand was assigned, the posterior probabilities were

obtained accordingly.

Results

Characteristics of the three gene fragments

For the mitochondrial tRNA/COII dataset, the numberof aligned sites was 760 base pairs (bp), but 702 bp were

used for the analyses after excluding primer sites. Amongthe selected 702 bp, 223 were variable and 157 were par-simony informative. The uncorrected sequence divergence

among taxa for tRNA/COII ranged from 0.1 to 10.1% withan average of 6.4%. Average proportions of T : C : A : Gwere 39 : 12 : 41 : 8 with a narrow standard deviation. For

the mitochondrial 12S/16S dataset, the number of alignedsites was 1680 bp, but 1600 bp were used for the analyses,excluding primer and ambiguous sites by GBLOCKS. Amongthe selected 1600 bp, 421 were variable and 205 were

parsimony informative. The uncorrected sequence diver-gence among taxa for 12S/16S ranged from 0 to 7.5% withan average of 3.6%. Average proportions of T : C : A : G

were 45 : 5 : 38 : 10 with a narrow standard deviation. Forthe nuclear EF1a dataset, the number of aligned sites was1100 bp, but 987 bp were used for the analyses, excluding

primer and ambiguous sites by GBLOCKS. Exon sites were805 bp and intron sites were 182 bp. Among the selected987 bp, 328 were variable and 217 were parsimony infor-mative. The uncorrected sequence divergence among taxa

for EF1a ranged from 0 to 11.6% with an average of 5.4%.Average proportions of T : C : A : G were 29 : 20 : 30 : 21with a narrow standard deviation. Sequences of distinct

samples of both Aphis fabae and A. newtoni were identical inthe two datasets of 12S/16S and EF1a.After aligning and combining the three fragments, the

complete combined dataset of three gene fragments con-sisted of 3289 bp, of which 927 were variable and 579 wereparsimony-informative. The uncorrected sequence diver-

gence among taxa for the complete dataset ranged from0.1 to 8.2% with an average of 4.7%.

Phylogenetic analyses

Analyses of the single datasets. Analyses of the singledatasets were performed by the BP analyses, following theresults of the MODELTEST analysis, the general time revers-

ible (GTR þ I þ G) model was selected as the best-fit modelfor each dataset of the tRNA/COII, 12S/16S and EF1a. In

the analyses of the three individual datasets, tRNA/COII,12S/16S and EF1a, the branch support values on each treewere insufficient to resolve relationships between lower

taxa, but the higher clades forming subtribal or tribal clusterwere highly supported. The monophyly of the tribe Aphi-dini, minus Cryptosiphum artemisiae, was supported clearlyin all three analyses (PB � 99). In the tRNA/COII, 12S/16S

and EF1a trees, C. artemisiae was related closely to twomyzine species in the tribe Macrosiphini. The subtribeAphidina formed a monophyletic group supported with

high statistical values in 12S/16S (PB ¼ 96) and EF1a(PB ¼ 100), and the clade of the subtribe Rhopalosiphinawas also highly supported in tRNA/COII (PB ¼ 100) and

12S/16S (PB ¼ 96). However, the positions of two species,A. crinosa andM. japonica, were not congruent between thetRNA/COII and EF1a trees. Aphis crinosa was positionedoutside of the Aphidini in the tRNA/COII tree, and M.

japonica was placed outside of the Rhopalosiphina in theEF1a tree. In the Aphidina, two Aphis clades, similar to theclade E and F below, were recovered in all trees. The

members of each Aphis clade in the 12S/16S tree werecongruent with those in the EF1a tree. Also, the analysesof double datasets (tRNA/COII þ 12S/16S, 12S/16S þEF1a and tRNA/COII þ EF1a) were better resolved withhigher supports than those of single datasets. However,most branches differed in support values in different trees,

and the tree topologies were incompletely congruent as wellas in the single datasets. Consequently, the analyses of thedual datasets are not presented here.

Analyses of the combined dataset. As the partition-homogeneity test found no significant phylogenetic conflictin each combination between the tRNA/COII, 12S/16S and

EF1a data (0.23 � P � 0.27), they were combined into onedataset for phylogenetic analyses. MP analysis yielded 12equally parsimonious trees, with a tree length of 2630

(CI ¼ 0.489, RI ¼ 0.574). A strict consensus cladogram ofthe 12 most parsimonious trees, with bootstrap P-values(PP) of the MP analysis and Decay Indices (DI), is shown in

Fig. 1 and Supporting Information Fig. S1. For MLanalyses, following the results of the MODELTEST analysis,the general time reversible (GTR þ I þ G) model wasselected as the most suitable for the combined dataset. ML

analysis produced a single tree that, although differingslightly from the MP tree (Fig. 1 and Supporting Informa-tion Fig. S1), was almost identical to the post-burnin

consensus tree from BP analysis. The ML tree with boot-strap P-values (PL) and PB of BP analysis is presented inFig. 2 and Supporting Information Fig. S2.

Except for C. artemisiae, the monophyly of 38 Aphidinispecies (clade A; PP/DI/PB/PL ¼ 100/39/100/100) (Figs 1, 2and Supporting Information Figs S1, S2) was well supportedagainst the four macrosiphine species used as outgroup taxa.

In the tribe Macrosiphini, C. artemisiae was clustered withB. brassicae and L. pseudobrassicae, supported by highP-values (clade B; PP/DI/PB/PL ¼ 100/22/100/100). Myzus



persicae and the clade B together formed again a mono-phyletic, myzine group (clade C; PP/DI/PB/PL ¼ 86/8/99/81). The tribe Aphidini was separated completely into twomonophyletic lineages, corresponding to subtribes Aphidina

and Rhopalosiphina.The clade of Aphidina (clade D; PP/DI/PB/PL ¼ 99/18/

100/100) had strong support and included three genera,

Aleurosiphon, Aphis and Toxoptera. In Aphidina, twomonophyletic groups of the genus Aphis were recognizedin the combined analysis as well as in the single analysis. Thegossypii group (species resembling A. gossypii; clade E; PP/

DI/PB/PL ¼ 99/15/100/99) consisted of 10 species, A. gos-sypii, A. taraxacicola, A. egomae, A. clerodendri, A. sumire,A. hypericiphaga, A. ichigo, A. ichigocola, A. glycines and A.

Fig. 1. Strict consensus tree of 12 equally parsimonious trees (length ¼ 2630) using the combined sequences of tRNA/COII (702 bp), 12S/16S

(1600 bp), and EF1a (987 bp). Numbers above branches indicate support values from bootstrap test with 1000 replicates, and numbers under

branches indicate Decay Index values. Letters enclosed in black circles are discussed in the text. Colour version available in Supporting

Information Fig. S1.



sanguisorbicola. Aphis gossypii was sister to the rest of cladeE, and had small genetic distances from A. taraxacicola,A. egomae and A. clerodendri. On the other hand, the clade

F (PP/DI/PB/PL ¼ 74/4/100/90) was composed of ninespecies; six species, A. fabae, A. fukii, A. hederae, A. newtoni,A. neospiraecola, A. rumicis, as the fabae group (species

resembling A. fabae; clade G; PP/DI/PB/PL ¼ 90/5/100/96),two species, A. spiraecola andA. kurosawai, as the spiraecola

group (species resembling A. spiraecola) and A. craccivora.In the fabae group, four species, A. fabae, A. fukii, A.hederae and A. newtoni formed a clade with strong support.

The spiraecola group was also highly supported (PB/PL ¼100/92) within clade F. Although relationships between thespiraecola group, fabae group and A. craccivora differed

between MP and ML trees, the clade F including the fabaegroup, spiraecola group and A. craccivora was highly

Fig. 2. The phylogenetic relationships inferred from maximum likelihood (ML) based on the GTR þ I þ G model using the combined

sequences of tRNA/COII (702 bp), 12S/16S (1600 bp) and EF1a (987 bp). Posterior probabilities of Bayesian phylogenetic (BP) analysis

followed by support values ofML bootstrap test with 100 replicates are given on branches. Bold branches emphasise clades supported by�99%

in BP analysis and by �75% in ML bootstrap test. Letters enclosed in black circles are discussed in the text. Colour version available in

Supporting Information Fig. S2.



supported in both trees (Figs 1, 2 and Supporting InformationFigs S1, S2).

Toxoptera odinae showed a close relationship to clade Eand clade F consisting of Aphis species-groups. In the MPtree, both Aphis (Bursaphis) oenotherae and T. odinae

formed a sister group to clade F. However, in the ML andBP trees, T. odinae still remained inside to the clade of cladeE þ clade F þ itself, even although A. oenotherae wasplaced outside of the clade. In addition to A. oenotherae,

four distinct Aphis species (A. crinosa, A. farinosa, A. horiiand A. nerii) and other two genera (Aleurosiphon andToxoptera) were positioned outside of the clade E þ clade

F þ T. odinae group.In the Rhopalosiphina, the clade H (PP/DI/PB/PL ¼ 0/9/

100/99), composed of three genera, Hyalopterus, Rhopalo-

siphum and Schizaphis, was highly supported. However,Melanaphis was placed outside of clade H, and did not showa significant relationship to other genera.

Discussion

Monophyly and relationships within Aphidini

The MP and ML trees supported the monophyly of theAphidina and Rhopalosiphina in the combined analyses(Figs 1, 2 and Supporting Information Figs S1, S2). Our

results confirm the mutually monophyletic relationships ofthe two subtribes presented in previous phylogenetic anal-yses based on tRNA/COII and EF1a (von Dohlen &Teulon, 2003; von Dohlen et al., 2006). In the single gene

analyses, the tRNA/COII, 12S/16S and EF1a resultsshowed different tree topology and different supportingvalues. These differences may be caused by different evolu-

tionary rates of each gene. In the case of the analysis oftRNA/COII, the node of Aphidini, excluding A. crinosa,was supported with a relatively low P-value (PB ¼ 74)

compared with the other analyses. Because tRNA/COIIsequences were likely to experience saturation in many ofthe deeper branching events, homoplasy probably ac-counted for the inability of these genes to show bootstrap

support even for the monophyly of Aphidina (von Dohlen &Teulon, 2003; von Dohlen et al., 2006). In contrast, theanalysis of EF1a supported the monophyly of Aphidina

with a relatively high P-value and could resolve the deeprelationships at the subtribal level. However, in the analysisof EF1a, the genusMelanaphiswas not included in the clade

of the rest Rhopalosiphina (PB ¼ 100) and it seemed to showdifferent evolutionary rates to the mitochondrial genes. The12S/16S supported the monophylies of the Aphidina and

Rhopalosiphina with the highest P-values for all threemarkers. In this study, the 12S/16S was relatively informativeand useful in confirming the divergences of tribal andsubtribal relationships, although also proven suitable for

reconstructing the subfamily or higher level phylogeny inthe superfamily Aphidoidea (von Dohlen & Moran, 2000).In accordance with the results of von Dohlen & Tuelon

(2003) and von Dohlen et al. (2006), we confirm that

Rhopalosiphina is a sister group of Aphidina within amono-phyletic Aphidini (Figs 1, 2 and Supporting Information

Figs S1, S2). Morphologically, Aphidini, comprising Aphi-dina and Rhopalosiphina, have been considered to beclearly different from Macrosiphini in two morphological

characteristics: the presence of a marginal tubercle onabdominal segments I and VII, and a relatively long distancebetween the stigmal pori of abdominal segments I and II(Stroyan, 1984; Heie, 1986). Ecologically, both Aphidina

and Rhopalosiphina are similar in that some species (e.g., A.sumire, M. japonica) are attended by ants that constructearthen shelters for the aphids (Blackman & Eastop, 2006).

It is reasonable to regard Rhopalosiphina as more closelyrelated to Aphidina than to Macrosiphini.The morphological characteristic subdividing the two

subtribes, Aphidina and Rhopalosiphina, is the position(vertical level) of the marginal tubercle relative to that of thestigmal porus on abdominal segment VII (Stroyan, 1984;Heie, 1986). Although the relative length of the processus

terminalis compared with antennal segment VI base has alsobeen used to diagnose the two subtribes, it may be homo-plastic because there are many exceptional species without

relation to the presence or absence of marginal tubercles onabdominal segment I and VII (Heie, 1986; Lee & Kim,2006). Ecologically, Aphidina have various primary hosts in

eudicots, whereas Rhopalosiphina are specific to Rosaceae(e.g., Prunus,Malus, Pyrus) (Blackman & Eastop, 1994; Leeet al., 2002; Blackman & Eastop, 2006; Lee & Kim, 2006;

von Dohlen, 2006). Additionally, secondary hosts of Rho-palosiphina species are limited to monocots, especiallyPoaceae, with few exceptions (Heie, 1986; Blackman &Eastop, 1994; Lee et al., 2002; Blackman & Eastop, 2006;

Lee & Kim, 2006), whereas Aphidina (e.g., Aphis, Toxop-tera) might have diversified secondary hosts excludingPoaceae. The monophylies of Aphidina and Rhopalosiphi-

na are reasonable in light of the current molecular data aswell as the morphological and ecological data of previousworks (Heie, 1986; Blackman & Eastop, 1994; Lee et al.,

2002; Blackman & Eastop, 2006; Lee & Kim, 2006; vonDohlen, 2006).

Comparison of the generic and species relationships withclassic classification

In the MP and ML trees (Figs 1, 2 and SupportingInformation Figs S1, S2), Cryptosiphum is more closely

related to Brevicoryne brassicae and Lipaphis pseudobrassi-cae than it is to Myzus persicae, which has been included inthe myzine group of Macrosiphini (von Dohlen et al., 2006).

Historically, Borner (1952) treated Cryptosiphum as forminga separate tribe of the subfamily Anuraphidinae (nowincluded in Macrosiphini), closely related to Anuraphis delGuercio and Dysaphis Borner, but it was relocated into the

subtribe Aphidini by Shaposhnikov (1964) and Eastop(1979) (as cited in Stroyan, 1984). Stroyan (1984) thenplaced it again in Macrosiphini according to Borner

(1952). More recently, aphid taxonomists have agreed that



Cryptosiphum should be placed in the subtribe Aphidina(Heie, 1986; Remaudiere & Remaudiere, 1997). Its morpho-

logical characteristics closely resemble those of the Macro-siphini, although some characteristics do correspond withthose of Aphidini (Heie, 1986). The key morphological

characteristics of the genus Cryptosiphum differ from thediagnosis of the subtribe Aphidina as follows: siphunculusand cauda shorter than width and almost indiscernible;marginal tubercles absent on abdominal segment I-VII.

Moreover,Cryptosiphum species have a diagnostic charactermatching the Macrosiphini as follows: the distance betweenstigmal pori on abdominal segments I and II is shorter than

3� diameter of stigmal porus, and usually shorter than 0.5�the distance between stigmal pori on abdominal segments IIand III (Stroyan, 1984; Heie, 1986). As shown from our

results as well these morphological characteristics, the genusCryptosiphum should be moved into the tribe Macrosiphini.Within the Aphidina, the MP and ML trees showed that

Aleurosiphon and Toxoptera tended to be located, along

with other distinct Aphis species, as outside of a larger groupof Aphidina species complexes (Figs 1, 2 and SupportingInformation Figs S1, S2). The three species of the genus

Toxoptera were scattered within Aphidina, and thus did notform a clade in MP and ML trees. Especially, T. odinaetended to be positioned into the monophyletic group,

consisting of clade E þ clade F þ itself, which was wellsupported (PB ¼ 98) in BP analysis.Toxoptera species may originate from different lineages,

as reflected in the divergent position of each species in thetrees (Figs 1, 2 and Supporting Information Figs S1, S2),and possibly should be considered as a non-monophyletic.Morphologically, the genus Toxoptera is characterized by

a stridulatory apparatus consisting of lateroventral ridgeson the abdomen and peg-like hairs on the hind tibiae(Blackman & Eastop, 2006). This character may be plesio-

morphic, however, because weak lateroventral ridges on theabdomen are found also on most Aphis species, and A. neriieven has a few peg-like hairs, albeit weakly developed and

irregularly located, on the hind tibiae (Blackman & Eastop,1994; Lee & Kim, 2006).Aphis crinosa, A. nerii, A. farinosa and A. horii, which are

distinct morphologically (i.e. not believed to be parts oflarger species complexes), were placed on outer nodesrelative to the clade of the other congeneric species (Figs 1,2 and Supporting Information Figs S1, S2). As shown from

the results, these four species might slightly diverge from theother consubgeneric species (clade E þ clade F) such as thesubgenus Bursaphis (A. [B.] oenotherae). Divergences for the

molecular sequences seemed to be related to their morpho-logical differences and unusual hosts compared with theother consubgeneric species. Morphologically, some char-

acteristics of A. crinosa differ from those of the congeners: ithas a body covered with heavy wax in life, more numerousand longer setae throughout the body, siphunculi withoutimbrication and apical flanges, of which length is as long as

or shorter than cauda, and relative distance between mar-ginal tubercle and spiracle on abdominal tergum VII isslightly shorter than that of other Aphis species. Comparing

its host relationship with the other Aphis species, A. crinosais unusual because it is monoecious and highly specific to

Ligustrum sp. in the family Oleaceae (distributed in Korea,China and Japan; Paik, 1969, 1972). Aphis nerii also hasunusual hosts included in the family Asclepiadaceae or

Apocynaceae and unusual morphological characteristicscompared with congeneric species: thick and long siphun-culi, big cauda, weakly developed stridulatory apparatussimilar to the genus Toxoptera (Blackman & Eastop, 1994,

2006; Lee & Kim, 2006). Like A. nerii, A. farinosa, livingmainly on Salix spp., and A. horii, living mainly onSambucus spp., are morphologically distinct from the other

congeneric species (e.g. long and slender siphunculi; Taka-hashi, 1966; Lee et al., 2002; Lee & Kim, 2006). Neverthe-less, we could not find conclusive evidence to resolve the

relationships between the four distinct Aphis species andother Aphidina genera (Aleurosiphon and Toxoptera)because their branch support values were very low in ourresults (Figs 1, 2 and Supporting Information Figs S1, S2).

In Rhopalosiphina, the statistical support (PB/PP ¼ 91/75) for Melanaphis þ clade H was rather lower than that ofthe clade H (Figs 1, 2 and Supporting Information Figs S1,

S2). Morphologically, Rhopalosiphum resembles Schizaphis,with cylindrical and slender siphunculi, and their medialvein of the forewing is only once-branched; in contrast,

Melanaphis is different from these two genera in having veryshort and truncated conical siphunculi and a forewing witha twice-branched medial vein (Stroyan, 1984; Heie, 1986).

Also, body shape of Hyalopterus, Rhopalosiphum andSchizaphis is vertically slenderer than Melanaphis (Lee &Kim, 2006). Although Melanaphis was placed closest to R.nymphaeae based on the tRNA/COII and 12S/16S within

Rhopalosiphina, it was clearly placed outside of the othergenera of Rhopalosiphina in the EF1a analysis. In thisstudy, EF1a results were more consistent with the morpho-

logical divergence of each genus of Rhopalosiphina than themitochondrial genes, and the morphological and ecologicalsimilarities are congruent with molecular similarities for the

generic relationships in Rhopalosiphina (von Dohlen &Teulon, 2003; von Dohlen et al., 2006).

Species-group hypotheses in the genus Aphis

The genus Aphis is a species-rich group with nine sub-

genera; 90% of the species are included in the subgenusAphis (Remaudiere & Remaudiere, 1997; Blackman &Eastop, 2006). Recent aphid taxonomists have assumed that

Aphis consisted of some major entities centred on polyph-agous and broadly distributed species (Stroyan, 1984; Heie,1986). These grouping concepts seemed to be derived from

the morphological similarity of species complexes, such asthe fabae and the frangulae complexes (Heie, 1986; Holman,1987). Stroyan (1984) discussed three complexes, A. fabae,A. frangulae Kaltenbach 1845, and A. narsturtii Kaltenbach

1843, each grouped by morphology and host relationships.Heie (1986) suggested that some groups composed ofclosely-related species in Aphis are associated with plant



families, such as the craccivora group feeding on the familyFabaceae. We show that most Aphis species in our analyses

are sorted into four species-groups centred on A. craccivora,A. fabae, A. gossypii (¼A. frangulae gossypii) and A.spiraecola (each was named ‘- group’ which means ‘species-

group resembling �’; Figs 1, 2 and Supporting InformationFigs S1, S2). The gossypii group in this study was treated asthe frangulae group or frangulae-like group in previousstudies (Stroyan, 1984; Heie, 1986). As Pashchenko (1997)

assembled some similar species host-specific on Spiraea, thespiraecola group was added to the concept of the Aphisspecies-group living on Spiraea and resembling A. spiraecola

in this study. Recently, Coeur d’acier et al. (2007) haveexamined the phylogenetic relationships of the those spe-cies-groups, the craccivora group (as black backed species),

fabae group (as black species), gosypii group (as frangulae-like species) and spiraecola group (as only A. spiraecola)within the genus Aphis, the mutual relationships of thosespecies-groups are substantially congruent with those of our

results even although different molecular sequences (COI/COII and cytochrome b) were used with some differentsamples ofAphis species, including regional species in Europe,

that mostly do not distribute in eastern Asia. Therefore, theAphis species-grouping hypotheses are likely to be reasonableand are useful in understanding the phylogenetic relationships

of Aphis.Each species included in the gossypii group (clade E)

shows some differences in coloration, host-specificity and

wax pattern in life, but they resemble each other in themacerated specimen as follows: setae relatively short inwhole body; coloration pattern similar on head, abdomenand appendages; cauda pale (at least paler than siphunculi)

with few setae (about eight or fewer); number of setae onabdominal tergum VIII, (mostly two) not variable (Stroyan,1984; Heie, 1986; Lee & Kim, 2006). Members of this

monophyletic species-group may have adopted differenthost plants, diverging from a recent ancestor.In the fabae group (clade G), four species, A. fabae, A.

fukii, A. hederae and A. newtoni, appeared to be veryclosely related (PP/DI/PB/PL ¼ 100/24/100/100) and infact they are morphologically very similar with black or

blackish-brown coloration and a slight wax covering in life(Heie, 1986; Figs 1, 2 and Supporting Information Figs S1,S2). Two additional species, A. neospiraeae and A. rumicis,which clustered with the above four species, also are darkly

coloured with slight wax, but they are distinct from theother four species, in having a dark abdominal tergum (asa result of polygonal plates) as observed in the macerated

specimen (Heie, 1986). Although we expected that A.neospiraeae would be closely related to A. spiraecola,sharing hosts in the genus Spirea and some morphological

similarities (Pashchenko, 1997), A. neospiraeae was relatedmore closely to A. rumicis than the spiraecola group.Overall, these six species of the fabae group are similar toeach other with black coloration in life, and have the

following morphological similarities: all body setae, butespecially those on the legs, relatively longer than in thegossypii; cauda black (concolourous with siphunculi) with

many setae (about 15 or more); number of setae on abdom-inal tergum VIII, numerous (mostly six) and variable (3–8).

Sister group to the fabae group was the clade consisting ofA. spiraecola and A. kurosawai (Fig. 2 and SupportingInformation Fig. 2), which share host plants, such as

Artemisia and Chrysanthemum, in the family Asteraceae(Lee et al., 2002; Lee & Kim, 2006). Aphis kurosawai,described originally from Japan, is distributed in far easternAsia, with its primary hosts still unknown (Takahashi, 1966;

Lee & Kim, 2006). Aphis kurosawai and A. spiraecolaresemble each other in almost all morphological character-istics except for body size (A. kurosawai is smaller), and

possibly originated from a recent common ancestor. Aphisspiraecola is also associated with A. spiraephaga F.P. Muller1961 and A. spiraephila Patch 1914 living on Spiraea

(Pashchenko, 1997), and these species, including A. kurosa-wai, have similar caudal characteristics: black coloration(concolourous with siphunculi), with about ten or moresetae and a slightly elongated ovoid shape (generally tongue-

or triangular-shaped in the fabae group or gossypi group).Based on morphological and ecological similarities, theyhave been classified to the spiraecola group. We recognized

that the two spiraecola group species were closer to the fabaegroup than to the gossypii group in spite of pale colorationof them. Also, the clade of the spiraecola group þ fabae

group is supported by low P-values (PB/PL ¼ 89/79) in BPandML analyses (Fig. 2 and Supporting Information Fig. 2).Aphis craccivora appeared to be closely related to the fabae

group and spiraecola group as sister taxon in clade F, evenalthough MP and ML trees showed slightly different topolo-gies (Figs 1, 2 and Supporting Information Figs S1, S2). Aswell as A. craccivora, A. craccae Linne 1758, A. coronillae

Ferrari 1872 and A. cytisorumHartig 1841 have been consid-ered as the part of the craccivora group by Heie (1986),prefering hosts in the family Fabaceae. The monophyly of

those species in the craccivora group, together with othermorphologically similar species, have been strongly supportedin the previous study (Coeur d’acier et al., 2007). Morpholog-

ical characteristics in the craccivora group include a darklypigmented cuticle on the entire dorsal abdomen, in themacerated specimen and black coloration in life (Heie,

1986).Like the caseof the spiraecolagroup,wedidnot confirmthe taxonomic position of the othermembers of the craccivoragroup, but may infer that the phylogenetic position of thecraccivora group is the same as that of A. craccivora itself.

Comparedwith the gossypii group, themonophyletic relation-ship of the craccivora, fabae and spiraecola groups seems to bereasonable because species in the clade F havemany setae and

dark coloration (Heie, 1986; Pashchenko, 1997; Lee & Kim,2006; Coeur d’acier et al., 2007). Therefore, we consider the(craccivora group þ fabae group þ spiraecola group) as orig-

inating from a different lineage of the gossypii group.Except for A. oenotherae of the subgenus Bursaphis and

four distinct species (A. crinosa, A. horii, A. nerii and A.farinosa), species of the genus Aphis appear to be separated

into two distinct lineages (clade E and clade F). Thesemolecular results agree with previous morphological anal-yses (Stroyan, 1984; Heie, 1986; Pashchenko, 1997). Our



results also suggest that the evolution of the genus Aphis, asa highly host-specific aphid group, is closely associated with

that of the host plants, as shown in other studies of the sub-family Aphidinae (von Dohlen &Moran, 2000; Blackman &Eastop, 2006; von Dohlen et al., 2006). Our molecular

phylogeny was relatively well congruent with previous speciesgrouping-hypotheses based on morphological and ecologi-cal concepts (Stroyan, 1984; Heie, 1986; Pashchenko, 1997;Coeur d’acier et al., 2007).

Supporting Information

Additional Supporting Information may be found in the

online version of this article from Wiley InterScience underDOI reference: doi: 10.1111/j.1365-3113.2008.00440.x

ST1 Collection information for species used in this study.ST2 Primers used forDNAamplification and sequencing.

ST3 GenBank sequence accession numbers.Figure S1 Figure 1, colour.Figure S2 Figure 2, colour.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)should be directed to the corresponding author for the article.

Acknowledgements

Many thanks to Dr Robert Foottit andMr. Eric Maw of theCanadian National Insect Collection, Ottawa, Canada, andDr Jaroslav Holman of the Institute of Entomology, Czech

Academy of Science, Ceske Budejovice, Czech, Dr Carol D.vonDohlen, InsectMuseum, Utah State University, U.S.A.,for the loan of Aphidini specimens from their regions for

comparison.We also thankDr Colin Favret, Chief Scientist,AphidNet, MD, U.S.A. for reading the manuscript andmany valuable comments. Special thanks to Dr Hyun Jeong

Choe, United States Forces Korea, for advice on phyloge-netic analyses, Mr. Woonhoon Lee, Seoul National Uni-versity, for assistance with the molecular experiments, and

Mrs. Youngboon Lee, Seoul National University, for pre-paring many aphid specimens. This study is partiallysupported by the Eco-technopia 21 project ‘‘Constructionof the Korean Tree of Life’’.

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Accepted 7 April 2008



a molecular phylogeny of the tribe aphidini (insecta: hemiptera: aphididae) based on the...

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