systematics molecular and morphological identiï¬cation of

SYSTEMATICS

Molecular and Morphological Identification of the Soybean Aphid andOther Aphis Species on the Primary Host Rhamnus davurica in Asia

HYOJOONG KIM,1 KIM A. HOELMER,2 WONHOON LEE,1 YOUNG-DAE KWON,3

AND SEUNGHWAN LEE1,4

Ann. Entomol. Soc. Am. 103(4): 532Ð543 (2010); DOI: 10.1603/AN09166

ABSTRACT The soybean aphid, Aphis glycines Matsumura, was recently introduced into NorthAmerica where it has become a serious pest of soybean, Glyine max (L.) Merr. In its native range ofnortheastern Asia, A. glycines undergoes host alternation between the soybean (summer host) andDahurian buckthorn, Rhamnus davurica Pallas (winter host). On the primary host, it is difÞcult todiscriminate A. glycines from coexisting, morphologically similar Aphis species, including seasonalpolymorphisms of each species (e.g., gynopara, ovipara, and male). Two widely used molecularmarkers, the mitochondrial cytochrome c oxidase I (COI) “barcode” region (658 bp) and the partialtRNA-leucine � cytochrome c oxidase II (tRNA/COII, 702 bp), were used to analyze 31 individualsof Aphis from R. davurica in Asia and compared with 26 closely related Aphis species. We found thatthree different species, Aphis gossypii Glover and two new and undescribed putative Aphis species,occur together with A. glycines on R. davurica. All these species were genetically close within thegossypii group. A study of 28 quantitative morphological characters showed thatA. glycines,A. gossypii,andoneof thenewspecieswerequite similarwithonlya fewcharactersdiffering signiÞcantlybetweenspecies.

KEYWORDS Aphis glycines, Aphis gossypii, host alternation, molecular identiÞcation, morphology

The soybean aphid, Aphis glycines Matsumura, is arecently invaded exotic pest in North America, whereit causes considerable economic losses every year(Ragsdale et al. 2004, 2007). Its origin is northeast Asia,i.e., China, Japan, and Korea, but it has been trans-ferred to other regions where it has sometimes estab-lished depending on the suitability of the climate(Venette and Ragsdale 2004). As an aphid with aheteroecious holocyclic life, it has an obligatory hostalternation between soybean,Glycinemax (L.) Merr.,as the secondary host and certainRhamnus spp. as theprimary host (Blackman and Eastop 1994, Ragsdale etal. 2004). Recently, two Rhamnus species, Rhamnuscathartica L. and Rhamnus alnifolia LÕHeritier, wereconÞrmed as suitable primary host plants in NorthAmerica (Voegtlin et al. 2004a, 2005; Yoo et al. 2005),whereas Rhamnus davurica Pallas and Rhamnus ja-ponicaMaximowicz are known as primary hosts in Asia(Takahashi et al. 1993, Wu et al. 2004). Although R.japonica is endemic to Japan, R. davurica is widelydistributed in Korea and China; thus, the soybeanaphid population in the latter countries is largely re-

lated to the distribution of R. davurica. Large popu-lations of the soybean aphid can easily be found on R.davurica in Korea and China (Wu et al. 2004).

On R. davurica, there are also other Aphis specieswhich occur together with A. glycines (Blackman andEastop 1994, Wu et al. 2004). The cotton or melonaphid, Aphis gossypii Glover, has been reported pre-viously as sharing R. davurica with A. glycines as aprimary host in northeastern Asia (Blackman and Eas-top 1994, Wu et al. 2004). However, the buckthornaphid,Aphis nasturtiiKaltenbach, andAphis frangulaeKaltenbach have not been reported from China, Ja-pan, and Korea (Hua 2000, Tadauchi and Inoue 2000,Blackman and Eastop 2006, Lee and Kim 2006). A.glycines andA. gossypiihave been frequently found onRhamnus at the same time during overwintering sea-sons (fall, winter, and spring), and they often formmixed colonies of overwintering generations on thesame leaves (Voegtlin et al. 2004b), contributing totheir being misidentiÞed as the same species. Al-though it has been reported thatA. glycinesdiffer fromA. gossypii in external coloration among the springmigrant, gynopara, male, and ovipara (Voegtlin et al.2004b), discrimination by coloration pattern is notonly unreliable for species identiÞcation due to thevariation within each species, but it is almost impos-sible to discriminate between the species based ontheir morphologies in macerated slide specimens dueto the lack of diagnostic characters. The situation isfurther complicated because of differences between

1 Department of Agricultural Biotechnology, Research Institute forAgriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea.

2 USDAÐARS, BeneÞcial Insect Introduction Research Unit, New-ark, DE 19711.

3 Gyeonggi-do Forestry Environment Research Institute, Osan,447-290, Republic of Korea.

4 Corresponding author, e-mail: [email protected].

0013-8746/10/0532Ð0543$04.00/0 � 2010 Entomological Society of America

sexually specialized morphs (i.e., gynopara, ovipara,male), overwintering eggs, and fundatrices of eachspecies that occur onRhamnus (Blackman and Eastop1994, 2006). These difÞculties hamper efforts to studythe biology and ecology of soybean aphid and to ÞndspeciÞc natural enemies ofA. glycines for its control onRhamnus species.

Molecular identiÞcation techniques provide notonly objective and reliable data in the form of DNAsequences to identifyAphis species, thereby serving tocorroborate morphological identiÞcations, but alsocan provide good evidence for constructing phyloge-netic relationships. Many molecular phylogeneticstudies on aphids have exploited useful primer sets andaccumulated comparable DNA sequences like the mi-tochondrial cytochrome c oxidase I (COI), cyto-chrome c oxidase II (COII), and the nuclear elonga-tion factor 1� (EF1�) for major aphid groups (Stern1994, Moran et al. 1999, Normark 2000, von Dohlen etal. 2006). In addition, DNA barcode studies have ac-cumulated the sequences of �650 bp of COI for �300species, making molecular identiÞcation workable foraphids (Foottit et al. 2008). In the genus Aphis, themolecular identiÞcation technique has effectivelysolved the identiÞcation problems due to cryptic mor-phological differences betweenA. gossypii andA. fran-gulae (Carletto et al. 2009), and the phylogenetic stud-ies have provided many useful sequences of major taxa(von Dohlen and Teulon 2003, Coeur dÕacier et al.2007, Kim and Lee 2008).

In this study, we used the COI “barcode” and partialtRNA-leucine � cytochrome c oxidase II (tRNA/COII) regions for identiÞcation of the various Aphissamples collected fromR.davurica in Asia. In addition,we attempt to Þnd useful morphological characters forthe species identiÞed by molecular methods. Based onthe results, we suggest methods for the accurate iden-tiÞcation of A. glycines collected from Rhamnus spe-cies.

Materials and Methods

Taxon Sampling.We examined 31 individual Aphisspecimens obtained from eight different collectionson R. davurica located at two sites near Seoul, Repub-lic of Korea, and two sites in the vicinity of Chang-baishan, Jilin, China (Table 1). Different morphs, e.g.,gynoparae, oviparae, and fundatrices, occurring on

the primary host were variously selected at one col-lection site, the Mulhyanggi Arboretum, as part of astudy of the seasonal dynamics and successful over-wintering of the populations. Live samples were ini-tially selected according to differences in colorationthat ranged from pale yellow to dusky green (Fig. 1).

Twenty-six closely related species in Aphis wereused in our analysis for character comparison, andSchizaphis graminum (Rondani) was included as anoutgroup (Table 2). Before the molecular work, thespecies were identiÞed based on the taxonomic keysand descriptions of Takahashi (1966), Stroyan (1984),Heie (1986), Pashchenko (1997), and Blackman andEastop (2006); and, if available, they also were com-pared with the specimens loaned from the CanadianNational Insect Collection, Ottawa, ON, Canada(Robert Foottit and Eric Maw) and the Institute ofCzech Academy of Science, Ceske Budejovice, CzechRepublic (Jaroslav Holman and Jan Havelka). For thecomparison of COI, several GenBank sequences of A.nasturtii, A. frangulae, and Aphis sp. ex Rhamnuswereadded (GenBank accessions below). To conÞrm theintraspeciÞc variation within A. glycines orA. gossypii,we used eight and six duplicate samples, respectively.All the aphid specimens used for molecular analyseswere collected and preserved in 95 or 99% ethanol,and the samples for maceration and slide prepara-tion were collected in 80% ethanol. All the voucherspecimens and samples are deposited in the InsectCollection of the College of Agriculture and LifeSciences, Seoul National University (SNU), Seoul,Korea.DNA Extraction, Amplification, and Sequencing.

Total genomic DNA was extracted from single individ-uals using a DNeasy_ Blood & Tissue kit (QIAGEN,Dusseldorf, Germany). For making voucher specimensfromtheDNA-extractedsamples,weusedanondestruc-tive DNA extraction protocol slightly modiÞed from themethod of Favret (2005). The entire body of the aphidwas left in the lysis buffer with protease K solution at55�Cfor24h,andtheclearedcuticlewasdehydratedandmounted directly onto a microscopic slide. The primersused for the polymerase chain reaction (PCR) ampliÞ-cations were as follows: LCO1490f (5�-GGTCAA-CAAATCATAAAGATATTGG-3�; Folmer et al. 1994)and HCO2198 (5�-TAAACTTCAGGGTGACCAAAA-AATCA-3�; Folmer et al. 1994) for COI and 2993� (5�-CATTCATATTCAGAATTACC-3�; Stern 1994) and

Table 1. Sample list of the Aphis species collected from R. davuricaa

IDNo. individual

(morph.)Collection locality Date Collector Voucher no.

K-1 7 (gy.) Mulhyanggi Arboretum, Osan, Gyeonggi, KR 25 Oct. 2005 K. Hoelmer and S. Lee 051025SH1K-2 4 (gy.) SNU Arboretum, Suwon, Gyeonggi, KR 27 Oct. 2005 K. Hoelmer and S. Lee 051027HJ1K-3 3 (gy.) Mulhyanggi Arboretum, Osan, Gyeonggi, KR 9 Oct. 2008 H. Kim 081009HJ1K-4 1 (al.) Mulhyanggi Arboretum, Osan, Gyeonggi, KR 19 May 2008 H. Kim 080519HJ1K-5 3 (gy.) and 5 (ov.) Mulhyanggi Arboretum, Osan, Gyeonggi, KR 1 Nov. 2008 H. Kim 081101HJ1K-6 2 (fu.) Mulhyanggi Arboretum, Osan, Gyeonggi, KR 23 April 2009 H. Kim 090423HJ12C-1 3 (ov.) Changbaishan district (1), Jilin, CN 25 Sept. 2008 H. Kim 080925HJ16C-2 3 (ov.) Changbaishan district (2), Jilin, CN 25 Sept. 2008 H. Kim 080925HJ41

a gy., gynopara; al., alata; ov., ovipara; fu., fundatrix; KR, Republic of Korea; CN, China.

July 2010 KIM ET AL.: IDENTIFICATION OF Aphis SPECIES ON R. davurica IN ASIA 533

A3772 (5�-GAGACCATTACTTGCTTTCAGTCATCT-3�;Normark1996) for tRNA/COII.The DNA fragmentsto be analyzed were ampliÞed using AccuPower_PCRPreMix (BIONEER, Corp., Daejeon, Korea) in 20-�lreaction mixtures containing 0.4 �M each primer and0.05 �g of genomic DNA template. PCR was per-formed using a GS482 thermocycler (Gene Technol-ogies, Ltd., Essex, United Kingdom) according to thefollowing procedure: initial denaturation at 95�C for 5min, followed by 34 cycles of 95�C for 30 s; annealingtemperature (43Ð45�C depending on the primer sets)for 30Ð50 s; and extension at 72�C for 30Ð60 s and Þnalextension at 72�C for 5 min. The primer-speciÞc an-nealing temperatures of each primer set were as fol-lows: 43�C for COI, 42Ð45�C for tRNA/COII. PCRproducts were visualized by electrophoresis on a 1.5%agarose gel, and if observing a single band, puriÞedusing QIAquick_PCR puriÞcation kit (QIAGEN,Inc.), and then sequenced directly on an automatedsequencer (ABI Prism_3730 XL DNA Analyzer, Ap-plied Biosystems, Foster City, CA) at National Instru-mentation Center for Environment Management,SNU. The sequences generated in this study were alldeposited to the National Center for BiotechnologyInformation (NCBI) GenBank as the following acces-sions: GQ904080ÐGQ904148 for COI and GQ904149ÐGQ904192 for tRNA/COII.

Sequences Used or Retrieved From NCBI GenBank.For precise species identiÞcation, we used 99 COI se-quences (EU701339ÐEU701422, EU930150ÐEU930163,and DQ499026) of melon aphid; Þve (EU701334-EU701338) of the soybean aphid; Þve (EU701459Ð701463) of the buckthorn aphid; one (EU930142) ofA.frangulae; and one (EU701485) of Aphis sp. ex Rham-nus. In addition, we retrieved 25 tRNA/COII se-quences (EU358824ÐEU358847 and EU358858) forAphis species andSchizaphis graminum(Rondani) andone (AM085407) of A. frangulae.SequenceAlignment andCharacterization.Raw se-

quences were examined and corrected using SeqMan-Pro version 7.1.0 (DNASTAR, Inc., Madison, WI). AllDNA sequences for each fragment were aligned usingClustal X version 2.0.11 (Thompson et al. 1997), withdefault settings. Pairwise distances, number of substi-tutions, and nucleotide compositions for COI andtRNA/COII were obtained using MEGA 4.0 (Kumaret al. 2008) based on the Kimura two-parameter (K2P)model.Data Analysis of COI and tRNA/COII. For the COI

barcode data set, neighbor-joining (NJ) analyses wereperformed using MEGA 4.0. The K2P model of nu-cleotide substitution (Kimura 1980) which is the bestmodel for species-level analysis with low distances(Hebert et al. 2003), was selected for the analyses.

Fig. 1. Aphis species on leaves of R. davurica. (A) A yellow gynopara and its nymphs. (B) Yellowish green oviparousnymphs. (C) A mixture of variously colored oviparous nymphs. (D) Dark green gynopara and grayish green nymphs. Basedon coloration, validated by morphological characters (refer to text), aphids in A and B are A. glycines; those in D are Aphissp.1; and C includes A. glycines, A. gossypii, and Aphis sp.1. (Online Þgure in color.)

534 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 103, no. 4

For the combined data set of the COI and tRNA/COII, maximum parsimony (MP) analyses were per-formed with PAUP*4.0b10 (Swofford 1998), by usingthe heuristic search procedure, with 1,000 randomadditions of sequences and 10 trees held at each pseu-doreplicate, and the tree bissection-reconnectionbranch swapping method. All characters were treatedas unordered and equally weighted for MP analysis.Bootstrapping of the MP analysis was conducted using1,000 replicates under the heuristic search procedurewith 10 random-addition sequences. Then, Bayesianphylogenetic (BP) analyses were performed usingMrBayes version 3.1.1 (Ronquist and Huelsenbeck2003). Corresponding to the ML analysis, the best-

Þtting nucleotide substitution models (GTR � I � �)and estimated parameters for each of the Þve parti-tions were selected using the hierarchical likelihoodratio test implemented in MrModeltest 2.0 (Nylander2004). Markov Chain Monte Carlo (MCMC) analysiswas run with one cold chain and three heated chains.The number of generations of MCMC and tree sam-pling frequency were 10 million generations and 100thgenerations, respectively. The critical value for thetopological convergence diagnostic was checked withMCMC options of “stoprule � yes” and “stopval �0.01” in preliminary tests. The burn-in parameter wasempirically estimated by plotting ln L against thegeneration number by using Tracer version 1.5 (Ram-

Table 2. Collection information for 26 closely related Aphis species used for species identificationa

Species Host plant Collection locality Date Collector Voucher no.

Aphis glycines Matsumura Glycine max Boryeong, CH, KR 9 Oct. 2003 H. Kim & S. Lee 031009SH30Hoengseong, GW, KR 3 Sept. 2004 H. Kim & S. Lee 040917HJ3Suwon, GG, KR 23 Sept. 2004 S. Lee 040923SH1Gangneung, GW, KR 3 Aug. 2005 H. Kim 050829HJ3Yeoncheon, GG, KR 21 July 2008 H. Kim 080721HJ20USDAÐARS, Newark, DE, US 21 Aug. 2008 K. Hoelmer 080821HM1Judge Morris Estate, Newark,

DE, US28 Aug. 2008 K. Hoelmer 080828HM2

Omahachi, Nagano, JP 21 July 2008 Y. Matsumoto 080721MM1Aphis gossypii Glover Citrus sinensis Jeju Island, KR 13 May 2003 H. Kim & S. Lee 030513HJ47

Perilla frutescens Paju, GG, KR 6 June 2003 H. Kim & S. Lee 030606SH3Lysimachia coreana Pyeongchang, GW, KR 25 June 2003 H. Kim & S. Lee 030625SH15Clinopodium multicanle Boryeong, GN, KR 9 Oct. 2003 H. Kim & S. Lee 031009SH034Capsella bursa-pastoris Taean, CH, KR 22 April 2005 H. Kim & S. Lee 050422SH7Lysimachia barystachys Yangyang, GW, KR 16 June 2005 H. Kim & S. Lee 050616SH58

Aphis argrimoniae(Shinji)

Agrimonia pilosa Changbaishan district, Jilin, CN 25 Sept. 2008 H. Kim 080925HJ29

Aphis clerodendriMatsumura

Clerodendrum trichotomum Boryeong, CH, KR 9 Oct. 2003 H. Kim & S. Lee 031009SH32

Aphis craccivora Koch Robinia hispida Gwanak, SL, KR 26 Oct. 2003 H. Kim 031026HJ1Aphis crinosa Paik Ligustrum obtusifolium Gwanak, SL, KR 25 April 2005 S. Lee 050425SH1Aphis egomae Shinji Perilla frutescens Hoengseong, GW, KR 9 Aug. 2005 H. Kim & S. Lee 050809HJ1Aphis fabae Scopoli Rumex crispus Jeju Island, KR 27 May 2004 H. Kim 040527HJ16Aphis farinosa J.F.

GmelinSalix koreensis Suwon, GG, KR 23 May 2003 H. Kim & S. Lee 030523SH14

Aphis fukii Shinji Petasites japonicus Jeju Island, KR 27 May 2004 H. Kim 040527HJ10Aphis hederae Kaltenbach Hedera japonica Namhae, GN, KR 7 June 2006 H. Kim & S. Lee 060407SH24Aphis horii Takahashi Sambucus sieboldiana Urleung Island, GN, KR 8 June 2004 H. Kim & S. Lee 040608HJ24Aphis hyperciphaga

PashshenkoHypericum ascyron Yangyang, GW, KR 16 June 2005 H. Kim & S. Lee 050616SH29

Aphis ichigo Shinji Rubus sp. Hongcheon, GW, KR 25 June 2003 H. Kim & S. Lee 030625SH53Aphis ichigocola Shinji Rubus parvifolius Jeju Island, KR 13 May 2003 H. Kim & S. Lee 030513SH10Aphis kurosawai

TakahashiArtemisia princeps Anyang, GG, KR 3 June 2005 H. Kim 050603HJ16

Aphis neospiraeaeTakahashi

Spiraea prunifolia Suwon, GG, KR 23 May 2003 H. Kim & S. Lee 030523SH25

Aphis nerii Boyer deFoscolombe

Metaplexis japonica Boryeong, CH, KR 9 Oct. 2003 H. Kim & S. Lee 031009SH25

Aphis newtoni Theobald Iris sanguinea Anyang, GG, KR 3 June 2005 H. Kim & S. Lee 050603SH10Aphis rumicis Linnaeus Rumex crispus Busan, GN, KR 13 May 2004 H. Kim 040513HJ10Aphis sanguisorbicola

TakahashiSanguisorba officinalis Pyeongchang, GW, KR 17 Sept. 2004 H. Kim & S. Lee 040917HJ6

Aphis sedi Kaltenbach Sedum sp. Dongdaemun, SL, KR 11 May 2003 H. Kim 030511HJ1Aphis spiraecola Patch Spiraea thunbergii Anyang, GG, KR 3 June 2005 H. Kim & S. Lee 050603HJ6Aphis sumire Moritsu Viola sp. Gunpo, GG, KR 13 May 2005 H. Kim & S. Lee 050513HJ21Aphis taraxacicola

(Brner)Taraxacum officinale Gunpo, GG, KR 13 May 2005 H. Kim & S. Lee 050513HJ11

Aphis oenotheraeOestlund

Oenothera odorata Hongcheon, GW, KR 25 June 2003 H. Kim & S. Lee 030625SH67

Schizaphis graminum(Rondani)

Oryza sativa Suwon, GG, KR 4 Aug. 2005 H. Kim & S. Lee 050804HJ2

aDE, Delaware; GG, Gyeonggi-do; GW, Gangwon-do; CH, Chungcheongnam-do; GN, Gyeongsangnam-do; SL, Seoul; KR, Republic of Korea;US, United States; CN, China; JP, Japan.


baut and Drummond 2007), and the trees correspond-ing to the Þrst 10% generations were discarded. ToconÞrm the parsimony network between the closelyrelated species nested within A. frangulae (see Re-sults), a minimum-spanning network was constructedfor the combined data set of COI and tRNA/COIIusing TCS version 1.21 (Clement et al. 2000), wherea 95% connection limit was used and gaps were treatedas missing data.Morphological Measurement and Test of Differ-ence. All specimens were identiÞed by comparisonwith the voucher specimens used for DNA extraction.

Except for the one alate specimen (K-4; Aphis sp.2),samples of both gynoparae and oviparae mounted onmicroscopic slides could be separated into three spe-ciÞc phenotypes (A. glycines, A. gossypii, and Aphissp.1) based on our molecular identiÞcation. Fundatrixforms were excluded from morphological measure-ment due to lack of a sufÞcient number of specimens.A morphometric data set was assembled by measur-ing/counting 28 characters and 12 ratios for 106 spec-imens including the voucher specimens (Table 3),which followed the recent biometric works on Aphis(Mier Durante et al. 2003, Kim et al. 2006). Measure-

Table 3. Morphometric data of gynoparae and oviparae of three different speciesa

Character

Gynopara Ovipara

Aphis glycines(n � 24)

Aphis gossypii(n � 16)

Aphis sp.1(n � 18)

Aphis glycines(n � 18)

Aphis gossypii(n � 10)

Aphis sp.1(n � 20)

Body color when alive Greenish yellow,pale green,green

Green, duskygreen

Green, duskygreen

Greenish yellow,yellow, green

Dusky green,grayish green

Dusky green,grayish green

Abdominal tergiteswith blacksclerotized bands

Absent or only VIIand VIII(irregular)

IÐVI (irregular),VII and VIII(large)

IÐVI (irregular),VII and VIII(large)

Length (mm)Body 1.812 (1.393Ð2.157) 1.603 (1.384Ð1.950) 1.542 (1.182Ð1.778) 1.324 (1.132Ð1.547) 1.310 (1.220Ð1.389) 1.315 (1.180Ð1.552)Whole antennae 1.266 (1.047Ð1.464) 1.112 (1.002Ð1.226) 0.970 (0.793Ð1.135) 0.687 (0.591Ð0.743) 0.674 (0.648Ð0.687) 0.631 (0.536Ð0.687)Ant.I 0.058 (0.047Ð0.069) 0.054 (0.039Ð0.060) 0.046 (0.035Ð0.063) 0.043 (0.032Ð0.049) 0.043 (0.038Ð0.048) 0.042 (0.033Ð0.048)Ant.II 0.053 (0.041Ð0.059) 0.051 (0.036Ð0.062) 0.047 (0.042Ð0.057) 0.036 (0.031Ð0.043) 0.036 (0.032Ð0.041) 0.036 (0.030Ð0.045)Ant.III 0.316 (0.252Ð0.375) 0.276 (0.245Ð0.324) 0.252 (0.211Ð0.288) 0.228 (0.166Ð0.267) 0.222 (0.199Ð0.235) 0.209 (0.171Ð0.235)Ant.IV 0.221 (0.157Ð0.283) 0.190 (0.159Ð0.211) 0.169 (0.141Ð0.207) * * *Ant.V 0.187 (0.144Ð0.222) 0.171 (0.142Ð0.198) 0.140 (0.114Ð0.163) 0.107 (0.048Ð0.122) 0.111 (0.085Ð0.129) 0.105 (0.090Ð0.129)Ant.VIb 0.116 (0.089Ð0.181) 0.103 (0.081Ð0.131) 0.080 (0.055Ð0.100) 0.079 (0.070Ð0.092) 0.091 (0.076Ð0.107) 0.078 (0.064Ð0.100)PT 0.314 (0.252Ð0.363) 0.267 (0.246Ð0.286) 0.236 (0.149Ð0.288) 0.195 (0.166Ð0.221) 0.170 (0.135Ð0.206) 0.161 (0.108Ð0.206)URS 0.088 (0.078Ð0.127) 0.091 (0.090Ð0.092) 0.086 (0.077Ð0.093) 0.077 (0.591Ð0.743) 0.076 (0.061Ð0.078) 0.075 (0.060Ð0.096)Hind femur 0.465 (0.374Ð0.537) 0.415 (0.331Ð0.493) 0.376 (0.300Ð0.415) 0.221 (0.173Ð0.262) 0.226 (0.165Ð0.271) 0.220 (0.165Ð0.271)Hind tibia 0.920 (0.765Ð1.104) 0.813 (0.699Ð0.951) 0.707 (0.557Ð0.839) 0.398 (0.354Ð0.445) 0.407 (0.355Ð0.476) 0.392 (0.031Ð0.476)2HT 0.086 (0.064Ð0.103) 0.080 (0.069Ð0.088) 0.075 (0.052Ð0.095) 0.066 (0.043Ð0.084) 0.071 (0.061Ð0.078) 0.069 (0.057Ð0.078)SIPH 0.205 (0.163Ð0.243) 0.193 (0.150Ð0.247) 0.161 (0.116Ð0.205) 0.117 (0.096Ð0.145) 0.109 (0.085Ð0.125) 0.101 (0.084Ð0.125)Cauda 0.123 (0.098Ð0.170) 0.106 (0.083Ð0.128) 0.087 (0.058Ð0.095) 0.081 (0.050Ð0.094) 0.097 (0.071Ð0.124) 0.094 (0.070Ð0.124)Setae on Ant.III 0.013 (0.008Ð0.016) 0.015 (0.012Ð0.018) 0.012 (0.008Ð0.017) 0.011 (0.008Ð0.015) 0.011 (0.008Ð0.012) 0.010 (0.007Ð0.012)Setae on AbdT.III 0.024 (0.015Ð0.030) 0.023 (0.017Ð0.025) 0.018 (0.010Ð0.026) 0.015 (0.012Ð0.018) 0.017 (0.014Ð0.019) 0.017 (0.014Ð0.020)

No. hairs onAnt.I 5 (4Ð6) 4 (3Ð6) 4 (3Ð5) 4 (3Ð5) 5 (3Ð6) 5 (3Ð6)Ant.II 4 (3Ð6) 4 (3Ð5) 4 (3Ð5) 3 (2Ð4) 3 (3Ð4) 3 (3Ð4)Ant.III 5 (3Ð12) 5 (3Ð8) 6 (3Ð9) 3 (2Ð4) 2 (2Ð5) 3 (3Ð5)AbdT.VII 4 (3Ð4) 4 (3Ð4) 4 5 (4Ð6) 5 (5Ð8) 6 (4Ð8)AbdT.VIII 2 2 2 5 (4Ð6) 5 (5Ð8) 6 (4Ð8)Median of GP 2 (2Ð3) 3 (2Ð4) 2 (2Ð3) 12 (11Ð14) 12 (12Ð18) 15 (12Ð19)Posterior margin

of GP9 (8Ð12) 8 (7Ð10) 10 (6Ð14) 16 (13Ð19) 13 (13Ð19) 15 (12Ð19)

Cauda 8 (6Ð9) 5 (4Ð6) 6 (6Ð8) 6 (5Ð8) 4 (4Ð6) 5 (4Ð6)No. rhinaria on

Ant.III 8 (6Ð12) 8 (6Ð12) 10 (6Ð13) 0 0 0Ant.IV 0 0 (0Ð1) 4 (1Ð5) 0 0 0Ant.V 0 0 0 (0Ð2) 0 0 0

Ratio (times)Whole antennae/

body0.70 (0.59Ð0.81) 0.70 (0.57Ð0.81) 0.63 (0.52Ð0.75) 0.52 (0.43Ð0.63) 0.52 (0.47Ð0.56) 0.48 (0.39Ð0.56)

PT/Ant.VIb 2.73 (1.80Ð3.18) 2.62 (1.92Ð3.30) 2.95 (1.96Ð3.31) 2.47 (2.27Ð2.76) 1.88 (1.35Ð2.71) 2.07 (1.35Ð2.71)PT/Ant.III 1.00 (0.71Ð1.22) 0.97 (0.85Ð1.09) 0.94 (0.60Ð1.17) 0.87 (0.67Ð1.12) 0.77 (0.57Ð0.98) 0.77 (0.53Ð0.98)URS/2HT 1.03 (0.76Ð1.49) 1.14 (1.03Ð1.33) 1.15 (0.90Ð1.48) 1.19 (0.91Ð1.86) 1.07 (0.81Ð1.30) 1.09 (0.81Ð1.33)URS/Ant.VIb 0.77 (0.49Ð1.09) 0.89 (0.69Ð1.12) 1.08 (0.86Ð1.56) 0.98 (0.80Ð1.24) 0.84 (0.67Ð1.07) 0.96 (0.72Ð1.22)SIPH/body 0.11 (0.09Ð0.15) 0.12 (0.10Ð0.14) 0.11 (0.08Ð0.13) 0.09 (0.07Ð0.11) 0.08 (0.07Ð0.09) 0.08 (0.05Ð0.09)SIPH/Ant.III 0.65 (0.53Ð0.80) 0.70 (0.55Ð0.89) 0.64 (0.49Ð0.75) 0.52 (0.40Ð0.79) 0.49 (0.38Ð0.53) 0.48 (0.38Ð0.56)SIPH/hind femur 0.44 (0.34Ð0.53) 0.46 (0.38Ð0.58) 0.43 (0.34Ð0.53) 0.53 (0.44Ð0.68) 0.49 (0.38Ð0.64) 0.47 (0.36Ð0.64)SIPH/cauda 1.70 (1.19Ð2.09) 1.83 (1.28Ð2.17) 1.90 (1.22Ð2.89) 1.49 (1.16Ð2.18) 1.14 (0.95Ð1.39) 1.09 (0.82Ð1.39)Cauda/width of

cauda1.64 (1.11Ð2.10) 1.50 (1.11Ð1.71) 1.34 (0.89Ð1.63) 1.17 (0.56Ð1.69) 1.35 (0.97Ð1.65) 1.34 (0.97Ð1.65)

Setae on Ant.III/Ant.IIIBD

0.61 (0.40Ð0.78) 0.69 (0.58Ð0.86) 0.63 (0.44Ð0.81) 0.74 (0.47Ð1.36) 0.59 (0.47Ð0.67) 0.57 (0.47Ð0.67)

Setae on AbdT.III/Ant.IIIBD

1.11 (0.75Ð1.43) 1.06 (0.85Ð1.48) 0.95 (0.59Ð1.24) 1.00 (0.75Ð1.64) 0.96 (0.74Ð1.13) 0.98 (0.74Ð1.18)

a Values are means, with min.-max. in parentheses. Abbreviations are explained in the text. A blank cell in the range columns means thatall measurements were identical.

*Ant.VI mostly fused to Ant.III in ovipara.


ments and observations were performed using ImageLab version 2.2.4.0 software (MCM Design, Hillerød,Denmark) and digital images were captured by acharge-coupled device SPOT Insight Color Mosaic14.2 (Diagnostic Instruments, Inc., Sterling Heights,MI), attached to a DM 4000B microscope (Leica Mi-crosystems GmbH, Wetzlar, Germany). Morphologi-cal terminology, measurement range, and location ofeach character follow Heie (1986) and Blackman andEastop (2006).

To determine the morphological characters usefulfor identiÞcation, differences of each character be-tween the three species were examined by multiplecomparisons of TukeyÐKramer test in analysis of vari-ance (ANOVA). Some characters without variationwere excluded from analysis. SigniÞcant thresholdswere set to 99% conÞdence level (P 0.01). Statisticalanalyses were performed using SPSS version 12.0(SPSS Inc., Chicago, IL).

Abbreviations for the characters used in the textare as follows: Ant.I, Ant.II, Ant.III, Ant.IV, Ant.V,Ant.VI, Ant.VIb for antennal segments I, II, III, IV,V, VI, and the base of Ant.VI, respectively; PT,processus terminalis; URS, ultimate rostral segment(i.e., the combined length of rostral segments IV andV); HFM, hind femur; HTB, hind tibia; 2HT, secondsegment of hind tarsus; AbdT.I, AbdT.II, AbdT.III,AbdT.IV, AbdT.V, AbdT.VI, AbdT.VII, AbdT.VIIIfor abdominal tergite I, II, III, IV, V, VI, VII, and VIII,respectively; SIPH, siphunculus; and GP, genitalplate.

Results

Characteristics of theCOI and tRNA/COII.For theCOI data set, 66 terminal taxa in total were analyzedand 720 aligned base pair (bp) sites were found, butonly 658 bp were used for the analyses after excludingprimer sites. Among the 658 bp selected, 175 werevariable and 127 were parsimony informative. TheK2P sequence divergence among taxa for tRNA/COIIranged from 0 to 12.6%, with an average of 5.0%.Average proportions of T:C:A:G were 41:14:35:10, witha narrow SD.

For the tRNA/COII data set, 59 terminal taxa intotal were analyzed, with 760 aligned bp sites found,but 702 bp were used for the analyses after excludingprimer sites. Among the 702 bp selected, 160 werevariable and 93 were parsimony informative. The K2Psequence divergence among taxa for tRNA/COIIranged from 0 to 9.0%, with an average of 3.6%. Av-erage proportions of T:C:A:G were 39:12:41:8, with anarrow SD.Molecular IdentificationUsingCOI andCombinedAnalysisUsingCOIand tRNA/COII.TheNJ tree fromthe COI analysis shows that all samples that werematched to A. glycines from either soybean (13 se-quences)orR.davurica(11 sequences)were identicalto each other (Fig. 2). The clade of A. glycines wasplaced between two relatively unrelated species,Aphis ichigo Shinji andAphis ichigocola Shinji, and wascompletely separated from those of the other Aphis

groups sampled on R. davurica, which means that A.glycines can be successfully discriminated from themby the COI barcode region. Except for the samplesidentiÞed as A. glycines, the remaining samples weredivided into four different groups: Aphis gossypii exRhamnus 1, Aphis gossypii ex Rhamnus 2, Aphis sp.2,and Aphis sp.1. A. gossypii ex Rhamnus 1 and 2 within“A. gossypii� closely related species” were congruentto four and 63 A. gossypii sequences, respectively,whereas the Aphis sp.1 and 2 differed 2.1 and 3.8%,respectively, from A. gossypii, and 4.6 and 3.5%, re-spectively, from A. glycines. This level of genetic di-vergence is considerably greater than the mean diver-gence of 1.9% seen among the species [e.g., Aphisargrimoniae (Shinji), Aphis clerodendri Matsumura,Aphis sumireMoritsu, andAphis taraxacicola (Borner)]within the clade that are sister to A. frangulae, whichsupports the treatment of Aphis sp.1 and 2 as differentspecies. In addition, these two groups did not matchthe buckthorn aphid, A. nasturtii, and Aphis sp. exRhamnus (EU701485) at all. The 13 samples of Aphissp.1 collected in both Korea and China were identical,which also suggests a new species distinct from A.gossypii and A. glycines, coexisting on R. davurica.Although we were not able to collect a comparablenumber of Aphis sp.2 samples, this species might useR. davurica as a secondary host because it was foundthat some colonies were initiated by migrants, alateviviparous females, on its young shoots in late May.All the recognized groups comprising our sampleswere seemingly located in the gossypii group (cladeA), whereas A. nasturtii is close to the fabae group(clade B).

In the combined analysis using the COI and tRNA/COII (Fig. 3), the tree inferred by both BP and MPanalyses shows a similar topology to that of COI, andall analyzed samples are located within the gossypiigroup (clade A). Species or samples that were notsequenced for tRNA/COII (e.g., A. nasturtii) wereexcluded from the analyses. Because the 19 samples ofA. glycines had no variation in the tRNA/COII se-quence, they clearly clustered. The phylogenetic treerobustly supports the idea that the Aphis sp.1 and 2could be different species coexisting withA. gossypiiand A. glycines on R. davurica. Because the nodesister to A. glycines were supported by 100% in PPand 91% in MP bootstrap analysis, Aphis sp.1 and 2were genetically closer to A. gossypii than A. gly-cines. Nested clade analysis (Fig. 4) shows that thesamples of A. gossypii were divided into four hap-lotypes and linked together by one mutational step,where the other closely related species were net-worked with the haplotypes of A. gossypii. In con-trast, Aphis sp.1 and 2 were not directly connectedwith A. gossypii, which were linked via 13 or 21missing haplotypes, respectively. However,A. tarax-acicola, A. clerodendri, and A. sedi that are not spe-ciÞc to Rhamnus were closely located to A. gossypiirather than Aphis sp.1 and 2.Morphological Identification and Test of CharacterDifference. Because of the possibility of overlappingmorphological variation among the different species,


the morphological characters of preserved cuticles ofspecimens identiÞed by molecular sequences werethen examined to Þnd morphological characters thatwould establish the identity of specimens not used forDNA sequencing. The results of 28 measured orcounted characters and 12 character ratios showedthat A. glycines, A. gossypii, and Aphis sp.1 were quite

similar in most characters (Table 3). There were notenough specimens collected of the putativeAphis sp.2to make a morphological comparison. In living spec-imens, both oviparae and gynoparae of A. glycineswere paler in coloration than the other two species,A.gossypii and Aphis sp.1. Gynoparae of A. glycines hadno or weakly sclerotized black bands on the abdominal

Fig. 2. Neighbor-joining tree of the COI data set consisting of 31 individuals from R. davurica and 29 Aphis species,including A. glycines (66 terminal taxa) obtained by MEGA 4.0. Sample identiÞers are explained in Table 1. Clade A is thegossypii group and clade B is the fabae group. The number inside a bracket indicates a number of duplication sequences, whichis followed by GenBank accession.


tergites, whereas A. gossypii and Aphis sp.1 generallyhad irregular bands on AbdT.IÐVI.

Statistical analyses using TukeyÐKramer test inANOVA showed that at least six characters (wholeantenna, Ant.V, PT, HTB, number of hairs on cauda,and number of rhinaria on Ant.III) could be useful toidentify gynoparae of the three species (Table 4). Thelengths of the whole antenna, Ant.V, PT, and HTB ofA. glycineswere slightly longer than those of two otherspecies. Gynoparae of Aphis sp.1 were distinguishabledue to the presence of secondary rhinaria on Ant.IVand Ant.V, in contrast with their absence inA. glycinesand A. gossypii, even though a few individuals of A.gossypii were observed bearing just one secondary

rhinarium on Ant.IV. However, signiÞcant characterswere not found in the analyses of oviparae (data notshown).

Discussion

A. glycines was clearly discriminated by the COIbarcode and the combined data set of COI and tRNA/COII sequences (Figs. 2 and 3). Our molecular anal-ysis showed that the samples ofA. glycineswere clearlyclustered distinctly apart from the clade of A. gossypiiand its related species. Species closely related to A.gossypii such as A. taraxacicola show very low molec-ular sequence differences for an independent species.

Fig. 3. The 50% majority rule consensus tree inferred by MrBayes using the combined data set of COI and tRNA/COII.Sample identiÞers are explained in Table 1. Clade A is the gossypii group and clade B is the fabae group. The number insidea bracket means a number of duplication sequences. Bold branches emphasize clades supported by 100% in posteriorprobability (PP). PP followed by supported values of MP bootstrap test with 1000 replicate are given on branches. If a cladeis not established by an analysis, the value is expressed as dashes (Ð).


Moreover, species in the gossypii group similarly showmorphologically reduced or regressive characterscompared with those of the fabae group (Lee and Kim2006). Together, this implies that the species in thegossypii group are very young taxa recently diversiÞedamong the genus Aphis.

Some species of the gossypii group are associatedwith the host family Rhamnaceae as their primary host(Blackman and Eastop 1994, 2006). Our study re-vealed that A. glycines, A. gossypii, A. frangulae, andAphis sp.1 using the genus Rhamnus as a primary hostwere grouped in clade A based on the molecular anal-yses. Moreover, host alternation between primary andsecondary hosts is not a common trait within the genusAphis (Blackman and Eastop 2006). The convergenceof host utilization may constrain some species cur-rently regarded as distinct into a species or speciescomplex. Indeed, for a long time A. gossypii has beentreated as a subspecies of A. frangulae due to such acongruence of primary host association as well as theirmorphological similarity (Stroyan 1984, Heie 1986).Similarly,A.gossypiiandAphis sp.1were indistinguish-able from each other except for one characteristic, thepresence of secondary rhinaria on Ant.IV and Ant.V,even though those species were separated by the mo-lecular markers used in this study. Nevertheless, weare reluctant to describeAphis sp.1 as a new species atthis time because its secondary host is unknown.

Based on our molecular analyses, the convergentrelationships of theseRhamnus-living species stronglysuggest that they originated from a common ancestorfeeding on Rhamnus or some other Rhamnaceae host.In particular, some species related to A. gossypii seemto show ongoing speciation related to the genusRhamnus.Certain EuropeanAphis species classiÞed inthe gossypii group have a host alternation with the

plants in Rhamnaceae (Cocuzza et al. 2008). TheAphis species that share Rhamnus as the primary hostbut that specialize on a different secondary host mayhave a similar mode of the aphid speciation, switchingto a secondary host, such as the genus Cryptomyzus(Dixon 1987, Moran 1992).

In contrast, although A. clerodendri, A. egomae, A.sedi, and A. taraxacicola show no primary host asso-ciation with Rhamnus, those species are geneticallyvery close toA. gossypii in COI and tRNA/COII results(Figs. 2 and 3). The nested clade analysis (Fig. 4)supports that these closely related species divergedfrom the different haplotypes ofA. gossypii, suggestingthat their diversiÞcation might have originated withthe population level isolation mainly by adapting todifferenthostplants.This intriguing resultmayexplainthe speciation mode within the gossypii group. Basedon this intimacy with A. gossypii, it may be hypothe-sized that they were separated by the loss of their hostalternation process that led to speciation (Moran1992).A. gossypiigenerally has a monoecious life cyclewith either holocycly or anholocycly, but there aresome reports of host alternation in parts of East Asiaand North America (Blackman and Eastop 2006, Liu etal. 2008a). Although it is still unclear whether a singlepopulation of A. gossypii is able to use two unrelatedhosts as primary and secondary hosts through hostalternation (Liu et al. 2008a,b),A. gossypiipopulationssampled on Malvaceae (Hibiscus spp.), considered tobe their primary host, showed higher genetic diversityand more genotypic variation than those collected onusual secondary hosts such as Cucurbitaeceae andSolanaceae (Charaabi et al. 2008). Presuming theseÞndings are the outcome of host specialization, thelow genetic diversity seen in populations on secondaryhosts suggests that host-related isolation may eventu-

Fig. 4. Parsimony network constructed by TCS using the combined data set. Numbers at nodes indicate the number ofnucleotide changes between haplotypes or species. The dashed nodes represent a single nucleotide change betweenhaplotypes or species. Small open circles signify possible missing haplotypes. Boxes signify the recognized clades in Fig. 3.


ally lead to speciation (Via et al. 2000, Raymond et al.2001). If the primary host is lost, the rate of speciationwill be increased by reproductive isolation (Moran1992). In addition, either monoecious holocycly oranholocyly in the host accelerates incipient specia-tion, even though these are temporary mechanismsthat only occur under certain conditions inA. gossypii(Dixon 1987, Moran 1992, Moran et al. 1999). In thegossypii group, this hypothesis is strongly supported inthat some species in the group show congruent use ofprimary hosts in the Rhamnaceae, especially the Fran-gula andRhamnusgenera (Cocuzza et al. 2008). More-over, that host alternation between primary and sec-ondary hosts is very rare within the genus Aphis andthat 15 species do so (Blackman and Eastop 2006)provides further support for this hypothesis.

Just as with our discovery of a putative Aphis sp.1coexisting with soybean aphid on Rhamnus in north-eastern Asia, we suspect a similar species complexmight exist on other Rhamnus species, e.g., Rhamnuscathartica, in North American regions during the over-wintering season. We expect molecular characteriza-tions to be useful in detecting new species related toA. glycines or A. gossypii in the future. Although ge-netic isolation leading to speciation generally requiresphysically isolated circumstances to prevent repro-duction to avoid possible hybridization between dif-ferent species, it is unusual and mysterious whyRhamnus-livingAphis species still share a primary host

during the sexual phase of the overwintering season.Thus, further study of Rhamnus-speciÞc species maybe helpful to reveal the speciation and evolution of therelated aphid species.

As a cautionary note, it is worth considering thatgenetic distance may be low between species (e.g.,1.6% as cited in Kankare et al. 2005) or relativelygreat within species for a variety of reasons (Ferguson2002). Resolution of cryptic species within the aphidparasitoid genus Aphelinus required as many as sixgene regions and corresponding laboratory cross mat-ing studies (Heraty et al. 2007). Thus, additional re-search on cryptic species within Aphis on Rhamnuswould ideally investigate population structure and in-clude mating studies where appropriate.

Acknowledgments

We are indebted to Robert Foottit, Eric Maw, JaroslavHolman, Jan Havelka for the loan of Aphis specimens fromtheir regions for comparison. We thank Gexia Qiao, LiyunJiang, and Xiaolei Huang for cooperative aphid collection inthe Changbaishan area in northeastern China. We also thankYoshiyuki Matsumoto for sending Japanese samples. Specialthanks to Youngboon Lee (Seoul National University) forpreparing many aphid specimens, to Wonyeol Jang for as-sistance with the measurement of specimens, and to YeyeunKim for assistance with the molecular work. This researchwas supported by the grants from the Technology Develop-ment Program for Agriculture and Forestry, Ministry for

Table 4. Multiple comparisons of character differences of gynoparae between three species by Tukey–Kramer test in ANOVAa

Character

P value of TukeyÐKramer test in ANOVAAll P values

0.01A. glycines vs.A. gossypii

A. glycines vs.Aphis sp.1

A.gossypii vs.Aphis sp.1

LengthBody 0.006 0.000 0.651Whole antennae 0.000 0.000 0.000 YesAnt.I 0.105 0.000 0.003Ant.II 0.414 0.002 0.107Ant.III 0.000 0.000 0.059Ant.IV 0.000 0.000 0.023Ant.V 0.004 0.000 0.000 YesAnt.VIb 0.013 0.000 0.000PT 0.000 0.000 0.008 YesURS 0.520 0.747 0.217Hind femur 0.002 0.000 0.023Hind tibia 0.000 0.000 0.001 Yes2HT 0.076 0.000 0.026SIPH 0.387 0.000 0.004Cauda 0.019 0.000 0.010Setae on Ant.III 0.124 0.176 0.003Setae on AbdT.III 0.801 0.001 0.012

No. hairs onAnt.I 0.007 0.000 0.043Ant.II 0.937 0.884 0.735Ant.III 0.653 0.861 0.399AbdT.VII 0.869 0.551 0.335Median of GP 0.027 0.987 0.029Posterior margin of GP 0.061 0.151 0.001

No. rhinaria onCauda 0.000 0.000 0.000 YesAnt.III 0.428 0.019 0.366Ant.IV 0.001 0.000 0.001 YesAnt.V 1.000 0.059 0.095

a Abbreviations are explained in the text.


Food, Agriculture, Forestry and Fisheries, Republic of Korea(500-20080241) and the project on survey and excavation ofKorean indigenous species of the National Institute of Bio-logical Resources under the Ministry of Environment, Korea.H.K. and S.L. also were supported by USDAÐARS speciÞccooperative agreement 58-1926-7-154 F.

References Cited

Blackman, R. L., and V. F. Eastop. 1994. Aphids on theworldÕs trees: an identiÞcation and information guide.CAB International, Wallingford, United Kingdom.

Blackman, R. L., and V. F. Eastop. 2006. Aphids on theworldÕs herbaceous plants and shrubs, vol. 2. The aphids.Wiley Ltd., Chichester, United Kingdom.

Carletto, J., A. Blin, and F. Vanlerberghe-Masutti. 2009.DNA-based discrimination between the sibling speciesAphis gossypii Glover and Aphis frangulae Kaltenbach.Syst. Entomol. 34: 307Ð314.

Charaabi, K., J. Carletto, P. Chavigny, M. Marrakchi, M.Makni, and F. Vanlerberghe-Masutti. 2008. Genotypicdiversity of the cotton-melon aphid Aphis gossypii(Glover) in Tunisia is structured by host plants. Bull.Entomol. Res. 98: 333Ð341.

Clement, M., D. Posada, and K. A. Crandall. 2000. TCS: acomputer program to estimate gene genealogies. Mol.Ecol. 9: 1657Ð1659.

Cocuzza, G., V. Cavalieri, and S. Barbagallo. 2008. Prelim-inary results in the taxonomy of the cryptic group Aphisfrangulae/gossypii obtained from mitochondrial DNA se-quences. Bull. Insectol. 61: 125Ð126.

Coeur d’acier, A., E. Jousselin, J. F. Martin, and J. Y. Rasplus.2007. Phylogeny of the genus Aphis Linnaeus, 1758 (Ho-moptera: Aphididae) inferred from mitochondrial DNAsequences. Mol. Phylogenet. Evol. 42: 598Ð611.

Dixon, A.F.G. 1987. The way of life of aphids: host speci-Þcity, speciation and distribution. pp. 197Ð207. In A. K.Minks and P. Harrewijn (eds.), Aphids their biology,natural enemies and control. Elsevier, Amsterdam, TheNetherlands.

Favret,C. 2005. A new non-destructive DNA extraction andspecimen clearing technique for aphids (Hemiptera).Proc. Entomol. Soc. Wash. 107: 469Ð470.

Ferguson, J.W.H. 2002. Ontheuseofgeneticdivergence foridentifying species. Biol. J. Linn. Soc. Lond. 75: 509Ð516.

Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrijenhoek.1994. DNA primers for ampliÞcation of mitochondrialcytochrome c oxidase subunit I from diverse metazoaninvertebrates. Mol. Mar. Biol. Biotechnol. 3: 294Ð299.

Foottit, R. G., H.E.L. Maw, C. D. von Dohlen, and P.D.N.Hebert. 2008. Species identiÞcation of aphids (Insecta:Hemiptera: Aphididae) through DNA barcodes. Mol.Ecol. Resour. 8: 1189Ð1201.

Hebert, P.D.N., A. Cywinska, S. L. Ball, and J. R. DeWaard.2003. Biological identiÞcations through DNA barcodes.Proc. R. Soc. Lond. B Biol. Sci. 270: 313Ð321.

Heie, O. E. 1986. The Aphidoidea (Hemiptera) of Fen-noscandia and Denmark. III. Family Aphididae: subfamilyPterocommatinae & tribe Aphidini of subfamily Aphidi-nae. Fauna Entomologica Scandinavica. E.J. Brill/Scan-dinavian Science Press Ltd., Leiden, The Netherlands.

Heraty, J. M., J. B.Woolley, K. R. Hopper, D. L. Hawks, J.W.Kim, and M. Buffington. 2007. Molecular phylogeneticsand reproductive incompatibility in a complex of crypticspecies of aphid parasitoids. Mol. Phylogenet. Evol. 45:480Ð493.

Hua, L.-Z. 2000. List of Chinese insects, vol. I. ZhongshanUniversity Press, Guangzhou, China.

Kankare, M., S. van Nouhuys, and I. Hanski. 2005. Geneticdivergence among host-speciÞc cryptic species inCotesiamelitaearum aggregate (Hymenoptera: Braconidae),parasitoids of checkerspot butterßies. Ann. Entomol. Soc.Am. 98: 382Ð394.

Kim, H., and S. Lee. 2008. A molecular phylogeny of thetribe Aphidini (Insecta: Hemiptera: Aphididae) based onthe mitochondrial tRNA/COII, 12S/16S and the nuclearEF1� genes. Syst. Entomol. 33: 711Ð721.

Kim,H.,W.Lee, and S. Lee. 2006. Three new records of thegenus Aphis (Hemiptera: Aphididae) from Korea. J. AsiaPac. Entomol. 9: 301Ð312.

Kimura, M. 1980. A simple method for estimating evolu-tionary rate of base substitutions through comparativestudies of nucleotide sequences. J. Mol. Evol. 16: 111Ð120.

Kumar, S., M. Nei, J. Dudley, and K. Tamura. 2008. MEGA:a biologist-centric software for evolutionary analysis ofDNA and protein sequences. Brief. Bioinform. 9: 299Ð306.

Lee, S., and H. Kim. 2006. Economic Insects of Korea 28(Insecta Koreana Suppl. 35), Aphididae: Aphidini(Hemiptera: Sternorrhyncha). National Institute of Ag-ricultural Science and Technology, Suwon, Korea.

Liu, X. D., B. P. Zhai, and X. X. Zhang. 2008b. Specializedhost-plant performance of the cotton aphid is altered byexperience. Ecol. Res. 23: 919Ð925.

Liu, X. D., B. P. Zhai, X. X. Zhang, and H. N. Gu. 2008a.Variability and genetic basis for migratory behaviour in aspring population of the aphid, Aphis gossypii Glover inthe Yangtze River Valley of China. Bull. Entomol. Res. 98:491Ð497.

Mier Durante, M. P., J. M. Nieto Nafrıa, and J. Ortego. 2003.Aphidini (Hemiptera: Aphididae) living on Senecio (As-teraceae), with description of a new genus and tree newspecies. Can. Entomol. 135: 187Ð212.

Moran, N. A. 1992. The evolution of aphid life cycles. Annu.Rev. Entomol. 37: 321Ð348.

Moran, N. A., M. E. Kaplan, M. J. Gelsey, T. G. Murphy, andE. A. Scholes. 1999. Phylogenetics and evolution of theaphid genus Uroleucon based on mitochondrial and nu-clear DNA sequences. Syst. Entomol. 24: 85Ð93.

Normark, B. B. 1996. Phylogeny and evolution of parthe-nogenetic weevils of the Aramigus tessellatus speciescomplex (Coleoptera: Curculionidae: Naupactini): evi-dence from mitochondrial DNA sequences. Evolution 50:734Ð745.

Normark, B. B. 2000. Molecular systematics and evolutionof the aphid family Lachnidae. Mol. Phylogenet. Evol. 14:131Ð140.

Nylander, J. 2004. MrModeltest 2.0. Program distributed bythe author. Evolutionary Biology Centre, Uppsala Uni-versity, Uppsala, Sweden.

Pashchenko, N. F. 1997. Aphid of the genus Aphis (Ho-moptera, Aphidinea, Aphididae) from the Russian fareast: communication 8. Entomol. Rev. 77: 871Ð882.

Ragsdale,D.W.,B.P.McCornack,R.C.Venette,B.D.Potter,I. V.Macrae, E.W.Hodgson,M.E.O’Neal, K.D. Johnson,R. J.O’Neil,C.D.Difonzo, et al. 2007. Economic thresh-old for soybean aphid (Hemiptera: Aphididae). J. Econ.Entomol. 100: 1258Ð1267.

Ragsdale, D. W., D. J. Voegtlin, and R. J. O’Neil. 2004. Soy-bean aphid biology in North America. Ann. Entomol. Soc.Am. 97: 204Ð208.

Rambaut, A., and A. J. Drummond. 2007. Tracer. (http://beast.bio.ed.ac.uk/tracer).

Raymond, B., J. B. Searle, and A. E. Douglas. 2001. On theprocesses shaping reproductive isolation in aphids of theAphis fabae (Scop.) complex (Aphididae: Homoptera).Biol. J. Linn. Soc. Lond. 74: 205Ð215.


Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3:Bayesian phylogenetic inference under mixed models.Bioinformatics 19: 1572Ð1574.

Stern, D. L. 1994. A phylogenetic analysis of soldier evolu-tion in the aphid family Hormaphididae. Proc. R. Soc.Lond. B Biol. Sci. 256: 203Ð209.

Stroyan,H.L.G. 1984. AphidsÑPterocommatinae and Aphidi-nae (Aphidini) Homoptera, Aphididae. Handbk. Ident.Br. Insects 2, pt. 6. Dramrite Printers Ltd., London, UnitedKingdom.

Swofford, D. L. 1998. PAUP*: phylogenetic analysis usingparsimony (* and other methods), version 4.0b10. Si-nauer, Sunderland, MA.

Tadauchi, O., and H. Inoue. 2000. On MOKUROKU Þlebased on “A Check List of Japanese Insects” on INTER-NET. Esakia 40: 81Ð84.

Takahashi, R. 1966. Description of some new and littleknown species of Aphis of Japan, with key to species.Trans. Am. Entomol. Soc. 92: 519Ð556.

Takahashi, S., M. Inaizumi, and K. Kawakami. 1993. Lifecycle of the soybean aphid Aphis glycinesMatsumura, inJapan. Jpn. J. Appl. Entomol. Zool. 37: 207Ð212.

Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin,and D. G. Higgins. 1997. The ClustalX windows inter-face: ßexible strategies for multiple sequence alignmentaided by quality analysis tools. Nucleic Acids Res. 24:4876Ð4882.

Venette, R. C., and D. W. Ragsdale. 2004. Assessing theinvasion by soybean aphid (Homoptera: Aphididae):where will it end? Ann. Entomol. Soc. Am. 97: 219Ð226.

Via, S., A. C. Bouck, and S. Skillman. 2000. Reproductiveisolation between divergent races of pea aphids on two

hosts. II. Selection against migrants and hybrids in theparental environments. Evolution 54: 1626Ð1637.

Voegtlin, D. J., R. J. O’Neil, and W. R. Graves. 2004a. Testsof suitability of overwintering hosts of Aphis glycines:identiÞcation of a new host association with Rhamnusalnifolia LÕHeritier. Ann. Entomol. Soc. Am. 97: 233Ð234.

Voegtlin,D. J., R. J.O’Neil,W.R.Graves,D.Lagos, andH.J.S.Yoo. 2005. Potential winter hosts of soybean aphid. Ann.Entomol. Soc. Am. 98: 690Ð693.

Voegtlin,D. J., S. E.Halbert, andG.X.Qiao. 2004b. A guideto separating Aphis glycines Matsumura and morpholog-ically similar species that share its hosts. Ann. Entomol.Soc. Am. 97: 227Ð232.

vonDohlen, C. D., andD.A.J. Teulon. 2003. Phylogeny andhistorical biogeography of New Zealand indigenousAphidini aphids (Hemiptera, Aphididae): an hypothesis.Ann. Entomol. Soc. Am. 96: 107Ð116.

vonDohlen, C. D., C. A. Rowe, and O. E. Heie. 2006. A testof morphological hypotheses for tribal and subtribal re-lationships of Aphidinae (Insecta: Hemiptera: Aphidi-dae) using DNA sequences. Mol. Phylogenet. Evol. 38:316Ð329.

Wu, Z. S., D. Schenk-Hamlin, W. Y. Zhan, D. W. Ragsdale,and G. E. Heimpel. 2004. The soybean aphid in China:a historical review. Ann. Entomol. Soc. Am. 97: 209Ð218.

Yoo, H.J.S., R. J. O’Neil, D. J. Voegtlin, and W. R. Graves.2005. Plant suitability of Rhamnaceae for soybean aphid(Homoptera: Aphididae). Ann. Entomol. Soc. Am. 98:926Ð930.

Received 14 November 2009; accepted 23 April 2010.


systematics molecular and morphological identiï¬cation of

Documents